Population patch clamp electrophysiology: a breakthrough technology for ion channel screening.

PubMed

Dale, Tim J; Townsend, Claire; Hollands, Emma C; Trezise, Derek J

2007-10-01

Population patch clamp (PPC) is a novel high throughput planar array electrophysiology technique that allows ionic currents to be recorded from populations of cells under voltage clamp. For the drug discovery pharmacologist, PPC promises greater speed and precision than existing methods for screening compounds at voltage-gated ion channel targets. Moreover, certain constitutively active or slow-ligand gated channels that have hitherto proved challenging to screen with planar array electrophysiology (e.g. SK/IK channels) are now more accessible. In this article we review early findings using PPC and provide a perspective on its likely impact on ion channel drug discovery. To support this, we include some new data on ion channel assay duplexing and on modulator assays, approaches that have thus far not been described.

Investigating ion channel conformational changes using voltage clamp fluorometry.

PubMed

Talwar, Sahil; Lynch, Joseph W

2015-11-01

Ion channels are membrane proteins whose functions are governed by conformational changes. The widespread distribution of ion channels, coupled with their involvement in most physiological and pathological processes and their importance as therapeutic targets, renders the elucidation of these conformational mechanisms highly compelling from a drug discovery perspective. Thanks to recent advances in structural biology techniques, we now have high-resolution static molecular structures for members of the major ion channel families. However, major questions remain to be resolved about the conformational states that ion channels adopt during activation, drug modulation and desensitization. Patch-clamp electrophysiology has long been used to define ion channel conformational states based on functional criteria. It achieves this by monitoring conformational changes at the channel gate and cannot detect conformational changes occurring in regions distant from the gate. Voltage clamp fluorometry involves labelling cysteines introduced into domains of interest with environmentally sensitive fluorophores and inferring structural rearrangements from voltage or ligand-induced fluorescence changes. Ion channel currents are monitored simultaneously to verify the conformational status. By defining real time conformational changes in domains distant from the gate, this technique provides unexpected new insights into ion channel structure and function. This review aims to summarise the methodology and highlight recent innovative applications of this powerful technique. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 Elsevier Ltd. All rights reserved.

An equivalent network representation of a clamped bimorph piezoelectric micromachined ultrasonic transducer with circular and annular electrodes using matrix manipulation techniques.

PubMed

Sammoura, Firas; Smyth, Katherine; Kim, Sang-Gook

2013-09-01

An electric circuit model for a clamped circular bimorph piezoelectric micromachined ultrasonic transducer (pMUT) was developed for the first time. The pMUT consisted of two piezoelectric layers sandwiched between three thin electrodes. The top and bottom electrodes were separated into central and annular electrodes by a small gap. While the middle electrode was grounded, the central and annular electrodes were biased with two independent voltage sources. The strain mismatch between the piezoelectric layers caused the plate to vibrate and transmit a pressure wave, whereas the received echo generated electric charges resulting from plate deformation. The clamped pMUT plate was separated into a circular and an annular plate, and the respective electromechanical transformation matrices were derived. The force and velocity vectors were properly selected using Hamilton's principle and the necessary boundary conditions were invoked. The electromechanical transformation matrix for the clamped circular pMUT was deduced using simple matrix manipulation techniques. The pMUT performance under three biasing schemes was elaborated: 1) central electrode only, 2) central and annular electrodes with voltages of the same magnitude and polarity, and 3) central and annular electrodes with voltages of the same magnitude and opposite polarity. The circuit parameters of the pMUT were extracted for each biasing scheme, including the transformer ratio, the clamped electric impedance, and the open-circuit mechanical impedance. Each pMUT scheme was characterized under different acoustic loadings using the theoretically developed model, which was verified with finite element modeling (FEM) simulation. The electrode size was optimized to maximize the electromechanical transformer ratio. As such, the developed model could provide more insight into the design, optimization, and characterization of pMUTs and allow for performance comparison with their cMUT counterparts.

Sodium influxes in internally perfused squid giant axon during voltage clamp.

PubMed

Atwater, I; Bezanilla, F; Rojas, E

1969-05-01

1. An experimental method for measuring ionic influxes during voltage clamp in the giant axon of Dosidicus is described; the technique combines intracellular perfusion with a method for controlling membrane potential.2. Sodium influx determinations were carried out while applying rectangular pulses of membrane depolarization. The ratio ;measured sodium influx/computed ionic flux during the early current' is 0.92 +/- 0.12.3. Plots of measured sodium influx and computed ionic flux during the early current against membrane potential are very similar. There was evidence that the membrane potential at which the sodium influx vanishes is the potential at which the early current reverses.

Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach

PubMed Central

Upadhye, Kalpesh V.; Candiello, Joseph E.; Davidson, Lance A.; Lin, Hai

2011-01-01

Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins. PMID:22174731

Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics.

PubMed

Beattie, Kylie A; Hill, Adam P; Bardenet, Rémi; Cui, Yi; Vandenberg, Jamie I; Gavaghan, David J; de Boer, Teun P; Mirams, Gary R

2018-03-24

Ion current kinetics are commonly represented by current-voltage relationships, time constant-voltage relationships and subsequently mathematical models fitted to these. These experiments take substantial time, which means they are rarely performed in the same cell. Rather than traditional square-wave voltage clamps, we fitted a model to the current evoked by a novel sum-of-sinusoids voltage clamp that was only 8 s long. Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions. The new model predicts the current under traditional square-wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically relevant series of action potential clamps is predicted extremely well. The short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell-cell variability in current kinetics for the first time. Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics - the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fitted a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells overexpressing hERG1a. The model was then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage clamps, and also a novel voltage clamp comprising a collection of physiologically relevant action potentials. We demonstrate that we can make predictive cell-specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell-cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches. © 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Sodium influxes in internally perfused squid giant axon during voltage clamp

PubMed Central

Atwater, I.; Bezanilla, F.; Rojas, E.

1969-01-01

1. An experimental method for measuring ionic influxes during voltage clamp in the giant axon of Dosidicus is described; the technique combines intracellular perfusion with a method for controlling membrane potential. 2. Sodium influx determinations were carried out while applying rectangular pulses of membrane depolarization. The ratio `measured sodium influx/computed ionic flux during the early current' is 0·92 ± 0·12. 3. Plots of measured sodium influx and computed ionic flux during the early current against membrane potential are very similar. There was evidence that the membrane potential at which the sodium influx vanishes is the potential at which the early current reverses. PMID:5767887

Voltage dependence of the rat chorda tympani response to Na+ salts: implications for the functional organization of taste receptor cells.

PubMed

Ye, Q; Heck, G L; DeSimone, J A

1993-07-01

1. Voltage-clamp and current-clamp data were obtained from a circumscribed region of the anterior rat lingual epithelium while simultaneously monitoring the afferent, stimulus-evoked, neural response from the same receptive field. 2. Chorda tympani (CT) responses at constant Na(+)-salt concentration were enhanced by submucosa negative voltage clamp and suppressed by positive voltage clamp. The complete CT response profile, including the time course of adaptation, was not uniquely determined by NaCl concentration alone. The response could be reproduced at different NaCl concentrations by applying a compensating voltage. 3. The form of the concentration and voltage dependence of the CT response indicates that the complete stimulus energy is the Na+ electrochemical potential difference across receptor cell apical membranes, and not Na+ concentration alone. This is the underlying principal behind the equivalence of chemical and electric taste for Na+ salts. 4. CT responses to sodium gluconate (25 and 200 mM) and 25 mM NaCl produced amiloride-insensitive components (AIC) of low magnitude. NaCl at 200 mM produced a significantly larger AIC. The AIC was voltage-clamp independent. The relative magnitude of the AIC was positively correlated with the transepithelial conductance of each salt. This suggests that the large AIC for 200 mM NaCl results from its relatively high permeability through the paracellular pathway. 5. Analysis of the CT response under voltage clamp revealed two anion effects on Na(+)-salt taste, both of which act through the paracellular shunt. 1) Anions modify the transepithelial potential (TP) across tight junctions and thereby modulate the cell receptor potential. This anion effect can be eliminated by voltage clamping the TP. 2) Sufficiently mobile anions facilitate electroneutral diffusion of Na+ salts through tight junctions. This effect is observed especially when Cl- is the anion and when the stimulus concentration favors NaCl influx, allowing Na+ to stimulate receptor cells from the submucosal side. Because the submucosal intercellular spaces are nearly isopotential regions, this effect is insensitive to voltage clamp of the TP. The large AIC associated with this anion effect is due to the low permeability of amiloride.

Separate Cl^- Conductances Activated by cAMP and Ca2+ in Cl^--Secreting Epithelial Cells

NASA Astrophysics Data System (ADS)

Cliff, William H.; Frizzell, Raymond A.

1990-07-01

We studied the cAMP- and Ca2+-activated secretory Cl^- conductances in the Cl^--secreting colonic epithelial cell line T84 using the whole-cell patch-clamp technique. Cl^- and K^+ currents were measured under voltage clamp. Forskolin or cAMP increased Cl^- current 2-15 times with no change in K^+ current. The current-voltage relation for cAMP-activated Cl^- current was linear from -100 to +100 mV and showed no time-dependent changes in current during voltage pulses. Ca2+ ionophores or increased pipette Ca2+ increased both Cl^- and K^+ currents 2-30 times. The Ca2+-activated Cl^- current was outwardly rectified, activated during depolarizing voltage pulses, and inactivated during hyperpolarizing voltage pulses. Addition of ionophore after forskolin further increased Cl^- conductance 1.5-5 times, and the current took on the time-dependent characteristics of that stimulated by Ca2+. Thus, cAMP and Ca2+ activate Cl^- conductances with different properties, implying that these second messengers activate different Cl^- channels or that they induce different conductive and kinetic states in the same Cl^- channel.

Tetrodotoxin-sensitive, voltage-dependent sodium currents in hair cells from the alligator cochlea.

PubMed

Evans, M G; Fuchs, P A

1987-10-01

We have used whole-cell patch clamp techniques to record from tall hair cells isolated from the apical half of the alligator cochlea. Some of these cells gave action potentials in response to depolarizing current injections. When the same cells were voltage clamped, large transient inward currents followed by smaller outward currents were seen in response to depolarizing steps. We studied the transient inward current after the outward current had been blocked by external tetraethylammonium (20 mM) or by replacing internal potassium with cesium. It was found to be a sodium current because it was abolished by either replacing external sodium with choline or by external application of tetrodotoxin (100 nM). The sodium current showed voltage-dependent activation and inactivation. Most of the spiking hair cells came from the apex of the cochlea, where they would be subject to low-frequency mechanical stimulation in vivo.

Preparation of Drosophila central neurons for in situ patch clamping.

PubMed

Ryglewski, Stefanie; Duch, Carsten

2012-10-15

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker(1,2). Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain(3,4) and ventral nerve cord of embryonic(5,6), larval(7,8,9,10), and adult Drosophila(11,12,13,14). A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5(15)), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

The Mechanism of Extracellular Stimulation of Nerve Cells on an Electrolyte-Oxide-Semiconductor Capacitor

PubMed Central

Schoen, Ingmar; Fromherz, Peter

2007-01-01

Extracellular excitation of neurons is applied in studies of cultured networks and brain tissue, as well as in neuroprosthetics. We elucidate its mechanism in an electrophysiological approach by comparing voltage-clamp and current-clamp recordings of individual neurons on an insulated planar electrode. Noninvasive stimulation of neurons from pedal ganglia of Lymnaea stagnalis is achieved by defined voltage ramps applied to an electrolyte/HfO2/silicon capacitor. Effects on the smaller attached cell membrane and the larger free membrane are distinguished in a two-domain-stimulation model. Under current-clamp, we study the polarization that is induced for closed ion channels. Under voltage-clamp, we determine the capacitive gating of ion channels in the attached membrane by falling voltage ramps and for comparison also the gating of all channels by conventional variation of the intracellular voltage. Neuronal excitation is elicited under current-clamp by two mechanisms: Rising voltage ramps depolarize the free membrane such that an action potential is triggered. Falling voltage ramps depolarize the attached membrane such that local ion currents are activated that depolarize the free membrane and trigger an action potential. The electrophysiological analysis of extracellular stimulation in the simple model system is a basis for its systematic optimization in neuronal networks and brain tissue. PMID:17098803

Real-Time Kinetic Modeling of Voltage-Gated Ion Channels Using Dynamic Clamp

PubMed Central

Milescu, Lorin S.; Yamanishi, Tadashi; Ptak, Krzysztof; Mogri, Murtaza Z.; Smith, Jeffrey C.

2008-01-01

We propose what to our knowledge is a new technique for modeling the kinetics of voltage-gated ion channels in a functional context, in neurons or other excitable cells. The principle is to pharmacologically block the studied channel type, and to functionally replace it with dynamic clamp, on the basis of a computational model. Then, the parameters of the model are modified in real time (manually or automatically), with the objective of matching the dynamical behavior of the cell (e.g., action potential shape and spiking frequency), but also the transient and steady-state properties of the model (e.g., those derived from voltage-clamp recordings). Through this approach, one may find a model and parameter values that explain both the observed cellular dynamics and the biophysical properties of the channel. We extensively tested the method, focusing on Nav models. Complex Markov models (10–12 states or more) could be accurately integrated in real time at >50 kHz using the transition probability matrix, but not the explicit Euler method. The practicality of the technique was tested with experiments in raphe pacemaker neurons. Through automated real-time fitting, a Hodgkin-Huxley model could be found that reproduced well the action potential shape and the spiking frequency. Adding a virtual axonal compartment with a high density of Nav channels further improved the action potential shape. The computational procedure was implemented in the free QuB software, running under Microsoft Windows and featuring a friendly graphical user interface. PMID:18375511

An equivalent circuit for small atrial trabeculae of frog.

PubMed Central

Jakobsson, E; Barr, L; Connor, J A

1975-01-01

An equivalent electrical circuit has been constructed for small atrial trabecula of frog in a double sucrose gap voltage clamp apparatus. The basic strategy in constructing the circuit was to derive the distribution of membrane capacitance and extracellular resistance from the preparation's response to small voltage displacements near the resting condition, when the membrane conductance is presumably quite low. Then standard Hodgkin-Huxley channels were placed in parallel with the capacitance and the results of voltage clamp experiments were simulated. The results suggest that the membranes of the preparation cannot in fact be clamped near the control voltage nor can the ionic currents be measured directly with reasonable accuracy by axon standards. It may or may not be a realizable goal in the future to define the preparation's electrical behavior well enough to permit the ultimate quantitative description of the membrane's specific ion conductances. The result of this paper suggest that if this goal is achieved using the double sucrose gap voltage clamp, it will be by a detailed quantitative accounting for substantial irreducible errors in voltage control, rather than by experimental achievement of good voltage control. PMID:1203441

Effects of tacrolimus on action potential configuration and transmembrane ion currents in canine ventricular cells.

PubMed

Szabó, László; Szentandrássy, Norbert; Kistamás, Kornél; Hegyi, Bence; Ruzsnavszky, Ferenc; Váczi, Krisztina; Horváth, Balázs; Magyar, János; Bányász, Tamás; Pál, Balázs; Nánási, Péter P

2013-03-01

Tacrolimus is a commonly used immunosuppressive agent which causes cardiovascular complications, e.g., hypertension and hypertrophic cardiomyopathy. In spite of it, there is little information on the cellular cardiac effects of the immunosuppressive agent tacrolimus in larger mammals. In the present study, therefore, the concentration-dependent effects of tacrolimus on action potential morphology and the underlying ion currents were studied in canine ventricular cardiomyocytes. Standard microelectrode, conventional whole cell patch clamp, and action potential voltage clamp techniques were applied in myocytes enzymatically dispersed from canine ventricular myocardium. Tacrolimus (3-30 μM) caused a concentration-dependent reduction of maximum velocity of depolarization and repolarization, action potential amplitude, phase-1 repolarization, action potential duration, and plateau potential, while no significant change in the resting membrane potential was observed. Conventional voltage clamp experiments revealed that tacrolimus concentrations ≥3 μM blocked a variety of ion currents, including I(Ca), I(to), I(K1), I(Kr), and I(Ks). Similar results were obtained under action potential voltage clamp conditions. These effects of tacrolimus developed rapidly and were fully reversible upon washout. The blockade of inward currents with the concomitant shortening of action potential duration in canine myocytes is the opposite of those observed previously with tacrolimus in small rodents. It is concluded that although tacrolimus blocks several ion channels at higher concentrations, there is no risk of direct interaction with cardiac ion channels when applying tacrolimus in therapeutic concentrations.

Correlation of open cell-attached and excised patch clamp techniques.

PubMed

Filipovic, D; Hayslett, J P

1995-11-01

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na+ channel, located on the apical surface of A6 cells, to determine whether the nystatin-induced open cell-attached patch could serve as an alternative configuration. Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and Erev was zero in symmetrical Na+ solutions, indicating similar Na+ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3-4pS) and Erev (75 mV), when the bath was perfused with a high K:low Na solution (ENa = 80 mV), were nearly equal in both patch configurations. Our results therefore indicate that the nystatin-induced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.

A self-strain feedback tuning-fork-shaped ionic polymer metal composite clamping actuator with soft matter elasticity-detecting capability for biomedical applications.

PubMed

Feng, Guo-Hua; Huang, Wei-Lun

2014-12-01

This paper presents a smart tuning-fork-shaped ionic polymer metal composite (IPMC) clamping actuator for biomedical applications. The two fingers of the actuator, which perform the clamping motion, can be electrically controlled through a unique electrode design on the IPMC material. The generated displacement or strain of the fingers can be sensed using an integrated soft strain-gage sensor. The IPMC actuator and associated soft strain gage were fabricated using a micromachining technique. A 13.5×4×2 mm(3) actuator was shaped from Nafion solution and a selectively grown metal electrode formed the active region. The strain gage consisted of patterned copper foil and polyethylene as a substrate. The relationship between the strain gage voltage output and the displacement at the front end of the actuator's fingers was characterized. The equivalent Young's modulus, 13.65 MPa, of the soft-strain-gage-integrated IPMC finger was analyzed. The produced clamping force exhibited a linear increasing rate of 1.07 mN/s, based on a dc driving voltage of 7 V. Using the developed actuator to clamp soft matter and simultaneously acquire its Young's modulus was achieved. This demonstrated the feasibility of the palpation function and the potential use of the actuator in minimally invasive surgery. Copyright © 2014 Elsevier B.V. All rights reserved.

Performance of Li-Ion Cells Under Battery Voltage Charge Control

NASA Technical Reports Server (NTRS)

Rao, Gopalakrishna M.; Vaidyanathan, Hari; Day, John H. (Technical Monitor)

2001-01-01

A study consisting of electrochemical characterization and Low-Earth-Orbit (LEO) cycling of Li-Ion cells from three vendors was initiated in 1999 to determine the cycling performance and to infuse the new technology in the future NASA missions. The 8-cell batteries included in this evaluation are prismatic cells manufactured by Mine Safety Appliances Company (MSA), cylindrical cells manufactured by SAFT and prismatic cells manufactured by Yardney Technical Products, Inc. (YTP). The three batteries were cycle tested in the LEO regime at 40% depth of discharge, and under a charge control technique that consists of battery voltage clamp with a current taper. The initial testing was conducted at 20 C; however, the batteries were cycled also intermittently at low temperatures. YTP 20 Ah cells consisted of mixed-oxide (Co and Ni) positive, graphitic carbon negative, LIPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was 32 V. The low temperature cycling tests started after 4575 cycles at 20 C. The cells were not capable of cycling. at low temperature since the charge acceptance at battery level was poor. There was a cell in the battery that showed too high an end-of-charge (EOC) voltage thereby limiting the ability to charge the rest of the cells in the battery. The battery has completed 6714 cycles. SAFT 12 Ah cells consisted of mixed-oxide (Co and NO positive, graphitic carbon negative, LiPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was for 30.8 V. The low temperature cycling tests started after 4594 cycles at 20 C. A cell that showed low end of discharge (EOD) and EOC voltages and three other cells that showed higher EOC voltages limited the charge acceptance at the selected voltage limit during charge. The cells were capable of cycling at 10 C and 0 C but the charge voltage limit had to be increased to 34.3 V (4.3 V per cell). The low temperature cycling may have induced poor chargeability since the voltage had to be increased to achieve the required charge input. The battery has completed 6226 cycles. MSA 10 Ah cells consisted of Co oxide positive, graphitic carbon negative, LiPF6 salt mixed with organic carbonate solvents. The battery voltage clamp was 30.8 V. The low temperature cycling tests were started after 2182 cycles at 20 C. The cells were capable of cycling at 10 C and 0 C. Like SAFT, the voltage limit on charge had to be increased to 36 V (4.5 V per cell). There was a cell (cell S/N 13) in the battery that showed poor performance features such as low EOD voltage and high EOC voltage. The battery has completed 3441 cycles. A reconditioning procedure that consisted of C15 charge to a taper current of C/100 and C/20 discharge improved the voltage behavior of SAFT and MSA cells with no significant effect on YTP cells. We have demonstrated that the charge operation with VT clamp at battery rather than at cell level is feasible for onboard Li-Ion battery operation.

Giga-seal formation alters properties of sodium channels of human myoballs.

PubMed

Fahlke, C; Rüdel, R

1992-03-01

The influence of giga-seal formation on the properties of the Na+ channels within the covered membrane patch was investigated with a whole-cell pipette and a patch pipette applied to the same cell. Current kinetics, current/voltage relation and channel densities were determined in three combinations: (i) voltage-clamping and current recording with the whole-cell pipette, (ii) voltage-clamping with the whole-cell pipette and current recording with the patch pipette and, (iii) voltage-clamping and current recording with the patch pipette. The Hodgkin-Huxley (1952) parameters tau m and tau h were smaller for the patch currents than for the whole cell, and the h infinity curve was shifted in the negative direction. The channel density was of the order of 10 times smaller. All effects were independent of the extracellular Ca2+ concentration. The capacitive current generated in the patch by the whole-cell Na+ current and its effect on the transmembrane voltage of the patch were evaluated. The kinetic parameters of the Na+ channels in the patch did not depend on whether the voltage was clamped with the whole-cell pipette or the patch pipette. Thus, the results are not due to spurious voltage.

Nicergoline inhibits T-type Ca2+ channels in rat isolated hippocampal CA1 pyramidal neurones.

PubMed Central

Takahashi, K.; Akaike, N.

1990-01-01

1. The effects of nicergoline on the T- and L-type Ca2+ currents in pyramidal cells freshly isolated from rat hippocampal CA1 region were investigated by use of a 'concentration-clamp' technique. The technique combines a suction-pipette technique, which allows intracellular perfusion under a single-electrode voltage-clamp, and rapid exchange of extracellular solution within 2 ms. 2. T-type Ca2+ currents were evoked by step depolarizations from a holding potential of -100 mV to potentials more positive than -70 to -60 mV, and reached a peak at about -30 mV in the current-voltage relationship. Activation and inactivation of T-type Ca2+ currents were highly potential-dependent. 3. Nicergoline and other Ca2+ antagonists dose-dependently blocked the T-type Ca2+ channel with an order of potency nicardipine greater than nicergoline greater than diltiazem. 4. The L-type Ca2+ channel was also blocked in the order nicardipine greater than nicergoline greater than diltiazem, although the T-type Ca2+ channel was more sensitive to nicergoline. 5. The inhibitory effects of nicergoline and nicardipine on the T-type Ca2+ current were voltage-, time-, and use-dependent, and the inhibition increased with a decrease in the external Ca2+ concentration. Diltiazem showed only a use-dependent block. PMID:2169937

Nicergoline inhibits T-type Ca2+ channels in rat isolated hippocampal CA1 pyramidal neurones.

PubMed

Takahashi, K; Akaike, N

1990-08-01

1. The effects of nicergoline on the T- and L-type Ca2+ currents in pyramidal cells freshly isolated from rat hippocampal CA1 region were investigated by use of a 'concentration-clamp' technique. The technique combines a suction-pipette technique, which allows intracellular perfusion under a single-electrode voltage-clamp, and rapid exchange of extracellular solution within 2 ms. 2. T-type Ca2+ currents were evoked by step depolarizations from a holding potential of -100 mV to potentials more positive than -70 to -60 mV, and reached a peak at about -30 mV in the current-voltage relationship. Activation and inactivation of T-type Ca2+ currents were highly potential-dependent. 3. Nicergoline and other Ca2+ antagonists dose-dependently blocked the T-type Ca2+ channel with an order of potency nicardipine greater than nicergoline greater than diltiazem. 4. The L-type Ca2+ channel was also blocked in the order nicardipine greater than nicergoline greater than diltiazem, although the T-type Ca2+ channel was more sensitive to nicergoline. 5. The inhibitory effects of nicergoline and nicardipine on the T-type Ca2+ current were voltage-, time-, and use-dependent, and the inhibition increased with a decrease in the external Ca2+ concentration. Diltiazem showed only a use-dependent block.

Niflumic acid alters gating of HCN2 pacemaker channels by interaction with the outer region of S4 voltage sensing domains.

PubMed

Cheng, Lan; Sanguinetti, Michael C

2009-05-01

Niflumic acid, 2-[[3-(trifluoromethyl)phenyl]amino]pyridine-3-carboxylic acid (NFA), is a nonsteroidal anti-inflammatory drug that also blocks or modifies the gating of many ion channels. Here, we investigated the effects of NFA on hyperpolarization-activated cyclic nucleotide-gated cation (HCN) pacemaker channels expressed in X. laevis oocytes using site-directed mutagenesis and the two-electrode voltage-clamp technique. Extracellular NFA acted rapidly and caused a slowing of activation and deactivation and a hyperpolarizing shift in the voltage dependence of HCN2 channel activation (-24.5 +/- 1.2 mV at 1 mM). Slowed channel gating and reduction of current magnitude was marked in oocytes treated with NFA, while clamped at 0 mV but minimal in oocytes clamped at -100 mV, indicating the drug preferentially interacts with channels in the closed state. NFA at 0.1 to 3 mM shifted the half-point for channel activation in a concentration-dependent manner, with an EC(50) of 0.54 +/- 0.068 mM and a predicted maximum shift of -38 mV. NFA at 1 mM also reduced maximum HCN2 conductance by approximately 20%, presumably by direct block of the pore. The rapid onset and state-dependence of NFA-induced changes in channel gating suggests an interaction with the extracellular region of the S4 transmembrane helix, the primary voltage-sensing domain of HCN2. Neutralization (by mutation to Gln) of any three of the outer four basic charged residues in S4, but not single mutations, abrogated the NFA-induced shift in channel activation. We conclude that NFA alters HCN2 gating by interacting with the extracellular end of the S4 voltage sensor domains.

Fabrication and characterization of a piezoelectric energy harvester with clamped-clamped beams

NASA Astrophysics Data System (ADS)

Cui, Yan; Yu, Menglin; Gao, Shiqiao; Kong, Xiangxin; Gu, Wang; Zhang, Ran; Liu, Bowen

2018-05-01

This work presents a piezoelectric energy harvester with clamped-clamped beams, and it is fabricated with MEMS process. When excited by sinusoidal vibration, the energy harvester has a sharp jumping down phenomenon and the measured frequency responses of the clamped-clamped beams structure show a larger bandwidth which is about 56Hz, more efficient than that with cantilever beams. When the exciting acceleration ac is 12m/s2, the energy harvester achieves to a maximum open-circuit voltage of 94mV on one beam. The load voltage is proportional to the load resistance, and it increased with the increase of load resistance. Connected four beams in series, the output power reaches the maximum value of 730 nW and the optimal load is 15KΩ to one beam.

Kinetics and components of the flash photocurrent of isolated retinal rods of the larval salamander, Ambystoma tigrinum.

PubMed Central

Cobbs, W H; Pugh, E N

1987-01-01

1. Membrane currents initiated by intense, 20 microseconds flashes (photocurrents) were recorded from isolated salamander rods by combined extracellular suction electrodes and intracellular tight-seal electrodes either in current or voltage clamp mode. The magnitudes (mean +/- 2 S.E.M.) of the maximal photoresponses recorded by the suction and by the intracellular electrode respectively were 40 +/- 5 pA (n = 18) and 35 +/- 7 mV (n = 8) for current clamp at zero current; 43 +/- 9 pA and 66 +/- 13 (n = 11) pA for voltage clamp at the zero-current holding potential, -24 +/- 3 mV. 2. Photocurrents initiated by flashes isomerizing 0.1% or more of the outer segment's rhodopsin achieved a saturated velocity and were 95% complete in less than 50 ms. The effect of incrementing flash intensity above 0.1% isomerization can be described as a translation of the photocurrent along the time axis towards the origin. Within the interval 0-50 ms the latter two-thirds of the velocity-saturated photocurrent is well described as a single-exponential decay. The decay was much faster in voltage clamp (2.8 +/- 1.2 ms, n = 11) than in current clamp mode (17 +/- 5 ms, n = 17). 3. The initial third of the velocity-saturated photocurrent, occurring over the interval from the flash to the onset of exponential decay, followed about the same time course in current and voltage clamp. The time interval occupied by this initial 'latent' phase decreased with increasing flash intensity and attained an apparent minimum of about 7 ms in response to flashes isomerizing 10% or more of the rhodopsin at ca. 22 degrees C. 4. The hypothesis that the decay of outer segment light-sensitive membrane current is the same in current and voltage clamp was supported by an analysis of the difference between outer segment currents measured successively in the two recording modes. First, the tail of the difference current decayed exponentially with a time constant approximately equal to R x C, where R and C are independently estimated slope resistance and capacitance of the rod. Secondly, the integral of the difference current, when divided by outer segment capacitance, closely approximated the hyperpolarizing light response measured under current clamp. Thus, displacement current accounted for the difference between photocurrents measured in current and voltage clamp.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2832596

Auxiliary quasi-resonant dc tank electrical power converter

DOEpatents

Peng, Fang Z.

2006-10-24

An auxiliary quasi-resonant dc tank (AQRDCT) power converter with fast current charging, voltage balancing (or charging), and voltage clamping circuits is provided for achieving soft-switched power conversion. The present invention is an improvement of the invention taught in U.S. Pat. No. 6,111,770, herein incorporated by reference. The present invention provides faster current charging to the resonant inductor, thus minimizing delay time of the pulse width modulation (PWM) due to the soft-switching process. The new AQRDCT converter includes three tank capacitors or power supplies to achieve the faster current charging and minimize the soft-switching time delay. The new AQRDCT converter further includes a voltage balancing circuit to charge and discharge the three tank capacitors so that additional isolated power supplies from the utility line are not needed. A voltage clamping circuit is also included for clamping voltage surge due to the reverse recovery of diodes.

Accurate measurement of junctional conductance between electrically coupled cells with dual whole-cell voltage-clamp under conditions of high series resistance.

PubMed

Hartveit, Espen; Veruki, Margaret Lin

2010-03-15

Accurate measurement of the junctional conductance (G(j)) between electrically coupled cells can provide important information about the functional properties of coupling. With the development of tight-seal, whole-cell recording, it became possible to use dual, single-electrode voltage-clamp recording from pairs of small cells to measure G(j). Experiments that require reduced perturbation of the intracellular environment can be performed with high-resistance pipettes or the perforated-patch technique, but an accompanying increase in series resistance (R(s)) compromises voltage-clamp control and reduces the accuracy of G(j) measurements. Here, we present a detailed analysis of methodologies available for accurate determination of steady-state G(j) and related parameters under conditions of high R(s), using continuous or discontinuous single-electrode voltage-clamp (CSEVC or DSEVC) amplifiers to quantify the parameters of different equivalent electrical circuit model cells. Both types of amplifiers can provide accurate measurements of G(j), with errors less than 5% for a wide range of R(s) and G(j) values. However, CSEVC amplifiers need to be combined with R(s)-compensation or mathematical correction for the effects of nonzero R(s) and finite membrane resistance (R(m)). R(s)-compensation is difficult for higher values of R(s) and leads to instability that can damage the recorded cells. Mathematical correction for R(s) and R(m) yields highly accurate results, but depends on accurate estimates of R(s) throughout an experiment. DSEVC amplifiers display very accurate measurements over a larger range of R(s) values than CSEVC amplifiers and have the advantage that knowledge of R(s) is unnecessary, suggesting that they are preferable for long-duration experiments and/or recordings with high R(s). Copyright (c) 2009 Elsevier B.V. All rights reserved.

A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

PubMed

Wang, Hong-Gang; Zhu, Wandi; Kanter, Ronald J; Silva, Jonathan R; Honeywell, Christina; Gow, Robert M; Pitt, Geoffrey S

2016-03-01

Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome. We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current. The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydrogen ion block of the sodium pore in squid giant axons

PubMed Central

1983-01-01

The block of squid axon sodium channels by H ions was studied using voltage-clamp and internal perfusion techniques. An increase in the concentration of internal permeant ions decreased the block produced by external H ions. The voltage dependence of the block was found to be nonmonotonic: it was reduced by both large positive and large negative potentials. The ability of internal ions to modify the block by external H+ is explained by a competition among these ions for a binding site within the pore. The nonmonotonic voltage dependence is consistent with this picture if the hydrogen ions are allowed to be permeant. PMID:6315859

Effects of pioglitazone on cardiac ion currents and action potential morphology in canine ventricular myocytes.

PubMed

Kistamás, Kornél; Szentandrássy, Norbert; Hegyi, Bence; Ruzsnavszky, Ferenc; Váczi, Krisztina; Bárándi, László; Horváth, Balázs; Szebeni, Andrea; Magyar, János; Bányász, Tamás; Kecskeméti, Valéria; Nánási, Péter P

2013-06-15

Despite its widespread therapeutical use there is little information on the cellular cardiac effects of the antidiabetic drug pioglitazone in larger mammals. In the present study, therefore, the concentration-dependent effects of pioglitazone on ion currents and action potential configuration were studied in isolated canine ventricular myocytes using standard microelectrode, conventional whole cell patch clamp, and action potential voltage clamp techniques. Pioglitazone decreased the maximum velocity of depolarization and the amplitude of phase-1 repolarization at concentrations ≥3 μM. Action potentials were shortened by pioglitazone at concentrations ≥10 μM, which effect was accompanied with significant reduction of beat-to-beat variability of action potential duration. Several transmembrane ion currents, including the transient outward K(+) current (Ito), the L-type Ca(2+) current (ICa), the rapid and slow components of the delayed rectifier K(+) current (IKr and IKs, respectively), and the inward rectifier K(+) current (IK1) were inhibited by pioglitazone under conventional voltage clamp conditions. Ito was blocked significantly at concentrations ≥3 μM, ICa, IKr, IKs at concentrations ≥10 μM, while IK1 at concentrations ≥30 μM. Suppression of Ito, ICa, IKr, and IK1 has been confirmed also under action potential voltage clamp conditions. ATP-sensitive K(+) current, when activated by lemakalim, was effectively blocked by pioglitazone. Accordingly, action potentials were prolonged by 10 μM pioglitazone when the drug was applied in the presence of lemakalim. All these effects developed rapidly and were readily reversible upon washout. In conclusion, pioglitazone seems to be a harmless agent at usual therapeutic concentrations. Copyright © 2013 Elsevier B.V. All rights reserved.

Perturbed atrial calcium handling in an ovine model of heart failure: Potential roles for reductions in the L-type calcium current

PubMed Central

Clarke, Jessica D.; Caldwell, Jessica L.; Horn, Margaux A.; Bode, Elizabeth F.; Richards, Mark A.; Hall, Mark C.S.; Graham, Helen K.; Briston, Sarah J.; Greensmith, David J.; Eisner, David A.; Dibb, Katharine M.; Trafford, Andrew W.

2015-01-01

Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during β-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca2 + and the response to β-adrenergic (β-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca2 + concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca2 + transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca2 + removal (kSR, by 32%), L-type Ca2 + current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca2 + content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca2 + current (ICa-L) in control cells reproduced both the decrease in Ca2 + transient amplitude and increase of SR Ca2 + content observed in voltage-clamped HF cells. During β-AR stimulation Ca2 + transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca2 + content was highest in HF cells during β-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca2 + transient amplitude and increased SR Ca2 + content observed in voltage-clamped cells. PMID:25463272

A monolithic patch-clamping amplifier with capacitive feedback.

PubMed

Prakash, J; Paulos, J J; Jensen, D N

1989-03-01

Patch-clamping is an established method for directly measuring ionic transport through cellular membranes with sufficient resolution to observe open/close transitions of individual channel molecules. This paper describes an alternative technique for patch-clamping which uses a capacitor as the transimpedance element. This approach eliminates bandwidth and saturation limitations experienced with resistive patch-clamping amplifiers. A complete monolithic design featuring an on-chip operational amplifier, a capacitor array with gain-ranging from 30 pF down to 0.03 pF, and reset and gain ranging switches has been fabricated using 5 microns CMOS technology. It is shown that the voltage noise of the CMOS operational amplifier limits the overall noise performance, but that performance competitive with conventional instruments can be achieved over a 10 kHz bandwidth, at least for small input capacitances (less than or equal to 5 pF). Results are presented along with an analysis and comparison of noise performance using both resistive and capacitive elements.

Elevated Neuronal Excitability Due to Modulation of the Voltage-Gated Sodium Channel Nav1.6 by Aβ1−42

PubMed Central

Wang, Xi; Zhang, Xiao-Gang; Zhou, Ting-Ting; Li, Na; Jang, Chun-Yan; Xiao, Zhi-Cheng; Ma, Quan-Hong; Li, Shao

2016-01-01

Aberrant increases in neuronal network excitability may contribute to the cognitive deficits in Alzheimer's disease (AD). However, the mechanisms underlying hyperexcitability are not fully understood. Such overexcitation of neuronal networks has been detected in the brains of APP/PS1 mice. In the present study, using current-clamp recording techniques, we observed that 12 days in vitro (DIV) primary cultured pyramidal neurons from P0 APP/PS1 mice exhibited a more prominent action potential burst and a lower threshold than WT littermates. Moreover, after treatment with Aβ1−42 peptide, 12 DIV primary cultured neurons showed similar changes, to a greater degree than in controls. Voltage-clamp recordings revealed that the voltage-dependent sodium current density of neurons incubated with Aβ1−42 was significantly increased, without change in the voltage-dependent sodium channel kinetic characteristics. Immunohistochemistry and western blot results showed that, after treatment with Aβ1−42, expressions of Nav and Nav1.6 subtype increased in cultured neurons or APP/PS1 brains compared to control groups. The intrinsic neuronal hyperexcitability of APP/PS1 mice might thus be due to an increased expression of voltage-dependent sodium channels induced by Aβ1−42. These results may illuminate the mechanism of aberrant neuronal networks in AD. PMID:27013956

Properties of voltage-activated Na+ and K+ currents in mouse hippocampal glial cells in situ and after acute isolation from tissue slices.

PubMed

Steinhäuser, C; Kressin, K; Kuprijanova, E; Weber, M; Seifert, G

1994-10-01

In the present study, we were interested in a quantitative analysis of voltage-activated channels in a subpopulation of hippocampal glial cells, termed "complex" cells. The patch-clamp technique in the whole-cell mode was applied to identified cells in situ and to glial cells acutely isolated from tissue slices. The outward current was composed of two components: a sustained and a transient current. The transient K+ channel had electrophysiological and pharmacological properties resembling those of the channel through which the A-currents pass. In addition, this glial A-type current possessed a significant Ca2+ dependence. The current parameters determined in situ or in isolated cells corresponded well. Due to space clamp problems in situ, properties of voltage-dependent Na+ currents were only analysed in suspended glial cells. The tetrodotoxin (TTX) sensitivity and the stationary and kinetic characteristics of this current were similar to corresponding properties of hippocampal neurons. These quantitative data demonstrate that at an early postnatal stage of central nervous system maturation, glial cells in situ express a complex pattern of voltage-gated ion channels. The results are compared to findings in other preparations and the possible consequences of transmitter-mediated channel modulation in glial cells are discussed.

Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Birinyi-Strachan, Liesl C.; Gunning, Simon J.; Lewis, Richard J.

2005-04-15

The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Na{sub v}) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Na{sub v} channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons,more » P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Na{sub v} currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP, and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Na{sub v} channel gating, observed clinically in response to ciguatera poisoning.« less

Crebanine inhibits voltage-dependent Na+ current in guinea-pig ventricular myocytes.

PubMed

Xiao-Shan, He; Qing, Lin; Yun-Shu, Ma; Ze-Pu, Yu

2014-01-01

To study the effects of crebanine on voltage-gated Na(+) channels in cardiac tissues. Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na(+) current was recorded using the whole cell voltage-clamp technique. Crebanine reversibly inhibited Na(+) current with an IC50 value of 0.283 mmol·L(-1) (95% confidence range: 0.248-0.318 mmol·L(-1)). Crebanine at 0.262 mmol·L(-1) caused a negative shift (about 12 mV) in the voltage-dependence of steady-state inactivation of Na(+) current, and retarded its recovery from inactivation, but did not affect its activation curve. In addition to blocking other voltage-gated ion channels, crebanine blocked Na(+) channels in guinea-pig ventricular myocytes. Crebanine acted as an inactivation stabilizer of Na(+) channels in cardiac tissues. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Nitric oxide augments voltage-activated calcium currents of crustacea (Idotea baltica) skeletal muscle.

PubMed

Erxleben, C; Hermann, A

2001-03-16

Invertebrate skeletal muscle contraction is regulated by calcium influx through voltage-dependent calcium channels in the sarcolemmal membrane. In present study we investigated the effects of nitric oxide (NO) donors on calcium currents of single skeletal muscle fibres from the marine isopod, Idotea baltica, using two-electrode voltage clamp recording techniques. The NO donors, S-nitrosocysteine, S-nitroso-N-acetyl-penicillamine or hydroxylamine reversibly increased calcium inward currents in a time dependent manner. The increase of the current was prevented by methylene blue. Our experiments suggest that NO increases calcium inward currents. NO, by acting on calcium ion channels in the sarcolemmal membrane, therefore, may directly be involved in the modulation of muscle contraction.

Poloxamer 188 decreases susceptibility of artificial lipid membranes to electroporation.

PubMed Central

Sharma, V; Stebe, K; Murphy, J C; Tung, L

1996-01-01

The effect of a nontoxic, nonionic block co-polymeric surface active agent, poloxamer 188, on electroporation of artificial lipid membranes made of azolectin, was investigated. Two different experimental protocols were used in our study: charge pulse and voltage clamp. For the charge pulse protocol, membranes were pulsed with a 10-micronsecond rectangular voltage waveform, after which membrane voltage decay was observed through an external 1-M omega resistance. For the voltage clamp protocol the membranes were pulsed with a waveform that consisted of an initial 10-microsecond rectangular phase, followed by a negative sloped ramp that decayed to zero in the subsequent 500 microseconds. Several parameters characterizing the electroporation process were measured and compared for the control membranes and membranes treated with 1.0 mM poloxamer 188. For both the charge pulse and voltage clamp experiments, the threshold voltage (amplitude of initial rectangular phase) and latency time (time elapsed between the end of rectangular phase and the onset of membrane electroporation) were measured. Membrane conductance (measured 200 microseconds after the initial rectangular phase) and rise time (tr; the time required for the porated membrane to reach a certain conductance value) were also determined for the voltage clamp experiments, and postelectroporation time constant (PE tau; the time constant for transmembrane voltage decay after onset of electroporation) for the charge pulse experiments. The charge pulse experiments were performed on 23 membranes with 10 control and 13 poloxamer-treated membranes, and voltage pulse experiments on 49 membranes with 26 control and 23 poloxamer-treated membranes. For both charge pulse and voltage clamp experiments, poloxamer 188-treated membranes exhibited a statistically higher threshold voltage (p = 0.1 and p = 0.06, respectively), and longer latency time (p = 0.04 and p = 0.05, respectively). Also, poloxamer 188-treated membranes were found to have a relatively lower conductance (p = 0.001), longer time required for the porated membrane to reach a certain conductance value (p = 0.05), and longer postelectroporation time constant (p = 0.005). Furthermore, addition of poloxamer 188 was found to reduce the membrane capacitance by approximately 4-8% in 5 min. These findings suggest that poloxamer 188 adsorbs into the lipid bilayers, thereby decreasing their susceptibility to electroporation. Images FIGURE 1 PMID:8968593

The Anion Paradox in Sodium Taste Reception: Resolution by Voltage-Clamp Studies

NASA Astrophysics Data System (ADS)

Ye, Qing; Heck, Gerard L.; Desimone, John A.

1991-11-01

Sodium salts are potent taste stimuli, but their effectiveness is markedly dependent on the anion, with chloride yielding the greatest response. The cellular mechanisms that mediate this phenomenon are not known. This "anion paradox" has been resolved by considering the field potential that is generated by restricted electrodiffusion of the anion through paracellular shunts between taste-bud cells. Neural responses to sodium chloride, sodium acetate, and sodium gluconate were studied while the field potential was voltage-clamped. Clamping at electronegative values eliminated the anion effect, whereas clamping at electropositive potentials exaggerated it. Thus, field potentials across the lingual epithelium modulate taste reception, indicating that the functional unit of taste reception includes the taste cell and its paracellular microenvironment.

Membrane currents underlying activity in frog sinus venosus

PubMed Central

Brown, Hilary F.; Giles, Wayne; Noble, Susan J.

1977-01-01

1. The spontaneous electrical activity of small strips of muscle from the sinus venosus region of the heart of Rana catesbeiana was investigated using the double sucrose gap technique. The voltage clamp was used to record the ionic currents underlying the pace-maker depolarization and the action potential. 2. The records of spontaneous electrical activity are very similar to those obtained from the sinus venosus using micro-electrodes. Moreover, the pace-maker activity is almost completely insensitive to tetrodotoxin (TTX) at 2·0 × 10-6 g/ml., which suggests that the pace-maker responses can be classified as primary, as opposed to follower pacing. 3. In response to short rectangular depolarizing voltage clamp pulses, only one inward current is activated. This current is almost completely insensitive to TTX but can be blocked by manganese ions. It appears, therefore, to be equivalent to the slow inward (Ca2+/Na+) current, Isi, of other cardiac tissues. The threshold for Isi is near to the maximum diastolic potential, indicating that it must be activated during the pace-maker depolarization. 4. Interruption of the normal pace-maker depolarization by rapid activation of the voltage clamp circuit reveals the time-dependent decay of outward current. This current reverses between -75 and -90 mV and, therefore, is probably carried mainly by potassium ions. 5. Outward current decay is not a simple exponential, and Hodgkin—Huxley analysis suggests that two distinct components of outward current may be present. One of these is activated in the potential range of the pace-maker depolarization and the other at more positive potentials. Both outward currents reach full, steady-state activation at about zero mV, i.e. within the `plateau' range of the sinus action potential. 6. These results are compared with other recently published voltage clamp data from the rabbit sino—atrial node. 7. A hypothesis for the generation of pace-maker activity is presented which involves (i) decay of outward current and (ii) activation of the slow inward current, Isi. PMID:303699

Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels.

PubMed

John, Victoria H; Dale, Tim J; Hollands, Emma C; Chen, Mao Xiang; Partington, Leanne; Downie, David L; Meadows, Helen J; Trezise, Derek J

2007-02-01

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.

Action of certain tropine esters on voltage-clamped lobster axon.

PubMed

Blaustein, M P

1968-03-01

Tropine p-tolylacetate (TPTA) and its quaternary analogue, tropine p-tolylacetate methiodide (TPTA MeI) decrease the early transient (Na) and late (K) currents in the voltage-clamped lobster giant axon. These agents, which block the nerve action potential, reduce the maximum Na and K conductance increases associated with membrane depolarization. They also slow the rate at which the sodium conductance is increased and shift the (normalized) membrane conductance vs. voltage curves in the direction of depolarization along the voltage axis. All these effects are qualitatively similar to those resulting from the action of procaine on the voltage-clamped axon. One unusual effect of the tropine esters, noticeable particularly at large depolarization steps, is that they cause the late, K current to reach a peak and then fall off with increasing pulse duration. This effect has not been reported to occur as a result of procaine action. Tropine p-chlorophenyl acetate (TPClphiA), which differs from TPTA only by the substitution of a p-Cl for a p-CH(3) group on the benzene ring, had a negligible effect on axonal excitability.

Action of Certain Tropine Esters on Voltage-Clamped Lobster Axon

PubMed Central

Blaustein, M. P.

1968-01-01

Tropine p-tolylacetate (TPTA) and its quaternary analogue, tropine p-tolylacetate methiodide (TPTA MeI) decrease the early transient (Na) and late (K) currents in the voltage-clamped lobster giant axon. These agents, which block the nerve action potential, reduce the maximum Na and K conductance increases associated with membrane depolarization. They also slow the rate at which the sodium conductance is increased and shift the (normalized) membrane conductance vs. voltage curves in the direction of depolarization along the voltage axis. All these effects are qualitatively similar to those resulting from the action of procaine on the voltage-clamped axon. One unusual effect of the tropine esters, noticeable particularly at large depolarization steps, is that they cause the late, K current to reach a peak and then fall off with increasing pulse duration. This effect has not been reported to occur as a result of procaine action. Tropine p-chlorophenyl acetate (TPClφA), which differs from TPTA only by the substitution of a p-Cl for a p-CH3 group on the benzene ring, had a negligible effect on axonal excitability. PMID:5648830

A low-voltage sense amplifier with two-stage operational amplifier clamping for flash memory

NASA Astrophysics Data System (ADS)

Guo, Jiarong

2017-04-01

A low-voltage sense amplifier with reference current generator utilizing two-stage operational amplifier clamp structure for flash memory is presented in this paper, capable of operating with minimum supply voltage at 1 V. A new reference current generation circuit composed of a reference cell and a two-stage operational amplifier clamping the drain pole of the reference cell is used to generate the reference current, which avoids the threshold limitation caused by current mirror transistor in the traditional sense amplifier. A novel reference voltage generation circuit using dummy bit-line structure without pull-down current is also adopted, which not only improves the sense window enhancing read precision but also saves power consumption. The sense amplifier was implemented in a flash realized in 90 nm flash technology. Experimental results show the access time is 14.7 ns with power supply of 1.2 V and slow corner at 125 °C. Project supported by the National Natural Science Fundation of China (No. 61376028).

M-currents and other potassium currents in bullfrog sympathetic neurones

PubMed Central

Adams, P. R.; Brown, D. A.; Constanti, A.

1982-01-01

1. Bullfrog lumbar sympathetic neurones were voltage-clamped in vitro through twin micro-electrodes. Four different outward (K+) currents could be identified: (i) a large sustained voltage-sensitive delayed rectifier current (IK) activated at membrane potentials more positive than -25 mV; (ii) a calcium-dependent sustained outward current (IC) activated at similar positive potentials and peaking at +20 to +60 mV; (iii) a transient current (IA) activated at membrane potentials more positive than -60 mV after a hyperpolarizing pre-pulse, but which was rapidly and totally inactivated at all potentials within its activation range; and (iv) a new K+ current, the M-current (IM). 2. IM was detected as a non-inactivating current with a threshold at -60 mV. The underlying conductance GM showed a sigmoidal activation curve between -60 and -10 mV, with half-activation at -35 mV and a maximal value (ḠM) of 84±14 (S.E.M.) nS per neurone. The voltage sensitivity of GM could be expressed in terms of a simple Boltzmann distribution for a single multivalent gating particle. 3. IM activated and de-activated along an exponential time course with a time constant uniquely dependent upon voltage, maximizing at ≃ 150 ms at -35 mV at 22 °C. 4. Instantaneous current—voltage (I/V) curves were approximately linear in the presence of IM, suggesting that the M-channels do not show appreciable rectification. However, the time- and voltage-dependent opening of the M-channels induced considerable rectification in the steady-state I/V curves recorded under both voltage-clamp and current-clamp modes between -60 and -25 mV. Both time- and voltage-dependent rectification in the voltage responses to current injection over this range could be predicted from the kinetic properties of IM. 5. It is suggested that IM exerts a strong potential-clamping effect on the behaviour of these neurones at membrane potentials subthreshold to excitation. PMID:6294290

The simulation on diode-clamped five-level converters common-mode voltage suppression with zero-vector PWM strategy

NASA Astrophysics Data System (ADS)

Zhang, Yonggao; Gao, Yanli; Long, Lizhong

2012-04-01

More and more researchers have great concern on the issue of Common-mode voltage (CMV) in high voltage large power converter. A novel common-mode voltage suppression scheme based on zero-vector PWM strategy (ZVPWM) is present in this paper. Taking a diode-clamped five-level converter as example, the principle of zero vector PWM common-mode voltage (ZCMVPWM) suppression method is studied in detail. ZCMVPWM suppression strategy is including four important parts, which are locating the sector of reference voltage vector, locating the small triangular sub-sector of reference voltage vector, reference vector synthesis, and calculating the operating time of vector. The principles of four important pars are illustrated in detail and the corresponding MATLAB models are established. System simulation and experimental results are provided. It gives some consultation value for the development and research of multi-level converters.

Relaxation of Isolated Ventricular Cardiomyocytes by a Voltage-Dependent Process

NASA Astrophysics Data System (ADS)

Bridge, John H. B.; Spitzer, Kenneth W.; Ershler, Philip R.

1988-08-01

Cell contraction and relaxation were measured in single voltage-clamped guinea pig cardiomyocytes to investigate the contribution of sarcolemmal Na+-Ca2+ exchange to mechanical relaxation. Cells clamped from -80 to 0 millivolts displayed initial phasic and subsequent tonic contractions; caffeine reduced or abolished the phasic and enlarged the tonic contraction. The rate of relaxation from tonic contractions was steeply voltage-dependent and was significantly slowed in the absence of a sarcolemmal Na+ gradient. Tonic contractions elicited in the absence of a Na+ gradient promptly relaxed when external Na+ was applied, reflecting activation of Na+-Ca2+ exchange. It appears that a voltage-dependent Na+-Ca2+ exchange can rapidly mechanically relax mammalian heart muscle.

Uncertainty quantification analysis of the dynamics of an electrostatically actuated microelectromechanical switch model

NASA Astrophysics Data System (ADS)

Snow, Michael G.; Bajaj, Anil K.

2015-08-01

This work presents an uncertainty quantification (UQ) analysis of a comprehensive model for an electrostatically actuated microelectromechanical system (MEMS) switch. The goal is to elucidate the effects of parameter variations on certain key performance characteristics of the switch. A sufficiently detailed model of the electrostatically actuated switch in the basic configuration of a clamped-clamped beam is developed. This multi-physics model accounts for various physical effects, including the electrostatic fringing field, finite length of electrodes, squeeze film damping, and contact between the beam and the dielectric layer. The performance characteristics of immediate interest are the static and dynamic pull-in voltages for the switch. Numerical approaches for evaluating these characteristics are developed and described. Using Latin Hypercube Sampling and other sampling methods, the model is evaluated to find these performance characteristics when variability in the model's geometric and physical parameters is specified. Response surfaces of these results are constructed via a Multivariate Adaptive Regression Splines (MARS) technique. Using a Direct Simulation Monte Carlo (DSMC) technique on these response surfaces gives smooth probability density functions (PDFs) of the outputs characteristics when input probability characteristics are specified. The relative variation in the two pull-in voltages due to each of the input parameters is used to determine the critical parameters.

Auxiliary resonant DC tank converter

DOEpatents

Peng, Fang Z.

2000-01-01

An auxiliary resonant dc tank (ARDCT) converter is provided for achieving soft-switching in a power converter. An ARDCT circuit is coupled directly across a dc bus to the inverter to generate a resonant dc bus voltage, including upper and lower resonant capacitors connected in series as a resonant leg, first and second dc tank capacitors connected in series as a tank leg, and an auxiliary resonant circuit comprising a series combination of a resonant inductor and a pair of auxiliary switching devices. The ARDCT circuit further includes first clamping means for holding the resonant dc bus voltage to the dc tank voltage of the tank leg, and second clamping means for clamping the resonant dc bus voltage to zero during a resonant period. The ARDCT circuit resonantly brings the dc bus voltage to zero in order to provide a zero-voltage switching opportunity for the inverter, then quickly rebounds the dc bus voltage back to the dc tank voltage after the inverter changes state. The auxiliary switching devices are turned on and off under zero-current conditions. The ARDCT circuit only absorbs ripples of the inverter dc bus current, thus having less current stress. In addition, since the ARDCT circuit is coupled in parallel with the dc power supply and the inverter for merely assisting soft-switching of the inverter without participating in real dc power transmission and power conversion, malfunction and failure of the tank circuit will not affect the functional operation of the inverter; thus a highly reliable converter system is expected.

Acidic pH modulation of Na+ channels in trigeminal mesencephalic nucleus neurons.

PubMed

Kang, In-Sik; Cho, Jin-Hwa; Choi, In-Sun; Kim, Do-Yeon; Jang, Il-Sung

2016-12-07

Cell bodies of trigeminal mesencephalic nucleus (Vmes) neurons are located within the central nervous system, and therefore, peripheral as well as central acidosis can modulate the excitability of Vmes neurons. Here, we report the effect of acidic pH on voltage-gated Na channels in acutely isolated rat Vmes neurons using a conventional whole-cell patch clamp technique. Acidic pH (pH 6.0) slightly but significantly shifted both the activation and steady-state fast inactivation relationships toward depolarized potentials. However, acidic pH (pH 6.0) had a minor effect on the inactivation kinetics of voltage-gated Na channels. Less sensitivity of voltage-gated Na channels to acidic pH may allow Vmes neurons to transduce the precise proprioceptive information even under acidic pH conditions.

Selective block of late Na+ current by local anaesthetics in rat large sensory neurones

PubMed Central

Baker, Mark D

2000-01-01

The actions of lignocaine and benzocaine on transient and late Na+ current generated by large diameter (⩾50 μm) adult rat dorsal root ganglion neurones, were studied using patch-clamp techniques.Both drugs blocked whole-cell late Na+ current in a concentration-dependent manner. At 200 ms following the onset of a clamp step from −110 to −40 mV, the apparent K for block of late Na+ current by lignocaine was 57.8±15 μM (mean±s.e.mean, n=4). The value for benzocaine was 24.9±3.3 μM, (mean±s.e.mean, n=3).The effect of lignocaine on transient current, in randomly selected neurones, appeared variable (n=8, half-block from ∼50 to 400 μM). Half-block by benzocaine was not attained, but both whole-cell (n=11) and patch data suggested a high apparent K,>250 μM. Transient current always remained after late current was blocked.The voltage-dependence of residual late current steady-state inactivation was not shifted by 20 μM benzocaine (n=3), whereas 200 μM benzocaine shifted the voltage-dependence of transient current steady-state inactivation by −18.7±5.9 mV (mean±s.e.mean, n=4).In current-clamp, benzocaine (250 μM) could block subthreshold, voltage-dependent inward current, increasing the threshold for eliciting action potentials, without preventing their generation (n=2).Block of late Na+ current by systemic local anaesthetic may play a part in preventing ectopic impulse generation in sensory neurones. PMID:10780966

MATLAB implementation of a dynamic clamp with bandwidth >125 KHz capable of generating INa at 37°C

PubMed Central

Clausen, Chris; Valiunas, Virginijus; Brink, Peter R.; Cohen, Ira S.

2012-01-01

We describe the construction of a dynamic clamp with bandwidth >125 KHz that utilizes a high performance, yet low cost, standard home/office PC interfaced with a high-speed (16 bit) data acquisition module. High bandwidth is achieved by exploiting recently available software advances (code-generation technology, optimized real-time kernel). Dynamic-clamp programs are constructed using Simulink, a visual programming language. Blocks for computation of membrane currents are written in the high-level matlab language; no programming in C is required. The instrument can be used in single- or dual-cell configurations, with the capability to modify programs while experiments are in progress. We describe an algorithm for computing the fast transient Na+ current (INa) in real time, and test its accuracy and stability using rate constants appropriate for 37°C. We then construct a program capable of supplying three currents to a cell preparation: INa, the hyperpolarizing-activated inward pacemaker current (If), and an inward-rectifier K+ current (IK1). The program corrects for the IR drop due to electrode current flow, and also records all voltages and currents. We tested this program on dual patch-clamped HEK293 cells where the dynamic clamp controls a current-clamp amplifier and a voltage-clamp amplifier controls membrane potential, and current-clamped HEK293 cells where the dynamic clamp produces spontaneous pacing behavior exhibiting Na+ spikes in otherwise passive cells. PMID:23224681

Implementation of a fast 16-Bit dynamic clamp using LabVIEW-RT.

PubMed

Kullmann, Paul H M; Wheeler, Diek W; Beacom, Joshua; Horn, John P

2004-01-01

The dynamic-clamp method provides a powerful electrophysiological tool for creating virtual ionic conductances in living cells and studying their influence on membrane potential. Here we describe G-clamp, a new way to implement a dynamic clamp using the real-time version of the Lab-VIEW programming environment together with a Windows host, an embedded microprocessor that runs a real-time operating system and a multifunction data-acquisition board. The software includes descriptions of a fast voltage-dependent sodium conductance, delayed rectifier, M-type and A-type potassium conductances, and a leak conductance. The system can also read synaptic conductance waveforms from preassembled data files. These virtual conductances can be reliably implemented at speeds < or =43 kHz while simultaneously saving two channels of data with 16-bit precision. G-clamp also includes utilities for measuring current-voltage relations, synaptic strength, and synaptic gain. Taking an approach built on a commercially available software/hardware platform has resulted in a system that is easy to assemble and upgrade. In addition, the graphical programming structure of LabVIEW should make it relatively easy for others to adapt G-clamp for new experimental applications.

Sodium efflux from voltage clamped squid giant axons.

PubMed Central

Landowne, D

1977-01-01

1. The efflux of radioactive sodium was measured from squid axons during simultaneous voltage clamp experiments such that it was possible to determine the efflux of sodium associated with a measured voltage clamp current. 2. The extra efflux of sodium associated with voltage clamp pulses increased linearly with the magnitude of the depolarization above 40 mV. A 100 mV pulse of sufficient duration to produce all of the sodium current increased the rate constant of efflux by about 10(-6). 3. Application of 100 nM tetrodotoxin eliminated the sodium current and the extra efflux of radioactive sodium. 4. Cooling the axon increased the extra efflux/voltage clamp pulse slightly with a Q10 of 1/1-1. On the same axons cooling increased the integral of the sodium current with a Q10 of 1/1-4. 5. Replacing external sodium with Tris, dextrose or Mg-mannitol reduced the extra efflux of sodium by about 50%. The inward sodium current was replaced with an outward current as expected. 6. Replacing external sodium with lithium also reduced the extra efflux by about 50% but the currents seen in lithium were slightly larger than those in sodium. 7. The effect of replacing external sodium was not voltage dependent. Cooling reduced the effect so that there was less reduction of efflux on switching to Tris ASW in the cold than in the warm. 8. The extra efflux of sodium into sodium-free ASW is approximately the same as the integral of the sodium current. Adding external sodium produces a deviation from the independence principle such that there is more exchange of sodium than predicted. Such a deviation from prediction was noted by Hodgkin & Huxley (1952c). 9. Using the equations of Hodgkin & Huxley (1952c) modified to include the deviation from independence reported in this paper and its temperature dependence, one can predict the temperature dependence of the sodium efflux associated with action potentials and obtain much better agreement than is possibly without these phenomena. 10. This deviation from independence in the sodium fluxes is the type expected from some kind of mixing and binding of sodium within the membrane phase. PMID:856999

Inertial piezoelectric linear motor driven by a single-phase harmonic wave with automatic clamping mechanism

NASA Astrophysics Data System (ADS)

He, Liangguo; Chu, Yuheng; Hao, Sai; Zhao, Xiaoyong; Dong, Yuge; Wang, Yong

2018-05-01

A novel, single-phase, harmonic-driven, inertial piezoelectric linear motor using an automatic clamping mechanism was designed, fabricated, and tested to reduce the sliding friction and simplify the drive mechanism and power supply control of the inertial motor. A piezoelectric bimorph and a flexible hinge were connected in series to form the automatic clamping mechanism. The automatic clamping mechanism was used as the driving and clamping elements. A dynamic simulation by Simulink was performed to prove the feasibility of the motor. The finite element method software COMSOL was used to design the structure of the motor. An experimental setup was built to validate the working principle and evaluate the performance of the motor. The prototype motor outputted a no-load velocity of 3.178 mm/s at a voltage of 220 Vp-p and a maximum traction force of 4.25 N under a preload force of 8 N. The minimum resolution of 1.14 μm was achieved at a driving frequency of 74 Hz, a driving voltage of 50 Vp-p, and a preload force of 0 N.

Verification of the windings axial clamping forces for high voltage power transformers by using passively mode-locked fiber lasers

NASA Astrophysics Data System (ADS)

Şchiopu, IonuÅ£ Romeo; ǎgulinescu, Andrei, Dr; Iordǎnescu, Raluca; Marinescu, Andrei

2015-02-01

The current paper describes an optoelectronic method for direct monitoring of the axial clamping forces both in static and in dynamic duty. As advantages of this method we can state that it can be applied both to new and refurbished transformers without performing constructive changes or affecting in any way the transformer safety in operation. For monitoring the axial clamping forces for high-voltage (HV) power transformers, we use an optical fiber that we integrate into the laser cavity of a passively mode-locked fiber laser (PMFL). To each axial clamp corresponds a solitonic optical spectrum that is changed at the periodical passing of the fundamental soliton pulse through the sensitive fiber inside the transformer. Moreover, as a specific characteristic, the laser stability is unique for each set of axial clamping forces. Other important advantages of using an optical fiber as compared to the classical approach in which electronic sensors are used consist in the good reliability and insulator properties of the optical fiber, avoiding any risk of fire or damage of the transformer.

Effect of electrical coupling on ionic current and synaptic potential measurements.

PubMed

Rabbah, Pascale; Golowasch, Jorge; Nadim, Farzan

2005-07-01

Recent studies have found electrical coupling to be more ubiquitous than previously thought, and coupling through gap junctions is known to play a crucial role in neuronal function and network output. In particular, current spread through gap junctions may affect the activation of voltage-dependent conductances as well as chemical synaptic release. Using voltage-clamp recordings of two strongly electrically coupled neurons of the lobster stomatogastric ganglion and conductance-based models of these neurons, we identified effects of electrical coupling on the measurement of leak and voltage-gated outward currents, as well as synaptic potentials. Experimental measurements showed that both leak and voltage-gated outward currents are recruited by gap junctions from neurons coupled to the clamped cell. Nevertheless, in spite of the strong coupling between these neurons, the errors made in estimating voltage-gated conductance parameters were relatively minor (<10%). Thus in many cases isolation of coupled neurons may not be required if a small degree of measurement error of the voltage-gated currents or the synaptic potentials is acceptable. Modeling results show, however, that such errors may be as high as 20% if the gap-junction position is near the recording site or as high as 90% when measuring smaller voltage-gated ionic currents. Paradoxically, improved space clamp increases the errors arising from electrical coupling because voltage control across gap junctions is poor for even the highest realistic coupling conductances. Furthermore, the common procedure of leak subtraction can add an extra error to the conductance measurement, the sign of which depends on the maximal conductance.

Origin of terminal voltage variations due to self-mixing in terahertz frequency quantum cascade lasers.

PubMed

Grier, Andrew; Dean, Paul; Valavanis, Alexander; Keeley, James; Kundu, Iman; Cooper, Jonathan D; Agnew, Gary; Taimre, Thomas; Lim, Yah Leng; Bertling, Karl; Rakić, Aleksandar D; Li, Lianhe H; Harrison, Paul; Linfield, Edmund H; Ikonić, Zoran; Davies, A Giles; Indjin, Dragan

2016-09-19

We explain the origin of voltage variations due to self-mixing in a terahertz (THz) frequency quantum cascade laser (QCL) using an extended density matrix (DM) approach. Our DM model allows calculation of both the current-voltage (I-V) and optical power characteristics of the QCL under optical feedback by changing the cavity loss, to which the gain of the active region is clamped. The variation of intra-cavity field strength necessary to achieve gain clamping, and the corresponding change in bias required to maintain a constant current density through the heterostructure is then calculated. Strong enhancement of the self-mixing voltage signal due to non-linearity of the (I-V) characteristics is predicted and confirmed experimentally in an exemplar 2.6 THz bound-to-continuum QCL.

Voltage and Current Clamp Transients with Membrane Dielectric Loss

PubMed Central

Fitzhugh, R.; Cole, K. S.

1973-01-01

Transient responses of a space-clamped squid axon membrane to step changes of voltage or current are often approximated by exponential functions of time, corresponding to a series resistance and a membrane capacity of 1.0 μF/cm2. Curtis and Cole (1938, J. Gen. Physiol. 21:757) found, however, that the membrane had a constant phase angle impedance z = z1(jωτ)-α, with a mean α = 0.85. (α = 1.0 for an ideal capacitor; α < 1.0 may represent dielectric loss.) This result is supported by more recently published experimental data. For comparison with experiments, we have computed functions expressing voltage and current transients with constant phase angle capacitance, a parallel leakage conductance, and a series resistance, at nine values of α from 0.5 to 1.0. A series in powers of tα provided a good approximation for short times; one in powers of t-α, for long times; for intermediate times, a rational approximation matching both series for a finite number of terms was used. These computations may help in determining experimental series resistances and parallel leakage conductances from membrane voltage or current clamp data. PMID:4754194

Equilibrium potential for the postsynaptic response in the squid giant synapse.

PubMed

Llinás, R; Joyner, R W; Nicholson, C

1974-11-01

The reversal potential for the EPSP in the squid giant synapse has been studied by means of an intracellular, double oil gap technique. This method allows the electrical isolation of a portion of the axon from the rest of the fiber and generates a quasi-isopotential segment. In order to make the input resistance of this nerve segment as constant as possible, the electroresponsive properties of the nerve membrane were blocked by intracellular injection of tetraethylammonium (TEA) and local extracellular application of tetrodotoxin (TTX). Thus, EPSP's could be evoked in the isolated segment with a minimal amount of electroresponsive properties. The reversal potential for the EPSP (EEPSP) was measured by recording the synaptic potential or the synaptic current during voltage clamping. The results indicate that EEPSP may vary from +15 to +25 mV, which is more positive than would be expected for a 1:1 conductance change for Na(+) and K(+) (approximately -15 mV) and too negative for a pure Na(+) conductance ((+)40 mV). This latter value (E(Na)) was directly determined in the voltage clamp experiments. The results suggest that the synaptic potential is probably produced by a permeability change to Na(+) to K(+) in a 4:1 ratio. No change in time-course was observed in the synaptic current at clamp levels of -100 and +90 mV. The implications of a variable ratio for Na(+)-K(+) permeability in subsynaptic-postsynaptic membranes are discussed.

Voltage equaliser for Li-Fe battery

NASA Astrophysics Data System (ADS)

Wu, Jinn-Chang; Jou, Hurng-Liahng; Chuang, Ping-Hao

2013-10-01

In this article, a voltage equaliser is proposed for a battery string with four Li-Fe batteries. The proposed voltage equaliser is developed from a flyback converter, which comprises a transformer, a power electronic switch and a resonant clamped circuit. The transformer contains a primary winding and four secondary windings with the same number of turns connected to each battery. The resonant clamped circuit is for recycling the energy of leakage inductance of the transformer and for performing zero-voltage switching (ZVS) of the power electronic switch. When the power electronic switch is switched on, the energy is stored in the transformer; and when the power electronic switch is switched off, the energy stored in the transformer will automatically charge the battery whose voltage is the lowest. In this way, the voltage of individual batteries in the battery string is balanced. The salient features of the proposed voltage equaliser are that only one switch is used, the energy stored in the leakage inductance of the transformer can be recycled and ZVS is obtained. A prototype is developed and tested to verify the performance of the proposed voltage equaliser. The experimental results show that the proposed voltage equaliser achieves the expected performance.

Development of a Piezoelectric Vacuum Sensing Component for a Wide Pressure Range

PubMed Central

Wang, Bing-Yu; Hsieh, Fan-Chun; Lin, Che-Yu; Chen, Shao-En; Chen, Fong-Zhi; Wu, Chia-Che

2014-01-01

In this study, we develop a clamped–clamped beam-type piezoelectric vacuum pressure sensing element. The clamped–clamped piezoelectric beam is composed of a PZT layer and a copper substrate. A pair of electrodes is set near each end. An input voltage is applied to a pair of electrodes to vibrate the piezoelectric beam, and the output voltage is measured at the other pair. Because the viscous forces on the piezoelectric beam vary at different air pressures, the vibration of the beam depends on the vacuum pressure. The developed pressure sensor can sense a wide range of pressure, from 6.5 × 10−6 to 760 Torr. The experimental results showed that the output voltage is inversely proportional to the gas damping ratio, and thus, the vacuum pressure was estimated from the output voltage. PMID:25421736

The amiodarone derivative KB130015 activates hERG1 potassium channels via a novel mechanism

PubMed Central

Gessner, Guido; Macianskiene, Regina; Starkus, John G.; Schönherr, Roland; Heinemann, Stefan H.

2010-01-01

Human ether à go-go related gene (hERG1) potassium channels underlie the repolarizing IKr current in the heart. Since they are targets of various drugs with cardiac side effects we tested whether the amiodarone derivative 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) blocks hERG1 channels like its parent compound. Using patch-clamp and two-electrode voltage-clamp techniques we found that KB130015 blocks native and recombinant hERG1 channels at high voltages, but it activates them at low voltages. The activating effect has an apparent EC50 value of 12 μM and is brought about by an about 4-fold acceleration of activation kinetics and a shift in voltage-dependent activation by −16 mV. Channel activation was not use-dependent and was independent of inactivation gating. KB130015 presumably binds to the hERG1 pore from the cytosolic side and functionally competes with hERG1 block by amiodarone, E4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl] -4-piperidinyl] carbonyl] phenyl] methanesulfonamide dihydrochloride), and sertindole. Vice versa, amiodarone attenuates hERG1 activation by KB130015. Based on synergic channel activation by mallotoxin and KB130015 we conclude that the hERG1 pore contains at least two sites for activators that are functionally coupled among each other and to the cavity-blocker site. KB130015 and amiodarone may serve as lead structures for the identification of hERG1 pore-interacting drugs favoring channel activation vs. block. PMID:20097192

Analysis of real-time numerical integration methods applied to dynamic clamp experiments.

PubMed

Butera, Robert J; McCarthy, Maeve L

2004-12-01

Real-time systems are frequently used as an experimental tool, whereby simulated models interact in real time with neurophysiological experiments. The most demanding of these techniques is known as the dynamic clamp, where simulated ion channel conductances are artificially injected into a neuron via intracellular electrodes for measurement and stimulation. Methodologies for implementing the numerical integration of the gating variables in real time typically employ first-order numerical methods, either Euler or exponential Euler (EE). EE is often used for rapidly integrating ion channel gating variables. We find via simulation studies that for small time steps, both methods are comparable, but at larger time steps, EE performs worse than Euler. We derive error bounds for both methods, and find that the error can be characterized in terms of two ratios: time step over time constant, and voltage measurement error over the slope factor of the steady-state activation curve of the voltage-dependent gating variable. These ratios reliably bound the simulation error and yield results consistent with the simulation analysis. Our bounds quantitatively illustrate how measurement error restricts the accuracy that can be obtained by using smaller step sizes. Finally, we demonstrate that Euler can be computed with identical computational efficiency as EE.

On the Nonlinear Dynamics of a Tunable Shock Micro-switch

NASA Astrophysics Data System (ADS)

Azizi, Saber; Javaheri, Hamid; Ghanati, Parisa

2016-12-01

A tunable shock micro-switch based on piezoelectric excitation is proposed in this study. This model includes a clamped-clamped micro-beam sandwiched with two piezoelectric layers throughout the entire length. Actuation of the piezoelectric layers via a DC voltage leads to an initial axial force in the micro-beam and directly affects on its overall bending stiffness; accordingly enables two-side tuning of both the trigger time and threshold shock. The governing motion equation, in the presence of an electrostatic actuation and a shock wave, is derived using Hamilton's principle. We employ the finite element method based on the Galerkin technique to obtain the temporal and phase responses subjected to three different shock waves including half sine, triangular and rectangular forms. Subsequently, we investigate the effect of the piezoelectric excitations on the threshold shock amplitude and trigger time.

Human Nav1.8: enhanced persistent and ramp currents contribute to distinct firing properties of human DRG neurons

PubMed Central

Han, Chongyang; Estacion, Mark; Huang, Jianying; Vasylyev, Dymtro; Zhao, Peng; Dib-Hajj, Sulayman D.

2015-01-01

Although species-specific differences in ion channel properties are well-documented, little has been known about the properties of the human Nav1.8 channel, an important contributor to pain signaling. Here we show, using techniques that include voltage clamp, current clamp, and dynamic clamp in dorsal root ganglion (DRG) neurons, that human Nav1.8 channels display slower inactivation kinetics and produce larger persistent current and ramp current than previously reported in other species. DRG neurons expressing human Nav1.8 channels unexpectedly produce significantly longer-lasting action potentials, including action potentials with half-widths in some cells >10 ms, and increased firing frequency compared with the narrower and usually single action potentials generated by DRG neurons expressing rat Nav1.8 channels. We also show that native human DRG neurons recapitulate these properties of Nav1.8 current and the long-lasting action potentials. Together, our results demonstrate strikingly distinct properties of human Nav1.8, which contribute to the firing properties of human DRG neurons. PMID:25787950

Human Na(v)1.8: enhanced persistent and ramp currents contribute to distinct firing properties of human DRG neurons.

PubMed

Han, Chongyang; Estacion, Mark; Huang, Jianying; Vasylyev, Dymtro; Zhao, Peng; Dib-Hajj, Sulayman D; Waxman, Stephen G

2015-05-01

Although species-specific differences in ion channel properties are well-documented, little has been known about the properties of the human Nav1.8 channel, an important contributor to pain signaling. Here we show, using techniques that include voltage clamp, current clamp, and dynamic clamp in dorsal root ganglion (DRG) neurons, that human Na(v)1.8 channels display slower inactivation kinetics and produce larger persistent current and ramp current than previously reported in other species. DRG neurons expressing human Na(v)1.8 channels unexpectedly produce significantly longer-lasting action potentials, including action potentials with half-widths in some cells >10 ms, and increased firing frequency compared with the narrower and usually single action potentials generated by DRG neurons expressing rat Na(v)1.8 channels. We also show that native human DRG neurons recapitulate these properties of Na(v)1.8 current and the long-lasting action potentials. Together, our results demonstrate strikingly distinct properties of human Na(v)1.8, which contribute to the firing properties of human DRG neurons.

An operational amplifier B1404UD1A-1 in the patch-clamp current-to-voltage converter.

PubMed

Korzun, A M; Rozinov, S V; Abashin, G I

1997-01-01

The applicability of the home-made operational amplifier B1404UD1A-1 in a patch-clamp current-to-voltage converter was analyzed. Its parameters (background noise, input bias current, and gain-bandwidth product) were estimated. Schematic solutions and practical recommendations for the use of this amplifier in a current-to-voltage converter were given. Based on the background noise and frequency parameters of the converter, we found that this device can be used for measuring ion channel currents with a high sensitivity and within a broad frequency range (0.055 pA, to 1 kHz; 0.4 pA, to 10 kHz). An example of the converter application in experiments is given.

Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

PubMed Central

Frolova, Sheyda R.; Gaiko, Olga; Tsvelaya, Valeriya A.; Pimenov, Oleg Y.; Agladze, Konstantin I.

2016-01-01

The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+) and calcium (Ca2+) currents and potentiation of net potassium (K+) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential. PMID:27015602

Electrical coupling of single cardiac rat myocytes to field-effect and bipolar transistors.

PubMed

Kind, Thomas; Issing, Matthias; Arnold, Rüdiger; Müller, Bernt

2002-12-01

A novel bipolar transistor for extracellular recording the electrical activity of biological cells is presented, and the electrical behavior compared with the field-effect transistor (FET). Electrical coupling is examined between single cells separated from the heart of adults rats (cardiac myocytes) and both types of transistors. To initiate a local extracellular voltage, the cells are periodically stimulated by a patch pipette in voltage clamp and current clamp mode. The local extracellular voltage is measured by the planar integrated electronic sensors: the bipolar and the FET. The small signal transistor currents correspond to the local extracellular voltage. The two types of sensor transistors used here were developed and manufactured in the laboratory of our institute. The manufacturing process and the interfaces between myocytes and transistors are described. The recordings are interpreted by way of simulation based on the point-contact model and the single cardiac myocyte model.

The properties of single cones isolated from the tiger salamander retina

PubMed Central

Attwell, David; Werblin, Frank S.; Wilson, Martin

1982-01-01

1. The properties of isolated single cones were studied using the voltage-clamp technique, with two micro-electrodes inserted under visual control. 2. Single cones had input resistances, when impaled with two electrodes, of up to 270 MΩ. This is probably lower than the true membrane resistance, because of damage by the impaling electrodes. The cone capacitance was about 85 pF. 3. The cone membrane contains a time-dependent current, IB, controlled by voltage, and a separate photosensitive current. 4. The gated current, IB, is an inward current with a reversal potential around -25 mV. It is activated by hyperpolarization over the range -30 to -80 mV, and at constant voltage obeys first order (exponential) kinetics. The gating time constant is typically 50 ms at the resting potential of -45 mV, rises to 170 ms at -70 mV, and decreases for further hyperpolarization. 5. The spectral sensitivity curve of the cone light response peaks at 620 nm wave-length, and is narrower than the nomogram for vitamin A2-based pigments. The light responses of isolated cones are spectrally univariant. 6. Voltage-clamped photocurrents were recorded at various membrane potentials, for light steps of various intensities. The photocurrent reversed at around -8 mV. The time course of the photocurrent, for a given intensity, was approximately independent of voltage (although its magnitude was voltage-dependent). The shape of the peak current—voltage relation of the light-sensitive current was independent of light intensity (although its magnitude was intensity-dependent). 7. These results can be explained if: (a) light simply changes the number of photosensitive channels open, without altering the properties of an open channel; (b) the reactions controlling the production of internal transmitter, the binding of internal transmitter to the photosensitive channels, and the closing and opening of the channels are unaffected by the electric field in the cone membrane, even though at least some of these reactions take place in the membrane. 8. IB plays only a small role in shaping the cone voltage response to light. ImagesPlate 1 PMID:7131315

Command-line cellular electrophysiology for conventional and real-time closed-loop experiments.

PubMed

Linaro, Daniele; Couto, João; Giugliano, Michele

2014-06-15

Current software tools for electrophysiological experiments are limited in flexibility and rarely offer adequate support for advanced techniques such as dynamic clamp and hybrid experiments, which are therefore limited to laboratories with a significant expertise in neuroinformatics. We have developed lcg, a software suite based on a command-line interface (CLI) that allows performing both standard and advanced electrophysiological experiments. Stimulation protocols for classical voltage and current clamp experiments are defined by a concise and flexible meta description that allows representing complex waveforms as a piece-wise parametric decomposition of elementary sub-waveforms, abstracting the stimulation hardware. To perform complex experiments lcg provides a set of elementary building blocks that can be interconnected to yield a large variety of experimental paradigms. We present various cellular electrophysiological experiments in which lcg has been employed, ranging from the automated application of current clamp protocols for characterizing basic electrophysiological properties of neurons, to dynamic clamp, response clamp, and hybrid experiments. We finally show how the scripting capabilities behind a CLI are suited for integrating experimental trials into complex workflows, where actual experiment, online data analysis and computational modeling seamlessly integrate. We compare lcg with two open source toolboxes, RTXI and RELACS. We believe that lcg will greatly contribute to the standardization and reproducibility of both simple and complex experiments. Additionally, on the long run the increased efficiency due to a CLI will prove a great benefit for the experimental community. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel crystallization method for visualizing the membrane localization of potassium channels.

PubMed Central

Lopatin, A N; Makhina, E N; Nichols, C G

1998-01-01

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel. PMID:9591643

A novel crystallization method for visualizing the membrane localization of potassium channels.

PubMed

Lopatin, A N; Makhina, E N; Nichols, C G

1998-05-01

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.

Phorbol ester impairs electrical excitation of rat pancreatic beta-cells through PKC-independent activation of KATP channels.

PubMed

Suga, S; Wu, J; Ogawa, Y; Takeo, T; Kanno, T; Wakui, M

2001-01-01

Phorbol 12-myristate 13-acetate (PMA) is often used as an activating phorbol ester of protein kinase C (PKC) to investigate the roles of the kinase in cellular functions. Accumulating lines of evidence indicate that in addition to activating PKC, PMA also produces some regulatory effects in a PKC-independent manner. In this study, we investigated the non-PKC effects of PMA on electrical excitability of rat pancreatic beta-cells by using patch-clamp techniques. In current-clamp recording, PMA (80 nM) reversibly inhibited 15 mM glucose-induced action potential spikes superimposed on a slow membrane depolarization and this inhibition can not be prevented by pre-treatment of the cell with a specific PKC inhibitor, bisindolylmaleimide (BIM, 1 microM). In the presence of a subthreshold concentration (5.5 mM) of glucose, PMA hyperpolarized beta-cells in a concentration-dependent manner (0.8-240 nM), even in the presence of BIM. Based on cell-attached single channel recordings, PMA increased ATP-sensitive K+ channel (KATP) activity. Based on inside-out patch-clamp recordings, PMA had little effect on KATP activity if no ATP was in the bath, while PMA restored KATP activity that was suppressed by 10 microM ATP in the bath. In voltage-clamp recording, PMA enhanced tolbutamide-sensitive membrane currents elicited by repetitive ramp pulses from -90 to -50 mV in a concentration-dependent manner, and this potentiation could not be prevented by pre-treatment of cell with BIM. 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a non-PKC-activating phorbol ester, mimicked the effect of PMA on both current-clamp and voltage-clamp recording configurations. With either 5.5 or 16.6 mM glucose in the extracellular solution, PMA (80 nM) increased insulin secretion from rat islets. However, in islets pretreated with BIM (1 microM), PMA did not increase, but rather reduced insulin secretion. In rat pancreatic beta-cells, PMA modulates insulin secretion through a mixed mechanism: increases insulin secretion by activation of PKC, and meanwhile decrease insulin secretion by impairing beta-cell excitability in a PKC-independent manner. The enhancement of KATP activity by reducing sensitivity of KATP to ATP seems to underlie the PMA-induced impairment of beta-cells electrical excitation in response to glucose stimulation.

ELECTRONIC MULTIPLIER

DOEpatents

Collier, D.M.; Meeks, L.A.; Palmer, J.P.

1961-01-31

S>An electronic multiplier is described for use in analog computers. Two electrical input signals are received; one controls the slope of a saw-tooth voltage wave while the other controls the time duration of the wave. A condenser and diode clamps are provided to sustain the crest voltage reached by the wave, and for storing that voltage to provide an output signal which is a steady d-c voltage.

A comparison of 15 Hz sine on-line and off-line magnetic stimulation affecting the voltage-gated sodium channel currents of prefrontal cortex pyramidal neurons

NASA Astrophysics Data System (ADS)

Zheng, Yu; Dong, Lei; Gao, Yang; Dou, Jun-Rong; Li, Ze-yan

2016-10-01

Combined with the use of patch-clamp techniques, repetitive transcranial magnetic stimulation (rTMS) has proven to be a noninvasive neuromodulation tool that can inhibit or facilitate excitability of neurons after extensive research. The studies generally focused on the method: the neurons are first stimulated in an external standard magnetic exposure device, and then moved to the patch-clamp to record electrophysiological characteristics (off-line magnetic exposure). Despite its universality, real-time observation of the effects of magnetic stimulation on the neurons is more effective (on-line magnetic stimulation). In this study, we selected a standard exposure device for magnetic fields acting on mouse prefrontal cortex pyramidal neurons, and described a new method that a patch-clamp setup was modified to allow on-line magnetic stimulation. By comparing the off-line exposure and on-line stimulation of the same magnetic field intensity and frequency affecting the voltage-gated sodium channel currents, we succeeded in proving the feasibility of the new on-line stimulation device. We also demonstrated that the sodium channel currents of prefrontal cortex pyramidal neurons increased significantly under the 15 Hz sine 1 mT, and 2 mT off-line magnetic field exposure and under the 1 mT and 2 mT on-line magnetic stimulation, and the rate of acceleration was most significant on 2 mT on-line magnetic stimulation. This study described the development of a new on-line magnetic stimulator and successfully demonstrated its practicability for scientific stimulation of neurons.

Non-equilibrium voltage noise generated by ion transport through pores.

PubMed

Frehland, E; Solleder, P

1985-01-01

In this paper, we describe a systematic approach to the theoretical analysis of non-equilibrium voltage noise that arises from ions moving through pores in membranes. We assume that an ion must cross one or two barriers in the pore in order to move from one side of the membrane to the other. In our analysis, we consider the following factors: a) surface charge as a variable in the kinetic equations, b) linearization of the kinetic equations, c) master equation approach to fluctuations. To analyze the voltage noise arising from ion movement through a two barrier (i.e., one binding site) pore, we included the effects of ions in the channel's interior on the voltage noise. The current clamp is considered as a white noise generating additional noise in the system. In contrast to what is found for current noise, at low frequencies the voltage noise intensity is reduced by increasing voltage across the membrane. With this approach, we demonstrate explicitly for the examples treated that, apart from additional noise generated by the current clamp, the non-equilibrium voltage fluctuations can be related to the current fluctuations by the complex admittance.

Robotic multi-well planar patch-clamp for native and primary mammalian cells

PubMed Central

Milligan, Carol J; Li, Jing; Sukumar, Piruthivi; Majeed, Yasser; Dallas, Mark L; English, Anne; Emery, Paul; Porter, Karen E; Smith, Andrew M; McFadzean, Ian; Beccano-Kelly, Dayne; Bahnasi, Yahya; Cheong, Alex; Naylor, Jacqueline; Zeng, Fanning; Liu, Xing; Gamper, Nikita; Jiang, Lin-Hua; Pearson, Hugh A; Peers, Chris; Robertson, Brian; Beech, David J

2009-01-01

Multi-well robotic planar patch-clamp has become common in drug development and safety programmes because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favoured method. Here we show the wider potential of the multi-well approach with the capability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints, and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by pre-programmed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 hr depending on the experimental design and yields 16-33 cell recordings. PMID:19197268

Ion pathways in the taste bud and their significance for transduction.

PubMed

DeSimone, J A; Ye, Q; Heck, G L

1993-01-01

Taste buds share a topology with ion-transporting epithelial and evidence now indicates that neural responses in rats to Na+ salts of differing anion are mediated by both transcellular and paracellular ion transport. Na+ exerts its effects mainly on the transcellular pathway. Neural responses to Na+ salts are enhanced by negative voltage clamp and suppressed by positive clamp in a manner indicating modulation of the apical membrane potential of receptor cells. Anion effects are mainly paracellular. Under zero current clamp increasing anion size reduces the neural response at constant Na+ concentration. Below about 50 mM this difference is entirely eliminated under voltage clamp. This suggests that paracellular transepithelial potentials normally create an anion difference. At higher concentrations the relatively high permeability of the paracellular shunt to Cl- permits sufficient electroneutral diffusion of NaCl below the tight junctions to stimulate cells that do not make direct contact with the oral cavity. In general, the sensitivity of a response to perturbations in the apical membrane potential indicates that some phase of Na+ salt taste transduction is accompanied by changes in an apical membrane channel conductance.

Toward a New Gold Standard for Early Safety: Automated Temperature-Controlled hERG Test on the PatchLiner®

PubMed Central

Polonchuk, Liudmila

2012-01-01

The Patchliner® temperature-controlled automated patch clamp system was evaluated for testing drug effects on potassium currents through human ether-à-go-go related gene (hERG) channels expressed in Chinese hamster ovary cells at 35–37°C. IC50 values for a set of reference drugs were compared with those obtained using the conventional voltage clamp technique. The results showed good correlation between the data obtained using automated and conventional electrophysiology. Based on these results, the Patchliner® represents an innovative automated electrophysiology platform for conducting the hERG assay that substantially increases throughput and has the advantage of operating at physiological temperature. It allows fast, accurate, and direct assessment of channel function to identify potential proarrhythmic side effects and sets a new standard in ion channel research for drug safety testing. PMID:22303293

The slow inward calcium current is responsible for a part of the contraction of patch-clamped rat myoballs.

PubMed

Rivet, M; Cognard, C; Raymond, G

1989-01-01

The slow inward calcium current and the contractile response were simultaneously recorded in voltage clamped (whole cell patch clamp recording) rat myoballs in primary culture. The shape of the contraction(T)/potential(V) relationship and the application of the inorganic calcium channel blocker cadmium (1.5 mM), which suppresses a part of the contractile activity, demonstrate the existence of two components of contraction. One of them is related to the slow calcium current.

Ion channel pharmacology under flow: automation via well-plate microfluidics.

PubMed

Spencer, C Ian; Li, Nianzhen; Chen, Qin; Johnson, Juliette; Nevill, Tanner; Kammonen, Juha; Ionescu-Zanetti, Cristian

2012-08-01

Automated patch clamping addresses the need for high-throughput screening of chemical entities that alter ion channel function. As a result, there is considerable utility in the pharmaceutical screening arena for novel platforms that can produce relevant data both rapidly and consistently. Here we present results that were obtained with an innovative microfluidic automated patch clamp system utilizing a well-plate that eliminates the necessity of internal robotic liquid handling. Continuous recording from cell ensembles, rapid solution switching, and a bench-top footprint enable a number of assay formats previously inaccessible to automated systems. An electro-pneumatic interface was employed to drive the laminar flow of solutions in a microfluidic network that delivered cells in suspension to ensemble recording sites. Whole-cell voltage clamp was applied to linear arrays of 20 cells in parallel utilizing a 64-channel voltage clamp amplifier. A number of unique assays requiring sequential compound applications separated by a second or less, such as rapid determination of the agonist EC(50) for a ligand-gated ion channel or the kinetics of desensitization recovery, are enabled by the system. In addition, the system was validated via electrophysiological characterizations of both voltage-gated and ligand-gated ion channel targets: hK(V)2.1 and human Ether-à-go-go-related gene potassium channels, hNa(V)1.7 and 1.8 sodium channels, and (α1) hGABA(A) and (α1) human nicotinic acetylcholine receptor receptors. Our results show that the voltage dependence, kinetics, and interactions of these channels with pharmacological agents were matched to reference data. The results from these IonFlux™ experiments demonstrate that the system provides high-throughput automated electrophysiology with enhanced reliability and consistency, in a user-friendly format.

Comparison of sarcolemmal calcium channel current in rabbit and rat ventricular myocytes.

PubMed Central

Yuan, W; Ginsburg, K S; Bers, D M

1996-01-01

1. Fundamental properties of Ca2+ channel currents in rat and rabbit ventricular myocytes were measured using whole cell voltage clamp. 2. In rat, as compared with rabbit myocytes, Ca2+ channel current (ICa) was half-activated at about 10 mV more negative potential, decayed slower, was half-inactivated (in steady state) at about 5 mV more positive potential, and recovered faster from inactivation. 3. These features result in a larger steady-state window current in rat, and also suggest that under comparable voltage clamp conditions, including action potential (AP) clamp, more Ca2+ influx would be expected in rat myocytes. 4. Ca2+ channel current carried by Na+ and Cs+ in the absence of divalent ions (Ins) also activated at more negative potential and decayed more slowly in rat. 5. The reversal potential for Ins was 6 mV more positive in rabbit, consistent with a larger permeability ratio (PNa/PCs) in rabbit than in rat. ICa also reversed at slightly more positive potentials in rabbit (such that PCa/PCs might also be higher). 6. Ca2+ influx was calculated by integration of ICa evoked by voltage clamp pulses (either square pulses or pulses based on recorded rabbit or rat APs). For a given clamp waveform, the Ca2+ influx was up to 25% greater in rat, as predicted from the fundamental properties of ICa and Ins. 7. However, the longer duration of the AP in rabbit myocytes compensated for the difference in influx, such that the integrated Ca2+ influx via ICa in response to the species-appropriate waveform was about twice as large as that seen in rat. PMID:8799895

The Nano-Patch-Clamp Array: Microfabricated Glass Chips for High-Throughput Electrophysiology

NASA Astrophysics Data System (ADS)

Fertig, Niels

2003-03-01

Electrophysiology (i.e. patch clamping) remains the gold standard for pharmacological testing of putative ion channel active drugs (ICADs), but suffers from low throughput. A new ion channel screening technology based on microfabricated glass chip devices will be presented. The glass chips contain very fine apertures, which are used for whole-cell voltage clamp recordings as well as single channel recordings from mammalian cell lines. Chips containing multiple patch clamp wells will be used in a first bench-top device, which will allow perfusion and electrical readout of each well. This scalable technology will allow for automated, rapid and parallel screening on ion channel drug targets.

Single Nisoldipine-Sensitive Calcium Channels in Smooth Muscle Cells Isolated from Rabbit Mesenteric Artery

NASA Astrophysics Data System (ADS)

Worley, Jennings F.; Deitmer, Joachim W.; Nelson, Mark T.

1986-08-01

Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.

Electrical Oscillations in Two-Dimensional Microtubular Structures

PubMed Central

Cantero, María del Rocío; Perez, Paula L.; Smoler, Mariano; Villa Etchegoyen, Cecilia; Cantiello, Horacio F.

2016-01-01

Microtubules (MTs) are unique components of the cytoskeleton formed by hollow cylindrical structures of αβ tubulin dimeric units. The structural wall of the MT is interspersed by nanopores formed by the lateral arrangement of its subunits. MTs are also highly charged polar polyelectrolytes, capable of amplifying electrical signals. The actual nature of these electrodynamic capabilities remains largely unknown. Herein we applied the patch clamp technique to two-dimensional MT sheets, to characterize their electrical properties. Voltage-clamped MT sheets generated cation-selective oscillatory electrical currents whose magnitude depended on both the holding potential, and ionic strength and composition. The oscillations progressed through various modes including single and double periodic regimes and more complex behaviours, being prominent a fundamental frequency at 29 Hz. In physiological K+ (140 mM), oscillations represented in average a 640% change in conductance that was also affected by the prevalent anion. Current injection induced voltage oscillations, thus showing excitability akin with action potentials. The electrical oscillations were entirely blocked by taxol, with pseudo Michaelis-Menten kinetics and a KD of ~1.29 μM. The findings suggest a functional role of the nanopores in the MT wall on the genesis of electrical oscillations that offer new insights into the nonlinear behaviour of the cytoskeleton. PMID:27256791

A nonlinear electrostatic potential change in the T-system of skeletal muscle detected under passive recording conditions using potentiometric dyes

PubMed Central

1990-01-01

Voltage-sensing dyes were used to examine the electrical behavior of the T-system under passive recording conditions similar to those commonly used to detect charge movement. These conditions are designed to eliminate all ionic currents and render the T-system potential linear with respect to the command potential applied at the surface membrane. However, we found an unexpected nonlinearity in the relationship between the dye signal from the T-system and the applied clamp potential. An additional voltage- and time-dependent optical signal appears over the same depolarizing range of potentials where change movement and mechanical activation occur. This nonlinearity is not associated with unblocked ionic currents and cannot be attributed to lack of voltage clamp control of the T-system, which appears to be good under these conditions. We propose that a local electrostatic potential change occurs in the T-system upon depolarization. An electrostatic potential would not be expected to extend beyond molecular distances of the membrane and therefore would be sensed by a charged dye in the membrane but not by the voltage clamp, which responds solely to the potential of the bulk solution. Results obtained with different dyes suggest that the location of the phenomena giving rise to the extra absorbance change is either intramembrane or at the inner surface of the T-system membrane. PMID:2299329

Analysis of the Effects of Calcium or Magnesium on Voltage-Clamp Currents in Perfused Squid Axons Bathed in Solutions of High Potassium

PubMed Central

Rojas, Eduardo; Taylor, Robert E.; Atwater, Illani; Bezanilla, Francisco

1969-01-01

Isolated axons from the squid, Dosidicus gigas, were internally perfused with potassium fluoride solutions. Membrane currents were measured following step changes of membrane potential in a voltage-clamp arrangement with external isosmotic solution changes in the order: potassium-free artificial seawater; potassium chloride; potassium chloride containing 10, 25, 40 or 50, mM calcium or magnesium; and potassium-free artificial seawater. The following results suggest that the currents measured under voltage clamp with potassium outside and inside can be separated into two components and that one of them, the predominant one, is carried through the potassium system. (a) Outward currents in isosmotic potassium were strongly and reversibly reduced by tetraethylammonium chloride. (b) Without calcium or magnesium a progressive increase in the nontime-dependent component of the currents (leakage) occurred. (c) The restoration of calcium or magnesium within 15–30 min decreases this leakage. (d) With 50 mM divalent ions the steady-state current-voltage curve was nonlinear with negative resistance as observed in intact axons in isosmotic potassium. (e) The time-dependent components of the membrane currents were not clearly affected by calcium or magnesium. These results show a strong dependence of the leakage currents on external calcium or magnesium concentration but provide no support for the involvement of calcium or magnesium in the kinetics of the potassium system. PMID:5823216

Analysis of the effects of calcium or magnesium on voltage-clamp currents in perfused squid axons bathed in solutions of high potassium.

PubMed

Rojas, E; Taylor, R E; Atwater, I; Bezanilla, F

1969-10-01

Isolated axons from the squid, Dosidicus gigas, were internally perfused with potassium fluoride solutions. Membrane currents were measured following step changes of membrane potential in a voltage-clamp arrangement with external isosmotic solution changes in the order: potassium-free artificial seawater; potassium chloride; potassium chloride containing 10, 25, 40 or 50, mM calcium or magnesium; and potassium-free artificial seawater. The following results suggest that the currents measured under voltage clamp with potassium outside and inside can be separated into two components and that one of them, the predominant one, is carried through the potassium system. (a) Outward currents in isosmotic potassium were strongly and reversibly reduced by tetraethylammonium chloride. (b) Without calcium or magnesium a progressive increase in the nontime-dependent component of the currents (leakage) occurred. (c) The restoration of calcium or magnesium within 15-30 min decreases this leakage. (d) With 50 mM divalent ions the steady-state current-voltage curve was nonlinear with negative resistance as observed in intact axons in isosmotic potassium. (e) The time-dependent components of the membrane currents were not clearly affected by calcium or magnesium. These results show a strong dependence of the leakage currents on external calcium or magnesium concentration but provide no support for the involvement of calcium or magnesium in the kinetics of the potassium system.

Alteration of the fast excitatory postsynaptic current by barium in voltage-clamped amphibian sympathetic ganglion cells.

PubMed Central

Connor, E. A.; Parsons, R. L.

1984-01-01

Barium-induced alterations in fast excitatory postsynaptic currents (e.p.s.cs) have been studied in voltage-clamped bullfrog sympathetic ganglion B cells. In the presence of 2-8 mM barium, e.p.s.c. decay was prolonged and in many cells the e.p.s.c. decay phase deviated from a single exponential function. The decay phase in these cases was more accurately described as the sum of two exponential functions. The frequency of occurrence of a complex decay increased both with increasing barium concentration and with hyperpolarization. Miniature e.p.s.c. decay also was prolonged in barium-treated cells. E.p.s.c. amplitude was not markedly affected by barium (2-8 mM) in cells voltage-clamped to -50 mV whereas at -90 mV there was a progressive increase in peak size with increasing barium concentration. In control cells the e.p.s.c.-voltage relationship was linear between -20 and -100 mV; however, this relationship became progressively non-linear with membrane hyperpolarization in barium-treated cells. The e.p.s.c. reversal potential was shifted to a more negative value in the presence of barium. There was a voltage-dependent increase in charge movement during the e.p.s.c. in barium-treated cells which was not present in control cells. We conclude that the voltage-dependent alteration in e.p.s.c. decay time course, peak amplitude and charge movement in barium-treated cells is due to a direct postsynaptic action of barium on the kinetics of receptor-channel gating in postganglionic sympathetic neurones. PMID:6333261

A new type of single-phase five-level inverter

NASA Astrophysics Data System (ADS)

Xu, Zhi; Li, Shengnan; Qin, Risheng; Zhao, Yanhang

2017-11-01

At present, Neutral Point Clamped (NPC) multilevel inverter is widely applied in new energy field. However, it has some disadvantages including low utilization rate of direct current (DC) voltage source and the unbalance of neutral potential. Therefore, a new single-phase five level inverter is proposed in this paper. It has two stage structure, the former stage is equivalent to three level DC/DC converter, and the back stage uses H bridge to realize inverter. Compared with the original central clamp type inverter, the new five level inverter can improve the utilization of DC voltage, and realize the neutral point potential balance with hysteresis comparator.

Calcium dependent current recordings in Xenopus laevis oocytes in microgravity

NASA Astrophysics Data System (ADS)

Wuest, Simon L.; Roesch, Christian; Ille, Fabian; Egli, Marcel

2017-12-01

Mechanical unloading by microgravity (or weightlessness) conditions triggers profound adaptation processes at the cellular and organ levels. Among other mechanisms, mechanosensitive ion channels are thought to play a key role in allowing cells to transduce mechanical forces. Previous experiments performed under microgravity have shown that gravity affects the gating properties of ion channels. Here, a method is described to record a calcium-dependent current in native Xenopus laevis oocytes under microgravity conditions during a parabolic flight. A 3-voltage-step protocol was applied to provoke a calcium-dependent current. This current increased with extracellular calcium concentration and could be reduced by applying extracellular gadolinium. The custom-made ;OoClamp; hardware was validated by comparing the results of the 3-voltage-step protocol to results obtained with a well-established two-electrode voltage clamp (TEVC). In the context of the 2nd Swiss Parabolic Flight Campaign, we tested the OoClamp and the method. The setup and experiment protocol worked well in parabolic flight. A tendency that the calcium-dependent current was smaller under microgravity than under 1 g condition could be observed. However, a conclusive statement was not possible due to the small size of the data base that could be gathered.

Barbiturates Block Sodium and Potassium Conductance Increases in Voltage-Clamped Lobster Axons

PubMed Central

Blaustein, M. P.

1968-01-01

Sodium pentobarbital and sodium thiopental decrease both the peak initial (Na) and late steady-state (K) currents and reduce the maximum sodium and potassium conductance increases in voltage-clamped lobster giant axons. These barbiturates also slow the rate at which the sodium conductance turns on, and shift the normalized sodium conductance vs. voltage curves in the direction of depolarization along the voltage axis. Since pentobarbital (pKa = 8.0) blocks the action potential more effectively at pH 8.5 than at pH 6.7, the anionic form of the drug appears to be active. The data suggest that these drugs affect the axon membrane directly, rather than secondarily through effects on intermediary metabolism. It is suggested that penetration of the lipid layer of the membrane by the nonpolar portion of the barbiturate molecules may cause the decrease in membrane conductances, while electrostatic interactions involving the anionic group on the barbiturate, divalent cations, and "fixed charges" in the membrane could account for the slowing of the rate of sodium conductance turn-on and the shift of the normalized conductance curves along the voltage axis. PMID:5648829

A novel high performance ESD power clamp circuit with a small area

NASA Astrophysics Data System (ADS)

Zhaonian, Yang; Hongxia, Liu; Li, Li; Qingqing, Zhuo

2012-09-01

A MOSFET-based electrostatic discharge (ESD) power clamp circuit with only a 10 ns RC time constant for a 0.18-μm process is proposed. A diode-connected NMOSFET is used to maintain a long delay time and save area. The special structure overcomes other shortcomings in this clamp circuit. Under fast power-up events, the gate voltage of the clamp MOSFET does not rise as quickly as under ESD events, the special structure can keep the clamp MOSFET thoroughly off. Under a falsely triggered event, the special structure can turn off the clamp MOSFET in a short time. The clamp circuit can also reject the power supply noise effectively. Simulation results show that the clamp circuit avoids fast false triggering events such as a 30 ns/1.8 V power-up, maintains a 1.2 μs delay time and a 2.14 μs turn-off time, and reduces to about 70% of the RC time constant. It is believed that the proposed clamp circuit can be widely used in high-speed integrated circuits.

Closed-form solution for static pull-in voltage of electrostatically actuated clamped-clamped micro/nano beams under the effect of fringing field and van der Waals force

NASA Astrophysics Data System (ADS)

Bhojawala, V. M.; Vakharia, D. P.

2017-12-01

This investigation provides an accurate prediction of static pull-in voltage for clamped-clamped micro/nano beams based on distributed model. The Euler-Bernoulli beam theory is used adapting geometric non-linearity of beam, internal (residual) stress, van der Waals force, distributed electrostatic force and fringing field effects for deriving governing differential equation. The Galerkin discretisation method is used to make reduced-order model of the governing differential equation. A regime plot is presented in the current work for determining the number of modes required in reduced-order model to obtain completely converged pull-in voltage for micro/nano beams. A closed-form relation is developed based on the relationship obtained from curve fitting of pull-in instability plots and subsequent non-linear regression for the proposed relation. The output of regression analysis provides Chi-square (χ 2) tolerance value equals to 1  ×  10-9, adjusted R-square value equals to 0.999 29 and P-value equals to zero, these statistical parameters indicate the convergence of non-linear fit, accuracy of fitted data and significance of the proposed model respectively. The closed-form equation is validated using available data of experimental and numerical results. The relative maximum error of 4.08% in comparison to several available experimental and numerical data proves the reliability of the proposed closed-form equation.

Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenberg, R.L.

1985-01-01

The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based onmore » molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.« less

Dynamics of internal pore opening in KV channels probed by a fluorescent unnatural amino acid

PubMed Central

Kalstrup, Tanja; Blunck, Rikard

2013-01-01

Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNA-synthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions. PMID:23630265

A novel frequency analysis method for assessing K(ir)2.1 and Na (v)1.5 currents.

PubMed

Rigby, J R; Poelzing, S

2012-04-01

Voltage clamping is an important tool for measuring individual currents from an electrically active cell. However, it is difficult to isolate individual currents without pharmacological or voltage inhibition. Herein, we present a technique that involves inserting a noise function into a standard voltage step protocol, which allows one to characterize the unique frequency response of an ion channel at different step potentials. Specifically, we compute the fast Fourier transform for a family of current traces at different step potentials for the inward rectifying potassium channel, K(ir)2.1, and the channel encoding the cardiac fast sodium current, Na(v)1.5. Each individual frequency magnitude, as a function of voltage step, is correlated to the peak current produced by each channel. The correlation coefficient vs. frequency relationship reveals that these two channels are associated with some unique frequencies with high absolute correlation. The individual IV relationship can then be recreated using only the unique frequencies with magnitudes of high absolute correlation. Thus, this study demonstrates that ion channels may exhibit unique frequency responses.

NO involvement in the inhibition of ghrelin on voltage-dependent potassium currents in rat hippocampal cells.

PubMed

Lu, Yong; Dang, Shaokang; Wang, Xu; Zhang, Junli; Zhang, Lin; Su, Qian; Zhang, Huiping; Lin, Tianwei; Zhang, Xiaoxiao; Zhang, Yurong; Sun, Hongli; Zhu, Zhongliang; Li, Hui

2018-01-01

Ghrelin is a peptide hormone that plays an important role in promoting appetite, regulating distribution and rate of use of energy, cognition, and mood disorders, but the relevant neural mechanisms of these function are still not clear. In this study, we examined the effect of ghrelin on voltage-dependent potassium (K + ) currents in hippocampal cells of 1-3 days SD rats by whole-cell patch-clamp technique, and discussed whether NO was involved in this process. The results showed that ghrelin significantly inhibited the voltage-dependent K + currents in hippocampal cells, and the inhibitory effect was more significant when l-arginine was co-administered. In contrast, N-nitro- l-arginine methyl ester increased the ghrelin inhibited K + currents and attenuated the inhibitory effect of ghrelin. While d-arginine (D-AA) showed no significant impact on the ghrelin-induced decrease in K + current. These results show that ghrelin may play a physiological role by inhibiting hippocampal voltage dependent K + currents, and the NO pathway may be involved in this process. Copyright © 2017 Elsevier B.V. All rights reserved.

Impact of Renal Hilar Control on Outcomes of Robotic Partial Nephrectomy: Systematic Review and Cumulative Meta-analysis.

PubMed

Cacciamani, Giovanni E; Medina, Luis G; Gill, Tania S; Mendelsohn, Alec; Husain, Fatima; Bhardwaj, Lokesh; Artibani, Walter; Sotelo, Renè; Gill, Inderbir S

2018-02-05

During robotic partial nephrectomy (RPN), various techniques of hilar control have been described, including on-clamp, early unclamping, selective/super-selective clamping, and completely-unclamped RPN. To evaluate the impact of various hilar control techniques on perioperative, functional, and oncological outcomes of RPN for tumors. We conducted a systematic literature review and meta-analysis of all comparative studies on various hilar control techniques during RPN using PubMed, Scopus, and Web of Science according to the Preferred Reporting Items for Systematic Review and Meta-analysis statement, and Methods and Guide for Effectiveness and Comparative Effectiveness Review of the Agency for Healthcare Research and Quality. Cumulative meta-analysis of comparative studies was conducted using Review Manager 5.3. Of 987 RPN publications in the literature, 19 qualified for this analysis. Comparison of off-clamp versus on-clamp RPN (n=9), selective clamping versus on-clamp RPN (n=3), super selective clamping versus on-clamp RPN (n=5), and early unclamped versus on-clamp (n=3) were reported. Patients undergoing RPN using off-clamp, selective/super selective, or early unclamp techniques had higher estimated blood loss compared with on-clamp RPN (weight mean difference [WMD]: 47.83, p=0.000, WMD: 41.06, p=0.02, and WMD: 37.50, p=0.47); however, this did not seem clinically relevant, since transfusion rates were similar (odds ratio [OR]: 0.98, p=0.95, OR: 0.72, p=0.7, and OR: 1.36, p=0.33, respectively). All groups appeared similar with regards to hospital stay, transfusions, overall and major complications, and positive cancer margin rates. Short- and long-term renal functional outcomes appeared superior in the off-clamp and super selective clamp groups compared with the on-clamp RPN cohort. Off-clamp, selective/super selective clamp, and early unclamp hilar control techniques are safe and feasible approaches for RPN surgery, with similar perioperative and oncological outcomes compared with on-clamp RPN. Minimizing global renal ischemia may provide superior renal function preservation. However, higher quality data are necessary for definitive conclusions in this regard. The objective of partial nephrectomy is to treat the cancer while maximizing renal function preservation. Clamping the main vessels is done primarily to reduce the blood loss during partial nephrectomy; however, vascular clamping can compromise kidney function. In order to avoid clamping, various techniques have been described. Our analysis showed that techniques that avoid main renal artery clamping during RPN are associated with better renal function preservation, yet deliver non-inferior perioperative and oncological outcomes as compared with robotic partial nephrectomy procedures that clamp the main vessels. Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Inhibition of mechanotransducer currents in crayfish sensory neuron by CGS 9343B, a calmodulin antagonist.

PubMed

Lin, J H; Rydqvist, B

2000-05-26

The effects of CGS 9343B (zaldaride maleate), a calmodulin antagonist, on mechanosensitive channels were examined in crayfish slowly adapting sensory neurons using the two-electrode voltage clamp technique. In addition to its inhibition of voltage-gated Na(+) and K(+) currents, CGS 9343B (<30 microM) blocked reversibly the receptor current in a dose-dependent and voltage-dependent manner with a dissociation constant (K(d)) of 26.8 microM. The time course of the block was 265 s. Within the extension range of 3-30%, the reduction in receptor current was stimulus-independent and the gating mechanisms were not affected. Extracellular Ca(2+) was not necessary for its blocking effects. No changes in passive muscle tension were observed in the presence of 20 microM CGS 9343B. These results suggest that CGS 9343B, as a calmodulin antagonist, can also block mechanosensitive channels, possibly by being incorporated into the lipid membrane and/or interacting with the channel protein.

Distinct pH regulation of slow and rapid anion channels at the plasma membrane of Arabidopsis thaliana hypocotyl cells.

PubMed

Colcombet, Jean; Lelièvre, Françoise; Thomine, Sébastien; Barbier-Brygoo, Hélène; Frachisse, Jean-Marie

2005-07-01

Variations in both intracellular and extracellular pH are known to be involved in a wealth of physiological responses. Using the patch-clamp technique on Arabidopsis hypocotyl cells, it is shown that rapid-type and slow-type anion channels at the plasma membrane are both regulated by pH via distinct mechanisms. Modifications of pH modulate the voltage-dependent gating of the rapid channel. While intracellular alkalinization facilitates channel activation by shifting the voltage gate towards negative potentials, extracellular alkalinization shifts the activation threshold to more positive potentials, away from physiological resting membrane potentials. By contrast, pH modulates slow anion channel activity in a voltage-independent manner. Intracellular acidification and extracellular alkalinization increase slow anion channel currents. The possible role of these distinct modulations in physiological processes involving anion efflux and modulation of extracellular and/or intracellular pH, such as elicitor and ABA signalling, are discussed.

Preferential inhibition of Ih in rat trigeminal ganglion neurons by an organic blocker.

PubMed

Janigro, D; Martenson, M E; Baumann, T K

1997-11-15

The potency and specificity of a novel organic Ih current blocker DK-AH 268 (DK, Boehringer) was studied in cultured rat trigeminal ganglion neurons using whole-cell patch-clamp recording techniques. In neurons current-clamped at the resting potential, the application of 10 microM DK caused a slight hyperpolarization of the membrane potential and a small increase in the threshold for action potential discharge without any major change in the shape of the action potential. In voltage-clamped neurons, DK caused a reduction of a hyperpolarization-activated current. Current subtraction protocols revealed that the time-dependent, hyperpolarization-activated currents blocked by 10 microM DK or external Cs+ (3 mM) had virtually identical activation properties, suggesting that DK and Cs+ caused blockade of the same current, namely Ih. The block of Ih by DK was dose-dependent. At the intermediate and higher concentrations of DK (10 and 100 microM) a decrease in specificity was observed so that time-independent, inwardly rectifying and noninactivating, voltage-gated outward potassium currents were also reduced by DK but to a much lesser extent than the time-dependent, hyperpolarization-activated currents. Blockade of the time-dependent, hyperpolarization-activated currents by DK appeared to be use-dependent since it required hyperpolarization for the effect to take place. Relief of DK block was also aided by membrane hyperpolarization. Since both the time-dependent current blocked by DK and the Cs+-sensitive time-dependent current behaved as Ih, we conclude that 10 microM DK can preferentially reduce Ih without a major effect on other potassium currents. Thus, DK may be a useful agent in the investigation of the function of Ih in neurons.

Cell-Detection Technique for Automated Patch Clamping

NASA Technical Reports Server (NTRS)

McDowell, Mark; Gray, Elizabeth

2008-01-01

A unique and customizable machinevision and image-data-processing technique has been developed for use in automated identification of cells that are optimal for patch clamping. [Patch clamping (in which patch electrodes are pressed against cell membranes) is an electrophysiological technique widely applied for the study of ion channels, and of membrane proteins that regulate the flow of ions across the membranes. Patch clamping is used in many biological research fields such as neurobiology, pharmacology, and molecular biology.] While there exist several hardware techniques for automated patch clamping of cells, very few of those techniques incorporate machine vision for locating cells that are ideal subjects for patch clamping. In contrast, the present technique is embodied in a machine-vision algorithm that, in practical application, enables the user to identify good and bad cells for patch clamping in an image captured by a charge-coupled-device (CCD) camera attached to a microscope, within a processing time of one second. Hence, the present technique can save time, thereby increasing efficiency and reducing cost. The present technique involves the utilization of cell-feature metrics to accurately make decisions on the degree to which individual cells are "good" or "bad" candidates for patch clamping. These metrics include position coordinates (x,y) in the image plane, major-axis length, minor-axis length, area, elongation, roundness, smoothness, angle of orientation, and degree of inclusion in the field of view. The present technique does not require any special hardware beyond commercially available, off-the-shelf patch-clamping hardware: A standard patchclamping microscope system with an attached CCD camera, a personal computer with an imagedata- processing board, and some experience in utilizing imagedata- processing software are all that are needed. A cell image is first captured by the microscope CCD camera and image-data-processing board, then the image data are analyzed by software that implements the present machine-vision technique. This analysis results in the identification of cells that are "good" candidates for patch clamping (see figure). Once a "good" cell is identified, a patch clamp can be effected by an automated patchclamping apparatus or by a human operator. This technique has been shown to enable reliable identification of "good" and "bad" candidate cells for patch clamping. The ultimate goal in further development of this technique is to combine artificial-intelligence processing with instrumentation and controls in order to produce a complete "turnkey" automated patch-clamping system capable of accurately and reliably patch clamping cells with a minimum intervention by a human operator. Moreover, this technique can be adapted to virtually any cellular-analysis procedure that includes repetitive operation of microscope hardware by a human.

Control method for peak power delivery with limited DC-bus voltage

DOEpatents

Edwards, John; Xu, Longya; Bhargava, Brij B.

2006-09-05

A method for driving a neutral point-clamped multi-level voltage source inverter supplying a synchronous motor is provided. A DC current is received at a neutral point-clamped multi-level voltage source inverter. The inverter has first, second, and third output nodes. The inverter also has a plurality of switches. A desired speed of a synchronous motor connected to the inverter by the first second and third nodes is received by the inverter. The synchronous motor has a rotor and the speed of the motor is defined by the rotational rate of the rotor. A position of the rotor is sensed, current flowing to the motor out of at least two of the first, second, and third output nodes is sensed, and predetermined switches are automatically activated by the inverter responsive to the sensed rotor position, the sensed current, and the desired speed.

Direct electro-optic effect in langasites and α-quartz

NASA Astrophysics Data System (ADS)

Ivanov, Vadim

2018-05-01

Strain-constant (clamped) electro-optic coefficients r11S of langasite La3Ga5SiO14 (LGS), langatate La3Ga5.5Ta0.5O14 (LGT), catangasite Ca3TaGa3Si2O14 (CTGS) and α-quartz are measured at 1540 nm in the frequency range of 3-25 MHz. Experimental ratio of clamped and unclamped electro-optic coefficients r11S/r11T is 0.97 for LGS, 0.91 for LGT, 0.31 for CTGS, and 0.49 for quartz. Most of direct electro-optic effect in LGS and LGT is associated with lanthanum ions: clamped electro-optic coefficient r11S in lanthanum-free CTGS is 14 times less than in LGS. Low piezoelectric contribution to unclamped electro-optic coefficient r11T makes LGS and LGT promising materials for electro-optic devices, whose performance can be deteriorated by piezoelectric effect, especially, for high-voltage optical voltage sensors.

Testing of Diode-Clamping in an Inductive Pulsed Plasma Thruster Circuit

NASA Technical Reports Server (NTRS)

Toftul, Alexandra; Polzin, Kurt A.; Martin, Adam K.; Hudgins, Jerry L.

2014-01-01

Testing of a 5.5 kV silicon (Si) diode and 5.8 kV prototype silicon carbide (SiC) diode in an inductive pulsed plasma thruster (IPPT) circuit was performed to obtain a comparison of the resulting circuit recapture efficiency,eta(sub r), defined as the percentage of the initial charge energy remaining on the capacitor bank after the diode interrupts the current. The diode was placed in a pulsed circuit in series with a silicon controlled rectifier (SCR) switch, and the voltages across different components and current waveforms were collected over a range of capacitor charge voltages. Reverse recovery parameters, including turn-off time and peak reverse recovery current, were measured and capacitor voltage waveforms were used to determine the recapture efficiency for each case. The Si fast recovery diode in the circuit was shown to yield a recapture efficiency of up to 20% for the conditions tested, while the SiC diode further increased recapture efficiency to nearly 30%. The data presented show that fast recovery diodes operate on a timescale that permits them to clamp the discharge quickly after the first half cycle, supporting the idea that diode-clamping in IPPT circuit reduces energy dissipation that occurs after the first half cycle

Voltage control of magnetism in NiZn ferrite/mica/PMNPT heterostructure with giant tunability and narrow linewidth

NASA Astrophysics Data System (ADS)

Wang, Xinjun; Chen, Yunpeng; Chen, Huaihao; Gao, Yuan; He, Yifan; Li, Menghui; Lin, Hwaider; Sun, Neville; Sun, Nian

2018-05-01

Recently, large magnetoelectric coupling of a spinel/piezoelectric heterostructure has been reported. However, the linewidth of the spinel is very large due to lattice mismatch when ferrite is directly deposited on piezoelectric substrates. This indicates a large magnetic loss, which impedes the spinel/piezoelectric heterostructure from useful device applications. Mica is a well-known 2D material, which can be split manually layer by layer without the substrate clamping effect. In this report, NiZn ferrite was deposited on a mica substrate by a spin-spray deposition technique. Spin-spray deposition is a wet chemical synthesis technique involving several chemical reactions for generating high-quality crystalline spinel ferrite films with various compositions directly from an aqueous solution. The thickness of ferrite is 2 μm, and the linewidth of the ferromagnetic resonance (FMR) is 115 Oe which is suitable for RF/microwave devices. The large FMR field tuning of 605 Oe was observed in NiZn ferrite/mica/PMN-PT heterostructures with minimal substrate clamping effect by reducing the thickness of the mica substrate. These multiferroic heterostructures exhibiting combined giant magnetoelectric coupling and narrow ferromagnetic resonance linewidth offer great opportunities for flexible RF magnetic devices.

Simultaneous recording of electrical activity and the underlying ionic currents in NG108-15 cells cultured on gold substrate.

PubMed

Acosta-García, Ma Cristina; Morales-Reyes, Israel; Jiménez-Anguiano, Anabel; Batina, Nikola; Castellanos, N P; Godínez-Fernández, R

2018-02-01

This paper shows the simultaneous recording of electrical activity and the underlying ionic currents by using a gold substrate to culture NG108-15 cells. Cells grown on two different substrates (plastic Petri dishes and gold substrates) were characterized quantitatively through scanning electron microscopy (SEM) as well as qualitatively by optical and atomic force microscopy (AFM). No significant differences were observed between the surface area of cells cultured on gold substrates and Petri dishes, as indicated by measurements performed on SEM images. We also evaluated the electrophysiological compatibility of the cells through standard patch-clamp experiments by analyzing features such as the resting potential, membrane resistance, ionic currents, etc. Cells grown on both substrates showed no significant differences in their dependency on voltage, as well as in the magnitude of the Na+ and K+ current density; however, cells cultured on the gold substrate showed a lower membrane capacitance when compared to those grown on Petri dishes. By using two separate patch-clamp amplifiers, we were able to record the membrane current with the conventional patch-clamp technique and through the gold substrate simultaneously. Furthermore, the proposed technique allowed us to obtain simultaneous recordings of the electrical activity (such as action potentials firing) and the underlying membrane ionic currents. The excellent conductivity of gold makes it possible to overcome important difficulties found in conventional electrophysiological experiments such as those presented by the resistance of the electrolytic bath solution. We conclude that the technique here presented constitutes a solution to the problem of the simultaneous recording of electrical activity and the underlying ionic currents, which for decades, had been solved only partially.

Emission control apparatus for internal combustion engines with a controllably disabled clamping circuit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asano, M.

1979-08-28

The invention discloses an emission control apparatus for internal combustion engine includes an exhaust composition sensor to sense the mixture ratio, a circuit for clamping the mixture ratio to a predetermined constant value to prevent the mixture from becoming too rich or too lean when a failure should occur in the control loop, for example, in the exhaust composition sensor failure and a circuit for interrupting the clamping circuit when the engine operating condition is such that the sensor is caused to produce low voltage signals although the sensor is functioning properly.

Voltage-clamp study of the activation currents and fast block to polyspermy in the egg of Xenopus laevis.

PubMed

Glahn, David; Nuccitelli, Richard

2003-04-01

Voltage-clamped mature, jelly-intact Xenopus eggs were used to carefully examine the ionic currents crossing the plasma membrane before, during, and after fertilization. The bulk of the fertilization current was transient, of large amplitude, and reversed at the predicted Cl- reversal potential. However, the large amplitude fertilization current was preceded by a small, step-like increase in holding current. This small increase in holding current is referred to in this paper as Ion to acknowledge its qualitative similarity to the Ion current previously described in the sea urchin. It was observed in both fertilized and artificially activated eggs, and was found to be unaffected by 10 mm tetra-ethyl ammonium (TEA), a concentration found to block K+ currents in Rana pipiens. Current-voltage relationships are presented for the large fertilization potential, and show that the fertilization currents have a marked outward rectification and are voltage sensitive. These properties are in contrast to the total lack of rectification and slight voltage sensitivity seen before or after the fertilization currents. The time required for sperm to fertilize the egg was found to be voltage dependent with a relatively more depolarized voltage requiring a longer time for fertilization to occur. The percentage of eggs blocked with varying potential levels was determined and this information was fitted to a modified Boltzmann equation having a midpoint of -9 mV.

Design and Comparison of Cascaded H-Bridge, Modular Multilevel Converter, and 5-L Active Neutral Point Clamped Topologies for Motor Drive Applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marzoughi, Alinaghi; Burgos, Rolando; Boroyevich, Dushan

This paper presents the design procedure and comparison of converters currently used in medium-voltage high-power motor drive applications. For this purpose, the cascaded H-bridge (CHB), modular multilevel converter (MMC), and five-level active neutral point clamped (5-L ANPC) topologies are targeted. The design is performed using 1.7-kV insulated gate bipolar transistors (IGBTs) for CHB and MMC converters, and utilizing 3.3- and 4.5-kV IGBTs for 5-L ANPC topology as normally done in industry. The comparison is done between the designed converter topologies at three different voltage levels (4.16, 6.9, and 13.8 kV, with only the first two voltage levels in case ofmore » the 5-L ANPC) and two different power levels (3 and 5 MVA), in order to elucidate the dependence of different parameters on voltage and power rating. Finally, the comparison is done from several points of view such as efficiency, capacitive energy storage, semiconductor utilization, parts count (for measure of reliability), and power density.« less

High voltage bus and auxiliary heater control system for an electric or hybrid vehicle

DOEpatents

Murty, Balarama Vempaty

2000-01-01

A control system for an electric or hybrid electric vehicle includes a vehicle system controller and a control circuit having an electric immersion heater. The heater is electrically connected to the vehicle's high voltage bus and is thermally coupled to a coolant loop containing a heater core for the vehicle's climate control system. The system controller responds to cabin heat requests from the climate control system by generating a pulse width modulated signal that is used by the control circuit to operate the heater at a duty cycle appropriate for the amount of cabin heating requested. The control system also uses the heater to dissipate excess energy produced by an auxiliary power unit and to provide electric braking when regenerative braking is not desirable and manual braking is not necessary. The control system further utilizes the heater to provide a safe discharge of a bank of energy storage capacitors following disconnection of the battery or one of the high voltage connectors used to transmit high voltage operating power to the various vehicle systems. The control circuit includes a high voltage clamping circuit that monitors the voltage on the bus and operates the heater to clamp down the bus voltage when it exceeds a pre-selected maximum voltage. The control system can also be used to phase in operation of the heater when the bus voltage exceeds a lower threshold voltage and can be used to phase out the auxiliary power unit charging and regenerative braking when the battery becomes fully charged.

Selective Arterial Clamping Versus Hilar Clamping for Minimally Invasive Partial Nephrectomy.

PubMed

Yezdani, Mona; Yu, Sue-Jean; Lee, David I

2016-05-01

Partial nephrectomy has become an accepted treatment of cT1 renal masses as it provides improved long-term renal function compared to radical nephrectomy (Campbell et al. J Urol. 182:1271-9, 2009). Hilar clamping is utilized to help reduce bleeding and improve visibility during tumor resection. However, concern over risk of kidney injury with hilar clamping has led to new techniques to reduce length of warm ischemia time (WIT) during partial nephrectomy. These techniques have progressed over the years starting with early hilar unclamping, controlled hypotension during tumor resection, selective arterial clamping, minimal margin techniques, and off-clamp procedures. Selective arterial clamping has progressed significantly over the years. The main question is what are the exact short- and long-term renal effects from increasing clamp time. Moreover, does it make sense to perform these more time-consuming or more complex procedures if there is no long-term preservation of kidney function? More recent studies have shown no difference in renal function 6 months from surgery when selective arterial clamping or even hilar clamping is employed, although there is short-term improved decline in estimated glomerular filtration rate (eGFR) with selective clamping and off-clamp techniques (Komninos et al. BJU Int. 115:921-8, 2015; Shah et al. 117:293-9, 2015; Kallingal et al. BJU Int. doi: 10.1111/bju.13192, 2015). This paper reviews the progression of total hilar clamping to selective arterial clamping (SAC) and the possible difference its use makes on long-term renal function. SAC may be attempted based on surgeon's decision-making, but may be best used for more complex, larger, more central or hilar tumors and in patients who have renal insufficiency at baseline or a solitary kidney.

Effects of acidic pH on voltage-gated ion channels in rat trigeminal mesencephalic nucleus neurons.

PubMed

Han, Jin-Eon; Cho, Jin-Hwa; Choi, In-Sun; Kim, Do-Yeon; Jang, Il-Sung

2017-03-01

The effects of acidic pH on several voltage-dependent ion channels, such as voltage-dependent K + and Ca 2+ channels, and hyperpolarization-gated and cyclic nucleotide-activated cation (HCN) channels, were examined using a whole-cell patch clamp technique on mechanically isolated rat mesencephalic trigeminal nucleus neurons. The application of a pH 6.5 solution had no effect on the peak amplitude of voltage-dependent K + currents. A pH 6.0 solution slightly, but significantly inhibited the peak amplitude of voltage-dependent K + currents. The pH 6.0 also shifted both the current-voltage and conductance-voltage relationships to the depolarization range. The application of a pH 6.5 solution scarcely affected the peak amplitude of membrane currents mediated by HCN channels, which were profoundly inhibited by the general HCN channel blocker Cs + (1 mM). However, the pH 6.0 solution slightly, but significantly inhibited the peak amplitude of HCN-mediated currents. Although the pH 6.0 solution showed complex modulation of the current-voltage and conductance-voltage relationships, the midpoint voltages for the activation of HCN channels were not changed by acidic pH. On the other hand, voltage-dependent Ca 2+ channels were significantly inhibited by an acidic pH. The application of an acidic pH solution significantly shifted the current-voltage and conductance-voltage relationships to the depolarization range. The modulation of several voltage-dependent ion channels by an acidic pH might affect the excitability of mesencephalic trigeminal nucleus neurons, and thus physiological functions mediated by the mesencephalic trigeminal nucleus could be affected in acidic pH conditions.

Electrodiffusion kinetics of ionic transport in a simple membrane channel.

PubMed

Valent, Ivan; Petrovič, Pavol; Neogrády, Pavel; Schreiber, Igor; Marek, Miloš

2013-11-21

We employ numerical techniques for solving time-dependent full Poisson-Nernst-Planck (PNP) equations in 2D to analyze transient behavior of a simple ion channel subject to a sudden electric potential jump across the membrane (voltage clamp). Calculated spatiotemporal profiles of the ionic concentrations and electric potential show that two principal exponential processes can be distinguished in the electrodiffusion kinetics, in agreement with original Planck's predictions. The initial fast process corresponds to the dielectric relaxation, while the steady state is approached in a second slower exponential process attributed to the nonlinear ionic redistribution. Effects of the model parameters such as the channel length, height of the potential step, boundary concentrations, permittivity of the channel interior, and ionic mobilities on electrodiffusion kinetics are studied. Numerical solutions are used to determine spatiotemporal profiles of the electric field, ionic fluxes, and both the conductive and displacement currents. We demonstrate that the displacement current is a significant transient component of the total electric current through the channel. The presented results provide additional information about the classical voltage-clamp problem and offer further physical insights into the mechanism of electrodiffusion. The used numerical approach can be readily extended to multi-ionic models with a more structured domain geometry in 2D or 3D, and it is directly applicable to other systems, such as synthetic nanopores, nanofluidic channels, and nanopipettes.

A patch clamp study on reconstituted calcium permeable channels of human sperm plasma membranes.

PubMed

Ma, X H; Shi, Y L

1999-10-01

Ionic flux is thought to be important in the initiating process of gamete interaction such as acrosome reaction. However, modern electrophysiological methods, intracellular recording and patch-clamping, are difficult to approach the ion channels in mammal sperm membrane of an intact sperm due to its small size. In this work, by reconstituting the channel protein into lipid bilayer, Ca2+ channels in human spermatozoa were investigated with voltage clamp technique. Membrane proteins isolated from human sperm of 12 healthy donors were incorporated into lipid bilayer via fusion. In a cis 50//trans 10 mmol/L CaCl2 solution system, two types of channel events with similar reversal potential near the value of a perfect Ca2+ electrode, and sensitive to nifedipine and verapamil, were observed. Their unit conductance was 40 and 25 pS respectively. Percentage of channel open time was not dependent to holding potential for the former. However, for the channels of 25 pS, the percentage increased when the holding potential was changed from -20 to 100 mV. Ca(2+)-permeable channels were also detected from the spermatozoon samples of two infertile donors. Abnormal open time of these channels indicates that there are some defects in the conformation of the channel protein of infertile sperm membrane.

Elucidation of pyrethroid and DDT receptor sites in the voltage-gated sodium channel.

PubMed

Zhorov, Boris S; Dong, Ke

2017-05-01

DDT and pyrethroid insecticides were among the earliest neurotoxins identified to act on voltage-gated sodium channels. In the 1960s, equipped with, at the time, new voltage-clamp techniques, Professor Narahashi and associates provided the initial evidence that DDT and allethrin (the first commercial pyrethroid insecticide) caused prolonged flow of sodium currents in lobster and squid giant axons. Over the next several decades, continued efforts by Prof. Narahashi's group as well as other laboratories led to a comprehensive understanding of the mechanism of action of DDT and pyrethroids on sodium channels. Fast forward to the 1990s, genetic, pharmacological and toxicological data all further confirmed voltage-gated sodium channels as the primary targets of DDT and pyrethroid insecticides. Modifications of the gating kinetics of sodium channels by these insecticides result in repetitive firing and/or membrane depolarization in the nervous system. This mini-review focuses on studies from Prof. Narahashi's pioneer work and more recent mutational and computational modeling analyses which collectively elucidated the elusive pyrethroid receptor sites as well as the molecular basis of differential sensitivities of insect and mammalian sodium channels to pyrethroids. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrophysiological responses of dissociated type I cells of the rabbit carotid body to cyanide.

PubMed Central

Biscoe, T J; Duchen, M R

1989-01-01

1. The carotid body is the major peripheral sensor of arterial PO2 in the mammal and is excited by cyanide (CN-). Type I cells, the presumed sites for transduction, were freshly dissociated from the carotid body of the adult rabbit and studied with the whole-cell patch clamp technique. 2. Type I cells were hyperpolarized by CN-, the action potential was shortened, and there was an increased after-hyperpolarization. 3. Under voltage clamp control, CN- increased a voltage-dependent outward current, which showed pronounced outward rectification. Tail currents increased by CN- reversed close to the predicted EK, the reversal potential of the CN--induced current depended on extracellular [K+], and the current was blocked by intracellular TEA+ and Cs+. 4. The i-V relation of the CN--induced conductance strongly mirrored that of voltage-gated Ca2+ entry, and the response was abolished by removal of extracellular Ca2+. We conclude that the increased gK is Ca2+ -dependent (gK(Ca]. 5. The Ca2+ current was attenuated by CN-, and showed an increased rate of inactivation. Thus, the increased gK(Ca) must result from an alteration in Ca2+ homeostasis independent of the Ca2+ current, and not an increased Ca2+ entry through voltage-activated channels. 6. Carbachol also hyperpolarized cells and increased a K+ conductance. 7. At depolarized holding potentials a steady-state outward current was increased by CN-. The current reversed close to EK, and was associated with increased current fluctuations. Noise analysis showed that a channel conductance of 3 pS carries the current. 8. The response to CN- was not impaired by the inclusion of 5 mM-MgATP in the patch pipette. 9. If signals to the CNS are initiated by the calcium-dependent release of transmitters from type I cells, transduction would appear to be the direct consequence of the energy dependence of Ca2+ homeostasis. PMID:2557439

C-Terminal residues in small potassium channel blockers OdK1 and OSK3 from scorpion venom fine-tune the selectivity.

PubMed

Kuzmenkov, Alexey I; Peigneur, Steve; Chugunov, Anton O; Tabakmakher, Valentin M; Efremov, Roman G; Tytgat, Jan; Grishin, Eugene V; Vassilevski, Alexander A

2017-05-01

We report isolation, sequencing, and electrophysiological characterization of OSK3 (α-KTx 8.8 in Kalium and Uniprot databases), a potassium channel blocker from the scorpion Orthochirus scrobiculosus venom. Using the voltage clamp technique, OSK3 was tested on a wide panel of 11 voltage-gated potassium channels expressed in Xenopus oocytes, and was found to potently inhibit Kv1.2 and Kv1.3 with IC 50 values of ~331nM and ~503nM, respectively. OdK1 produced by the scorpion Odontobuthus doriae differs by just two C-terminal residues from OSK3, but shows marked preference to Kv1.2. Based on the charybdotoxin-potassium channel complex crystal structure, a model was built to explain the role of the variable residues in OdK1 and OSK3 selectivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X).

PubMed

Thomas, Dierk; Hammerling, Bettina C; Wimmer, Anna-Britt; Wu, Kezhong; Ficker, Eckhard; Kuryshev, Yuri A; Scherer, Daniel; Kiehn, Johann; Katus, Hugo A; Schoels, Wolfgang; Karle, Christoph A

2004-12-01

The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, I(Kr). The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and I(Kr) currents was investigated in this study. hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native I(Kr) currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC(50) values of 1.0 and 13.2 muM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of -20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 muM BIM I reduced native I(Kr) currents by 69.2% and lead to action potential prolongation. In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and I(Kr) currents.

The blockade of excitation/contraction coupling by nifedipine in patch-clamped rat skeletal muscle cells in culture.

PubMed

Cognard, C; Rivet, M; Raymond, G

1990-04-01

The effects of the dihydropyridine derivative, nifedipine, well known as a blocker of calcium channels, were tested on cultured rat myoballs. Membrane currents and contractions were simultaneously recorded by means of the patch-clamp technique and a photoelectric transducing method. High concentrations of nifedipine (5 microM) inhibited the contractile responses and inward calcium current (ICa) elicited by long depolarizations. In the absence of ICa (1.5 mM cadmium in the bath), nifedipine inhibited both the ICa-independent contractile component and the outward current, supposed to depend on the intracellular calcium released during contraction. At low concentrations (0.5 microM) the blocking effects of nifedipine could be strongly enhanced by shifting the membrane potential towards less negative values (-60 mV) for 50 s prior to the test pulse. A blocking effect of nifedipine, at a usually ineffective concentration (0.1 microM), could also be observed when long-lasting (3 min) prepulses to 0 mV were applied from a reference membrane potential of -60 mV. This effect could be relieved by long-lasting cell hyperpolarizations (-90 mV). The blocking effects of nifedipine unrelated to ICa could be interpreted as an action on a molecule (voltage sensor) in the T-tubule membrane involved in the excitation/contraction coupling process and as a preferential binding of the dihydropyridine derivative on the inactivated form of this molecule, favored by the weak negative potentials or long-lasting depolarizations. The results provide data in favor of the existence of strong similarities between the calcium channels and voltage sensors since their operation was inhibited in a voltage-dependent manner by nifedipine.

High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE.

PubMed

Li, Tianbo; Lu, Gang; Chiang, Eugene Y; Chernov-Rogan, Tania; Grogan, Jane L; Chen, Jun

2017-01-01

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.

Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

PubMed Central

Hristov, Kiril L.; Smith, Amy C.; Parajuli, Shankar P.; Malysz, John

2013-01-01

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility. PMID:24352333

Validation of a patch clamp screening protocol that simultaneously measures compound activity in multiple states of the voltage-gated sodium channel Nav1.2.

PubMed

Liu, Yi; Beck, Edward J; Flores, Christopher M

2011-12-01

Hyperactivity of voltage-gated sodium channels underlies, at least in part, a range of pathological states, including pain and epilepsy. Selective blockers of these channels may offer effective treatment of such disorders. Currently employed methods to screen for sodium channel blockers, however, are inadequate to rationally identify mechanistically diverse blockers, limiting the potential range of indications that may be treated by such agents. Here, we describe an improved patch clamp screening assay that increases the mechanistic diversity of sodium channel blockers being identified. Using QPatch HT, a medium-throughput, automated patch clamp system, we tested three common sodium channel blockers (phenytoin, lidocaine, and tetrodotoxin) with distinct mechanistic profiles at Nav1.2. The single-voltage protocol employed in this assay simultaneously measured the compound activity in multiple states, including the slow inactivated state, of the channel. A long compound incubation period (10 s) was introduced during channel inactivation to increase the probability of identifying "slow binders." As such, phenytoin, which preferentially binds with slow kinetics to the fast inactivated state, exhibited significantly higher potency than that obtained from a brief exposure (100 ms) used in typical assays. This assay also successfully detected the use-dependent block of tetrodotoxin, a well-documented property of this molecule yet unobserved in typical patch clamp protocols. These results indicate that the assay described here can increase the likelihood of identification and mechanistic diversity of sodium channel blockers from a primary screen. It can also be used to efficiently guide the in vitro optimization of leads that retain the desired mechanistic properties. © MARY ANN LIEBERT, INC.

A Comparison of the Performance and Application Differences Between Manual and Automated Patch-Clamp Techniques

PubMed Central

Yajuan, Xiao; Xin, Liang; Zhiyuan, Li

2012-01-01

The patch clamp technique is commonly used in electrophysiological experiments and offers direct insight into ion channel properties through the characterization of ion channel activity. This technique can be used to elucidate the interaction between a drug and a specific ion channel at different conformational states to understand the ion channel modulators’ mechanisms. The patch clamp technique is regarded as a gold standard for ion channel research; however, it suffers from low throughput and high personnel costs. In the last decade, the development of several automated electrophysiology platforms has greatly increased the screen throughput of whole cell electrophysiological recordings. New advancements in the automated patch clamp systems have aimed to provide high data quality, high content, and high throughput. However, due to the limitations noted above, automated patch clamp systems are not capable of replacing manual patch clamp systems in ion channel research. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and differentiated cells that derive from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Therefore, further improvements in flexibility with regard to cell types and data quality will broaden the applications of the automated patch clamp systems in both academia and industry. PMID:23346269

Novel Application of Stem Cell-Derived Neurons to Evaluate the Time-and Dose-Dependent Progression of Excitotoxic Injury

DTIC Science & Technology

2013-05-14

enzymes . At sufficiently high doses of glutamate, this process culminates in excitogenic cell death [1]. Treatments to mitigate neuronal damage during...To evaluate the potential for therapeutic screening, we assessed the effect of several small molecule antagonists on excitotoxicity in a moderate...C. Current clamp recordings showing repeated overshooting action potentials are evoked by injection of a 75 pA current. D. Voltage-clamp recordings

Operational amplifier with adjustable frequency response.

PubMed

Gulisek, D; Hencek, M

1978-01-01

The authors describe an operational amplifier with an adjustable frequency response and its use in membrane physiology, using the voltage clamp and current clamp method. The amplifier eliminates feedback poles causing oscillation. It consists of a follower with a high input resistance in the form of a tube and of an actual amplifier with an adjustable frequency response allowing the abolition of clicks by one pole and of oscillation by two poles in the 500 Hz divided by infinity range. Further properties of the amplifier: a long-term voltage drift of 1 mv, a temperature voltage drift of 0.5 mv/degrees K, input resistance greater than 1 GOhm, amplification greater than 80 dB, output +/- 12 v, 25 ma, noise, measured from the width of the oscilloscope track in the presence of a ray of normal brightness, not exceeding 50 muv in the 0-250 kHz band, f1 = 1 MHz. A short report on the amplifier was published a few years ago (Gulísek and Hencek 1973).

A curved electrode electrostatic actuator designed for large displacement and force in an underwater environment

NASA Astrophysics Data System (ADS)

Preetham, B. S.; Lake, Melinda A.; Hoelzle, David J.

2017-09-01

There is a need for the development of large displacement (O (10-6) m) and force (O (10-6) N) electrostatic actuators with low actuation voltages (<  ±8 V) for underwater bio-MEMS applications. In this paper, we present the design, fabrication, and characterization of a curved electrode electrostatic actuator in a clamped-clamped beam configuration meant to operate in an underwater environment. Our curved electrode actuator is unique in that it operates in a stable manner past the pull-in instability. Models based on the Rayleigh-Ritz method accurately predict the onset of static instability and the displacement versus voltage function, as validated by quasistatic experiments. We demonstrate that the actuator is capable of achieving a large peak-to-peak displacement of 19.5 µm and force of 43 µN for a low actuation voltage of less than  ±8 V and is thus appropriate for underwater bio-MEMS applications.

Further characterization of the effect of ethanol on voltage-gated Ca(2+) channel function in developing CA3 hippocampal pyramidal neurons.

PubMed

Morton, Russell A; Valenzuela, C Fernando

2016-02-15

Developmental ethanol exposure damages the hippocampus, a brain region involved in learning and memory. Alterations in synaptic transmission and plasticity may play a role in this effect of ethanol. We previously reported that acute and repeated exposure to ethanol during the third trimester-equivalent inhibits long-term potentiation of GABAA receptor-dependent synaptic currents in CA3 pyramidal neurons through a mechanism that depends on retrograde release of brain-derived neurotrophic factor driven by activation of voltage-gated Ca(2+) channels (Zucca and Valenzuela, 2010). We found evidence indicating that voltage-gated Ca(2+) channels are inhibited in the presence of ethanol, an effect that may play a role in its mechanism of action. Here, we further investigated the acute effect of ethanol on the function of voltage-gated Ca(2+) channels in CA3 pyramidal neurons using Ca(2+) imaging techniques. These experiments revealed that acute ethanol exposure inhibits voltage-gated Ca(2+) channels both in somatic and proximal dendritic compartments. To investigate the long-term consequences of ethanol on voltage-gated Ca(2+) channels, we used patch-clamp electrophysiological techniques to assess the function of L-type voltage-gated Ca(2+) channels during and following ten days of vapor ethanol exposure. During ethanol withdrawal periods, the function of these channels was not significantly affected by vapor chamber exposure. Taken together with our previous findings, our results suggest that 3(rd) trimester-equivalent ethanol exposure transiently inhibits L-type voltage-gated Ca(2+) channel function in CA3 pyramidal neurons and that compensatory mechanisms restore their function during ethanol withdrawal. Transient inhibition of these channels by ethanol may be, in part, responsible for the hippocampal abnormalities associated with developmental exposure to this agent. Copyright © 2015 Elsevier B.V. All rights reserved.

Charge movement in a fast twitch skeletal muscle from rat.

PubMed

Simon, B J; Beam, K G

1983-02-01

Voltage-dependent charge movement in the rat omohyoid muscle was investigated using the three microelectrode voltage clamp technique. The charge that moved during a depolarization from the holding potential (-90 mV) to the test potential, V, increased with increasing V, saturating around 0 mV. The charge vs. voltage relationship was well fitted by Q = Q(max)/{1 + exp[-(V - V)/k]}, with Q(max) = 28.5 nC/muF, V = -34.2 mV, and k = 8.7 mV. Repolarization of the fiber from the test potential back to the holding potential caused an equal but opposite amount of charge to move. The kinetics of ON charge movement could be well described by a model developed for frog muscle by Horowicz and Schneider (1981b), which suggests that rat and frog charge movements are similar. This model failed to describe the kinetics of OFF charge movement for steps in potential from 0 mV to test potentials of -10 to -90 mV. OFF-charge movement rose to a peak more slowly and decayed more slowly than predicted by the theory.

Timing discriminator using leading-edge extrapolation

DOEpatents

Gottschalk, Bernard

1983-01-01

A discriminator circuit to recover timing information from slow-rising pulses by means of an output trailing edge, a fixed time after the starting corner of the input pulse, which is nearly independent of risetime and threshold setting. This apparatus comprises means for comparing pulses with a threshold voltage; a capacitor to be charged at a certain rate when the input signal is one-third threshold voltage, and at a lower rate when the input signal is two-thirds threshold voltage; current-generating means for charging the capacitor; means for comparing voltage capacitor with a bias voltage; a flip-flop to be set when the input pulse reaches threshold voltage and reset when capacitor voltage reaches the bias voltage; and a clamping means for discharging the capacitor when the input signal returns below one-third threshold voltage.

Nav1.7-A1632G Mutation from a Family with Inherited Erythromelalgia: Enhanced Firing of Dorsal Root Ganglia Neurons Evoked by Thermal Stimuli.

PubMed

Yang, Yang; Huang, Jianying; Mis, Malgorzata A; Estacion, Mark; Macala, Lawrence; Shah, Palak; Schulman, Betsy R; Horton, Daniel B; Dib-Hajj, Sulayman D; Waxman, Stephen G

2016-07-13

Voltage-gated sodium channel Nav1.7 is a central player in human pain. Mutations in Nav1.7 produce several pain syndromes, including inherited erythromelalgia (IEM), a disorder in which gain-of-function mutations render dorsal root ganglia (DRG) neurons hyperexcitable. Although patients with IEM suffer from episodes of intense burning pain triggered by warmth, the effects of increased temperature on DRG neurons expressing mutant Nav1.7 channels have not been well documented. Here, using structural modeling, voltage-clamp, current-clamp, and multielectrode array recordings, we have studied a newly identified Nav1.7 mutation, Ala1632Gly, from a multigeneration family with IEM. Structural modeling suggests that Ala1632 is a molecular hinge and that the Ala1632Gly mutation may affect channel gating. Voltage-clamp recordings revealed that the Nav1.7-A1632G mutation hyperpolarizes activation and depolarizes fast-inactivation, both gain-of-function attributes at the channel level. Whole-cell current-clamp recordings demonstrated increased spontaneous firing, lower current threshold, and enhanced evoked firing in rat DRG neurons expressing Nav1.7-A1632G mutant channels. Multielectrode array recordings further revealed that intact rat DRG neurons expressing Nav1.7-A1632G mutant channels are more active than those expressing Nav1.7 WT channels. We also showed that physiologically relevant thermal stimuli markedly increase the mean firing frequencies and the number of active rat DRG neurons expressing Nav1.7-A1632G mutant channels, whereas the same thermal stimuli only increase these parameters slightly in rat DRG neurons expressing Nav1.7 WT channels. The response of DRG neurons expressing Nav1.7-A1632G mutant channels upon increase in temperature suggests a cellular basis for warmth-triggered pain in IEM. Inherited erythromelalgia (IEM), a severe pain syndrome characterized by episodes of intense burning pain triggered by warmth, is caused by mutations in sodium channel Nav1.7, which are preferentially expressed in sensory and sympathetic neurons. More than 20 gain-of-function Nav1.7 mutations have been identified from IEM patients, but the question of how warmth triggers episodes of pain in IEM has not been well addressed. Combining multielectrode array, voltage-clamp, and current-clamp recordings, we assessed a newly identified IEM mutation (Nav1.7-A1632G) from a multigeneration family. Our data demonstrate gain-of-function attributes at the channel level and differential effects of physiologically relevant thermal stimuli on the excitability of DRG neurons expressing mutant and WT Nav1.7 channels, suggesting a cellular mechanism for warmth-triggered pain episodes in IEM patients. Copyright © 2016 the authors 0270-6474/16/367512-12$15.00/0.

Isolated and soft-switched power converter

DOEpatents

Peng, Fang Zheng; Adams, Donald Joe

2002-01-01

An isolated and soft-switched power converter is used for DC/DC and DC/DC/AC power conversion. The power converter includes two resonant tank circuits coupled back-to-back through an isolation transformer. Each resonant tank circuit includes a pair of resonant capacitors connected in series as a resonant leg, a pair of tank capacitors connected in series as a tank leg, and a pair of switching devices with anti-parallel clamping diodes coupled in series as resonant switches and clamping devices for the resonant leg. The power converter is well suited for DC/DC and DC/DC/AC power conversion applications in which high-voltage isolation, DC to DC voltage boost, bidirectional power flow, and a minimal number of conventional switching components are important design objectives. For example, the power converter is especially well suited to electric vehicle applications and load-side electric generation and storage systems, and other applications in which these objectives are important. The power converter may be used for many different applications, including electric vehicles, hybrid combustion/electric vehicles, fuel-cell powered vehicles with low-voltage starting, remote power sources utilizing low-voltage DC power sources, such as photovoltaics and others, electric power backup systems, and load-side electric storage and generation systems.

Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells

PubMed Central

Venkatachalam, Veena; Kralj, Joel M.; Dib-Hajj, Sulayman D.; Waxman, Stephen G.; Cohen, Adam E.

2013-01-01

Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators. PMID:24391999

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

PubMed

Banciu, Adela; Banciu, Daniel Dumitru; Mustaciosu, Cosmin Catalin; Radu, Mihai; Cretoiu, Dragos; Xiao, Junjie; Cretoiu, Sanda Maria; Suciu, Nicolae; Radu, Beatrice Mihaela

2018-05-09

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine myometrium and their gene expression regulation by beta-estradiol in pregnant conditions. Further exploration of the calcium signaling in TCs and its modulation by estrogen hormones will contribute to the understanding of labor and pregnancy mechanisms and to the development of effective strategies to reduce the risk of premature birth.

Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes?

PubMed

Hardy, M E L; Lawrence, C L; Standen, N B; Rodrigo, G C

2006-01-01

Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6+/-13.5 vs. 296.2+/-16.2 ms; p<0.01). Despite this effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically or with a patch electrode (91+/-46 vs. 81+/-20 nM). These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.

A Study of 4-level DC-DC Boost Inverter with Passive Component Reduction Consideration

NASA Astrophysics Data System (ADS)

Kasiran, A. N.; Ponniran, A.; Harimon, M. A.; Hamzah, H. H.

2018-04-01

This study is to analyze design principles of boost inductor and capacitor used in the 4-level DC-DC boost converter to realize size reduction of passive component referring to their attributes. The important feature of this circuit is that most of the boost-up energy is transferred from the capacitor-clamped to the output side which the small inductance can be used at the input side. The inductance of the boost inductor is designed by referring the inductor current ripple. On the other hand, the capacitance of the capacitor-clamped is designed by considering voltage stress on semiconductor devices and also the used switching frequency. Besides that, according to the design specifications, the required inductance in 4-level DC-DC boost converter is decreased compared to a conventional conventional DC-DC boost converter. Meanwhile, voltage stress on semiconductor device is depending on the maximum voltage ripple of the capacitor-clamped. A 50 W 4-level DC-DC boost converter prototype has been constructed. The results show that the inductor current ripple was 1.15 A when the inductors, 1 mH and 0.11 mH were used in the conventional and 4-level DC-DC boost converters, respectively. Thus, based on the experimental results, it shows that the reduction of passive components by referring to their attributes in 4-level DC-DC boost converter is achieved. Moreover, the decreasing of voltage stress on the semiconductor devices is an advantage for the selection of low ON-resistance of the devices which will contribute to the reduction of the semiconductor conduction loss. The integration result of boost converter and H-bridge inverter is also shown.

Acetylcholine-activated ionic currents in parasympathetic neurons of bullfrog heart.

PubMed

Tateishi, N; Kim, D K; Akaike, N

1990-05-01

1. The electrical and pharmacologic properties of acetylcholine (ACh)-induced current (IACh) were studied in the parasympathetic neurons isolated from bullfrog heart with the use of the concentration-clamp technique, which allows intracellular perfusion and rapid change of external solution within 2 ms under the single-electrode voltage-clamp condition. 2. The IACh consisted of an initial transient peak component and a successive steady-state plateau component. Both currents increased in a sigmoidal fashion with increasing ACh concentration. The dissociation constant (Kd value) and the Hill coefficient for each component were 2.2 X 10(-5) M and 1.6, respectively. 3. In the K(+)-free solution, the reversal potential (EACh) of IACh was close to the Na+ equilibrium potential (ENa). The current-voltage (I-V) relation showed inward rectification at positive potentials. 4. Nicotine mimicked only the peak component of IACh. However both peak and steady-state components were blocked nonselectively by the nicotinic blockers d-tubocurarine and hexamethonium. 5. Carbamylcholine (CCh) mimicked the steady-state component of IACh. The steady-state component was selectively inhibited by atropine at concentrations 1,000 times lower than that required for inhibition of the peak component. The steady state was blocked equally by either pirenzepine (M1 blocker) or AF-DX-116 (M2 blocker). 6. It was concluded that the IACh consisted of a peak component having double exponential activation and inactivation, mediated through the nicotinic actions, and a steady-state component having no inactivation, mediated through the muscarinic action.

Synergistic Inhibition of Delayed Rectifier K+ and Voltage-Gated Na+ Currents by Artemisinin in Pituitary Tumor (GH3) Cells.

PubMed

So, Edmund Cheung; Wu, Sheng-Nan; Wu, Ping-Ching; Chen, Hui-Zhen; Yang, Chia-Jung

2017-01-01

Artemisinin (ART) is an anti-malarial agent reported to influence endocrine function. Effects of ART on ionic currents and action potentials (APs) in pituitary tumor (GH3) cells were evaluated by patch clamp techniques. ART inhibited the amplitude of delayed-rectifier K+ current (IK(DR)) in response to membrane depolarization and accelerated the process of current inactivation. It exerted an inhibitory effect on IK(DR) with an IC50 value of 11.2 µM and enhanced IK(DR) inactivation with a KD value of 14.7 µM. The steady-state inactivation curve of IK(DR) was shifted to hyperpolarization by 10 mV. Pretreatment of chlorotoxin (1 µM) or iloprost (100 nM) did not alter the magnitude of ART-induced inhibition of IK(DR) in GH3 cells. ART also decreased the peak amplitude of voltage-gated Na+ current (INa) with a concentration-dependent slowing in inactivation rate. Application of KMUP-1, an inhibitor of late INa, was effective at reversing ART-induced prolongation in inactivation time constant of INa. Under current-clamp recordings, ART alone reduced the amplitude of APs and prolonged the duration of APs. Under ART exposure, the inhibitory actions on both IK(DR) and INa could be a potential mechanisms through which this drug influences membrane excitability of endocrine or neuroendocrine cells appearing in vivo. © 2017 The Author(s). Published by S. Karger AG, Basel.

Quantifying Insulin Sensitivity and Entero-Insular Responsiveness to Hyper- and Hypoglycemia in Ferrets

PubMed Central

Sui, Hongshu; Yi, Yaling; Yao, Jianrong; Liang, Bo; Sun, Xingshen; Hu, Shanming; Uc, Aliye; Nelson, Deborah J.; Ode, Katie Larson; Philipson, Louis H.; Engelhardt, John F.; Norris, Andrew W.

2014-01-01

Ferrets are an important emerging model of cystic fibrosis related diabetes. However, there is little documented experience in the use of advanced techniques to quantify aspects of diabetes pathophysiology in the ferret. Glycemic clamps are the gold standard technique to assess both insulin sensitivity and insulin secretion in humans and animal models of diabetes. We therefore sought to develop techniques for glycemic clamps in ferrets. To assess insulin sensitivity, we performed euglycemic hyperinsulinemic clamps in 5–6 week old ferrets in the anesthetized and conscious states. To assess insulin secretion, we performed hyperglycemic clamps in conscious ferrets. To evaluate responsiveness of ferret islet and entero-insular hormones to low glucose, a portion of the hyperglycemic clamps were followed by a hypoglycemic clamp. The euglycemic hyperinsulinemic clamps demonstrated insulin responsiveness in ferrets similar to that previously observed in humans and rats. The anesthetic isoflurane induced marked insulin resistance, whereas lipid emulsion induced mild insulin resistance. In conscious ferrets, glucose appearance was largely suppressed at 4 mU/kg/min insulin infusion, whereas glucose disposal was progressively increased at 4 and 20 mU/kg/min insulin. Hyperglycemic clamp induced first phase insulin secretion. Hypoglycemia induced a rapid diminishment of insulin, as well as a rise in glucagon and pancreatic polypeptide levels. The incretins GLP-1 and GIP were affected minimally by hyperglycemic and hypoglycemic clamp. These techniques will prove useful in better defining the pathophysiology in ferrets with cystic fibrosis related diabetes. PMID:24594704

Quantifying insulin sensitivity and entero-insular responsiveness to hyper- and hypoglycemia in ferrets.

PubMed

Sui, Hongshu; Yi, Yaling; Yao, Jianrong; Liang, Bo; Sun, Xingshen; Hu, Shanming; Uc, Aliye; Nelson, Deborah J; Ode, Katie Larson; Philipson, Louis H; Engelhardt, John F; Norris, Andrew W

2014-01-01

Ferrets are an important emerging model of cystic fibrosis related diabetes. However, there is little documented experience in the use of advanced techniques to quantify aspects of diabetes pathophysiology in the ferret. Glycemic clamps are the gold standard technique to assess both insulin sensitivity and insulin secretion in humans and animal models of diabetes. We therefore sought to develop techniques for glycemic clamps in ferrets. To assess insulin sensitivity, we performed euglycemic hyperinsulinemic clamps in 5-6 week old ferrets in the anesthetized and conscious states. To assess insulin secretion, we performed hyperglycemic clamps in conscious ferrets. To evaluate responsiveness of ferret islet and entero-insular hormones to low glucose, a portion of the hyperglycemic clamps were followed by a hypoglycemic clamp. The euglycemic hyperinsulinemic clamps demonstrated insulin responsiveness in ferrets similar to that previously observed in humans and rats. The anesthetic isoflurane induced marked insulin resistance, whereas lipid emulsion induced mild insulin resistance. In conscious ferrets, glucose appearance was largely suppressed at 4 mU/kg/min insulin infusion, whereas glucose disposal was progressively increased at 4 and 20 mU/kg/min insulin. Hyperglycemic clamp induced first phase insulin secretion. Hypoglycemia induced a rapid diminishment of insulin, as well as a rise in glucagon and pancreatic polypeptide levels. The incretins GLP-1 and GIP were affected minimally by hyperglycemic and hypoglycemic clamp. These techniques will prove useful in better defining the pathophysiology in ferrets with cystic fibrosis related diabetes.

Timing discriminator using leading-edge extrapolation

DOEpatents

Gottschalk, B.

1981-07-30

A discriminator circuit to recover timing information from slow-rising pulses by means of an output trailing edge, a fixed time after the starting corner of the input pulse, which is nearly independent of risetime and threshold setting is described. This apparatus comprises means for comparing pulses with a threshold voltage; a capacitor to be charged at a certain rate when the input signal is one-third threshold voltage, and at a lower rate when the input signal is two-thirds threshold voltage; current-generating means for charging the capacitor; means for comparing voltage capacitor with a bias voltage; a flip-flop to be set when the input pulse reaches threshold voltage and reset when capacitor voltage reaches the bias voltage; and a clamping means for discharging the capacitor when the input signal returns below one-third threshold voltage.

Use of vacuum tubes in test instrumentation for measuring characteristics of fast high-voltage semiconductor devices

NASA Technical Reports Server (NTRS)

Berning, D.

1981-01-01

Circuits are described that permit measurement of fast events occurring in power semiconductors. These circuits were developed for the dynamic characterization of transistors used in inductive-load switching applications. Fast voltage clamping using vacuum diodes is discussed, and reference is made to a unique circuit that was built for performing nondestructive, reverse-bias, second-breakdown tests on transistors.

A complete dc characterization of a constant-frequency, clamped-mode, series-resonant converter

NASA Technical Reports Server (NTRS)

Tsai, Fu-Sheng; Lee, Fred C.

1988-01-01

The dc behavior of a clamped-mode series-resonant converter is characterized systematically. Given a circuit operating condition, the converter's mode of operation is determined and various circuit parameters are calculated, such as average inductor current (load current), rms inductor current, peak capacitor voltage, rms switch currents, average diode currents, switch turn-on currents, and switch turn-off currents. Regions of operation are defined, and various circuit characteristics are derived to facilitate the converter design.

Presynaptic membrane potential affects transmitter release in an identified neuron in Aplysia by modulating the Ca2+ and K+ currents.

PubMed

Shapiro, E; Castellucci, V F; Kandel, E R

1980-01-01

We have examined the relationships between the modulation of transmitter release and of specific ionic currents by membrane potential in the cholinergic interneuron L10 of the abdominal ganglion of Aplysia californica. The presynaptic cell body was voltage-clamped under various pharmacological conditions and transmitter release from the terminals was assayed simultaneously by recording the synaptic potentials in the postsynaptic cell. When cell L10 was voltage-clamped from a holding potential of -60 mV in the presence of tetrodotoxin, graded transmitter release was evoked by depolarizing command pulses in the membrane voltage range (-35 mV to + 10 mV) in which the Ca(2+) current was also increasing. Depolarizing the holding potential of L10 results in increased transmitter output. Two ionic mechanisms contribute to this form of plasticity. First, depolarization inactivates some K(+) channels so that depolarizing command pulses recruit a smaller K(+) current. In unclamped cells the decreased K(+) conductance causes spike-broadening and increased influx of Ca(2+) during each spike. Second, small depolarizations around resting potential (-55 mV to -35 mV) activate a steady-state Ca(2+) current that also contributes to the modulation of transmitter release, because, even with most presynaptic K(+) currents blocked pharmacologically, depolarizing the holding potential still increases transmitter release. In contrast to the steady-state Ca(2+) current, the transient inward Ca(2+) current evoked by depolarizing clamp steps is relatively unchanged from various holding potentials.

Differential effects of five 'classical' scorpion {beta}-toxins on rNa{sub v}1.2a and DmNav1 provide clues on species-selectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bosmans, Frank; Martin-Eauclaire, Marie-France; Tytgat, Jan

2007-01-01

In general, scorpion {beta}-toxins have been well examined. However, few in-depth studies have been devoted to species selectivity and affinity comparisons on the different voltage-activated Na{sup +} channels since they have become available as cloned channels that can be studied in heterologous expression systems. As a result, their classification is largely historical and dates from early in vivo experiments on mice and cockroach and fly larvae. In this study, we aimed to provide an updated overview of selectivity and affinity of scorpion {beta}-toxins towards voltage-activated Na{sup +} channels of vertebrates or invertebrates. As pharmacological tools, we used the classic {beta}-toxinsmore » AaHIT, Css II, Css IV, Css VI and Ts VII and tested them on the neuronal vertebrate voltage-activated Na{sup +} channel, rNa{sub v}1.2a. For comparison, its invertebrate counterpart, DmNav1, was also tested. Both these channels were expressed in Xenopus laevis oocytes and the currents measured with the two-electrode voltage-clamp technique. We supplemented this data with several binding displacement studies on rat brain synaptosomes. The results lead us to propose a general classification and a novel nomenclature of scorpion {beta}-toxins based on pharmacological activity.« less

A Voltage Dependent Non-Inactivating Na+ Channel Activated during Apoptosis in Xenopus Oocytes

PubMed Central

Englund, Ulrika H.; Gertow, Jens; Kågedal, Katarina; Elinder, Fredrik

2014-01-01

Ion channels in the plasma membrane are important for the apoptotic process. Different types of voltage-gated ion channels are up-regulated early in the apoptotic process and block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are up-regulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na+ current increased three-fold. This current was activated at voltages more positive than 0 mV (midpoint of the open-probability curve was +55 mV) and showed almost no sign of inactivation during a 1-s pulse. The current was resistant to the Na+-channel blockers tetrodotoxin (1 µM) and amiloride (10 µM), while the Ca2+-channel blocker verapamil (50 µM) in the bath solution completely blocked the current. The intracellular Na+ concentration increased in staurosporine-treated oocytes, but could be prevented by replacing extracellular Na+ whith either K+ or Choline+. Prevention of this influx of Na+ also prevented the STS-induced up-regulation of the caspase-3 activity, suggesting that the intracellular Na+ increase is required to induce apoptosis. Taken together, we have found that a voltage dependent Na+ channel is up-regulated during apoptosis and that influx of Na+ is a crucial step in the apoptotic process in Xenopus oocytes. PMID:24586320

Active signal conduction through the sensory dendrite of a spider mechanoreceptor neuron.

PubMed

Gingl, Ewald; French, Andrew S

2003-07-09

Rapid responses to sensory stimulation are crucial for survival. This must be especially true for mechanical stimuli containing temporal information, such as vibration. Sensory transduction occurs at the tips of relatively long sensory dendrites in many mechanoreceptors of both vertebrates and invertebrates, but little is known about the electrical properties of these crucial links between transduction and action potential generation. The VS-3 slit-sense organ of the spider Cupiennius salei contains bipolar mechanosensory neurons that allow voltage-clamp recording from the somata, whereas mechanotransduction occurs at the tips of 100- to 200-microm-long sensory dendrites. We studied the properties of VS-3 sensory dendrites using three approaches. Voltage-jump experiments measured the spread of voltage outward from the soma by observing total mechanically transduced charge recovered at the soma as a function of time after a voltage jump. Frequency-response measurements between pseudorandom mechanical stimulation and somatic membrane potential estimated the passive cable properties of the dendrite for voltage spread in the opposite direction. Both of these sets of data indicated that the dendritic cable would significantly attenuate and retard a passively propagated receptor potential. Finally, current-clamp observations of receptor potentials and action potentials indicated that action potentials normally start at the distal dendrites and propagate regeneratively to the soma, reducing the temporal delay of passive conduction.

LabPatch, an acquisition and analysis program for patch-clamp electrophysiology.

PubMed

Robinson, T; Thomsen, L; Huizinga, J D

2000-05-01

An acquisition and analysis program, "LabPatch," has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/.

QPatch: the missing link between HTS and ion channel drug discovery.

PubMed

Mathes, Chris; Friis, Søren; Finley, Michael; Liu, Yi

2009-01-01

The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.

The passive cable properties of hair cell stereocilia and their contribution to somatic capacitance measurements.

PubMed

Breneman, Kathryn D; Highstein, Stephen M; Boyle, Richard D; Rabbitt, Richard D

2009-01-01

Somatic measurements of whole-cell capacitance are routinely used to understand physiologic events occurring in remote portions of cells. These studies often assume the intracellular space is voltage-clamped. We questioned this assumption in auditory and vestibular hair cells with respect to their stereocilia based on earlier studies showing that neurons, with radial dimensions similar to stereocilia, are not always isopotential under voltage-clamp. To explore this, we modeled the stereocilia as passive cables with transduction channels located at their tips. We found that the input capacitance measured at the soma changes when the transduction channels at the tips of the stereocilia are open compared to when the channels are closed. The maximum capacitance is felt with the transducer closed but will decrease as the transducer opens due to a length-dependent voltage drop along the stereocilium length. This potential drop is proportional to the intracellular resistance and stereocilium tip conductance and can produce a maximum capacitance error on the order of fF for single stereocilia and pF for the bundle.

Sodium and potassium conductance changes during a membrane action potential.

PubMed

Bezanilla, F; Rojas, E; Taylor, R E

1970-12-01

1. A method for turning a membrane potential control system on and off in less than 10 musec is described. This method was used to record membrane currents in perfused giant axons from Dosidicus gigas and Loligo forbesi after turning on the voltage clamp system at various times during the course of a membrane action potential.2. The membrane current measured just after the capacity charging transient was found to have an almost linear relation to the controlled membrane potential.3. The total membrane conductance taken from these current-voltage curves was found to have a time course during the action potential similar to that found by Cole & Curtis (1939).4. The instantaneous current voltage curves were linear enough to make it possible to obtain a good estimate of the individual sodium and potassium channel conductances, either algebraically or by clamping to the sodium, or potassium, reversal potentials. Good general agreement was obtained with the predictions of the Hodgkin-Huxley equations.5. We consider these results to constitute the first direct experimental demonstration of the conductance changes to sodium and potassium during the course of an action potential.

Properties of Single K+ and Cl− Channels in Asclepias tuberosa Protoplasts 1

PubMed Central

Schauf, Charles L.; Wilson, Kathryn J.

1987-01-01

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K+ and Cl−, respectively. Whole cell K+ currents activated exponentially during step depolarizations, while voltage-dependent Cl− channels were activated by hyperpolarizations. Single K+ channel conductance was 40 ± 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs+ and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 ± 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn2+ and ethacrynic acid. Whole-cell Cl− currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development. Images Fig. 5 PMID:16665712

Use of the Satinsky clamp for hilar clamping during robotic partial nephrectomy: indications, technique, and multi-center outcomes.

PubMed

Abdullah, Newaj; Rahbar, Haider; Barod, Ravi; Dalela, Deepansh; Larson, Jeff; Johnson, Michael; Mass, Alon; Zargar, Homayoun; Kaouk, Jihad; Allaf, Mohamad; Bhayani, Sam; Stifelman, Michael; Rogers, Craig

2017-03-01

A Satinsky clamp may be a backup option for hilar clamping during robotic partial nephrectomy (RPN) if there are challenges with application of bulldog clamps, but there are potential safety concerns. We evaluate outcomes of RPN using Satinsky vs. bulldog clamps, and provide tips for safe use of the Satinsky as a backup option. Using a multi-center database, we identified 1073 patients who underwent RPN between 2006 and 2013, and had information available about method of hilar clamping (bulldog clamp vs. Satinsky clamp). Patient baseline characteristics, tumor features, and perioperative outcomes were compared between the Satinsky and bulldog clamp groups. A Satinsky clamp was used for hilar clamping in 94 (8.8 %) RPN cases, and bulldog clamps were used in 979 (91.2 %) cases. The use of a Satinsky clamp was associated with greater operative time (198 vs. 175 min, p < 0.001), estimated blood loss (EBL, 200 vs. 100 ml, p < 0.001), warm ischemia time (WIT, 20 vs. 19 min, p = 0.036), transfusion rate (12.8 vs. 4.8 %, p = 0.001), and hospital stay (3 vs. 2 days, p < 0.001). Tumor characteristics and number of renal vessels were similar between groups. There were six intraoperative complications in the Satinsky clamp group, but none were directly related to the Satinsky clamp. On multivariable analysis, the use of the Satinsky clamp was not associated with increase in intraoperative or Clavien ≥3 postoperative complications, positive surgical margin rate or percentage change in estimated glomerular filtration rate. A Satinsky clamp can be a backup option for hilar clamping during challenging RPN cases, but requires careful technique, and was rarely necessary.

Whole-cell patch clamp recording of voltage-sensitive Ca²+ channel currents: heterologous expression systems and dissociated brain neurons.

PubMed

Hainsworth, Atticus H; Randall, Andrew D; Stefani, Alessandro

2005-01-01

Voltage-sensitive Ca(2+) channels (VSCC) play a central role in an extensive array of physiological processes. Their importance in cellular function arises from their ability both to sense membrane voltage and to conduct Ca(2+) ions, two facets that couple membrane excitability to a key intracellular second messenger. Through this relationship, activation of VSCCs is tightly coupled to the gamut of cellular functions dependent on intracellular Ca(2+), including muscle contraction, energy metabolism, gene expression, and exocytotic/endocytotic cycling.

Analysis of the dynamic avalanche of carrier stored trench bipolar transistor (CSTBT) during clamped inductive turn-off transient

NASA Astrophysics Data System (ADS)

Xue, Peng; Fu, Guicui

2017-03-01

The dynamic avalanche has a huge impact on the switching robustness of carrier stored trench bipolar transistor (CSTBT). The purpose of this work is to investigate the CSTBT's dynamic avalanche mechanism during clamped inductive turn-off transient. At first, with a Mitsubishi 600 V/150 A CSTBT and a Infineon 600 V/200 A field stop insulated gate bipolar transistor (FS-IGBT) utilized, the clamped inductive turn-off characteristics are obtained by double pulse test. The unclamped inductive switching (UIS) test is also utilized to identify the CSTBT's clamping voltage under dynamic avalanche condition. After the test data analysis, it is found that the CSTBT's dynamic avalanche is abnormal and can be triggered under much looser condition than the conventional buffer layer IGBT. The comparison between the FS-IGBT and CSTBT's experimental results implies that the CSTBT's abnormal dynamic avalanche phenomenon may be induced by the carrier storage (CS) layer. Based on the semiconductor physics, the electric field distribution and dynamic avalanche generation in the depletion region are analyzed. The analysis confirms that the CS layer is the root cause of the CSTBT's abnormal dynamic avalanche mechanism. Moreover, the CSTBT's negative gate capacitance effect is also investigated to clarify the underlying mechanism of the gate voltage bump observed in the test. In the end, the mixed-mode numerical simulation is utilized to reproduce the CSTBT's dynamic avalanche behavior. The simulation results validate the proposed dynamic avalanche mechanisms.

Anatomic partial nephrectomy: technique evolution.

PubMed

Azhar, Raed A; Metcalfe, Charles; Gill, Inderbir S

2015-03-01

Partial nephrectomy provides equivalent long-term oncologic and superior functional outcomes as radical nephrectomy for T1a renal masses. Herein, we review the various vascular clamping techniques employed during minimally invasive partial nephrectomy, describe the evolution of our partial nephrectomy technique and provide an update on contemporary thinking about the impact of ischemia on renal function. Recently, partial nephrectomy surgical technique has shifted away from main artery clamping and towards minimizing/eliminating global renal ischemia during partial nephrectomy. Supported by high-fidelity three-dimensional imaging, novel anatomic-based partial nephrectomy techniques have recently been developed, wherein partial nephrectomy can now be performed with segmental, minimal or zero global ischemia to the renal remnant. Sequential innovations have included early unclamping, segmental clamping, super-selective clamping and now culminating in anatomic zero-ischemia surgery. By eliminating 'under-the-gun' time pressure of ischemia for the surgeon, these techniques allow an unhurried, tightly contoured tumour excision with point-specific sutured haemostasis. Recent data indicate that zero-ischemia partial nephrectomy may provide better functional outcomes by minimizing/eliminating global ischemia and preserving greater vascularized kidney volume. Contemporary partial nephrectomy includes a spectrum of surgical techniques ranging from conventional-clamped to novel zero-ischemia approaches. Technique selection should be tailored to each individual case on the basis of tumour characteristics, surgical feasibility, surgeon experience, patient demographics and baseline renal function.

Flip the tip: an automated, high quality, cost-effective patch clamp screen.

PubMed

Lepple-Wienhues, Albrecht; Ferlinz, Klaus; Seeger, Achim; Schäfer, Arvid

2003-01-01

The race for creating an automated patch clamp has begun. Here, we present a novel technology to produce true gigaseals and whole cell preparations at a high rate. Suspended cells are flushed toward the tip of glass micropipettes. Seal, whole-cell break-in, and pipette/liquid handling are fully automated. Extremely stable seals and access resistance guarantee high recording quality. Data obtained from different cell types sealed inside pipettes show long-term stability, voltage clamp and seal quality, as well as block by compounds in the pM range. A flexible array of independent electrode positions minimizes consumables consumption at maximal throughput. Pulled micropipettes guarantee a proven gigaseal substrate with ultra clean and smooth surface at low cost.

Electrical Identification and Selective Microstimulation of Neuronal Compartments Based on Features of Extracellular Action Potentials

NASA Astrophysics Data System (ADS)

Radivojevic, Milos; Jäckel, David; Altermatt, Michael; Müller, Jan; Viswam, Vijay; Hierlemann, Andreas; Bakkum, Douglas J.

2016-08-01

A detailed, high-spatiotemporal-resolution characterization of neuronal responses to local electrical fields and the capability of precise extracellular microstimulation of selected neurons are pivotal for studying and manipulating neuronal activity and circuits in networks and for developing neural prosthetics. Here, we studied cultured neocortical neurons by using high-density microelectrode arrays and optical imaging, complemented by the patch-clamp technique, and with the aim to correlate morphological and electrical features of neuronal compartments with their responsiveness to extracellular stimulation. We developed strategies to electrically identify any neuron in the network, while subcellular spatial resolution recording of extracellular action potential (AP) traces enabled their assignment to the axon initial segment (AIS), axonal arbor and proximal somatodendritic compartments. Stimulation at the AIS required low voltages and provided immediate, selective and reliable neuronal activation, whereas stimulation at the soma required high voltages and produced delayed and unreliable responses. Subthreshold stimulation at the soma depolarized the somatic membrane potential without eliciting APs.

Voltage-gated Na+ currents in human dorsal root ganglion neurons

PubMed Central

Zhang, Xiulin; Priest, Birgit T; Belfer, Inna; Gold, Michael S

2017-01-01

Available evidence indicates voltage-gated Na+ channels (VGSCs) in peripheral sensory neurons are essential for the pain and hypersensitivity associated with tissue injury. However, our understanding of the biophysical and pharmacological properties of the channels in sensory neurons is largely based on the study of heterologous systems or rodent tissue, despite evidence that both expression systems and species differences influence these properties. Therefore, we sought to determine the extent to which the biophysical and pharmacological properties of VGSCs were comparable in rat and human sensory neurons. Whole cell patch clamp techniques were used to study Na+ currents in acutely dissociated neurons from human and rat. Our results indicate that while the two major current types, generally referred to as tetrodotoxin (TTX)-sensitive and TTX-resistant were qualitatively similar in neurons from rats and humans, there were several differences that have important implications for drug development as well as our understanding of pain mechanisms. DOI: http://dx.doi.org/10.7554/eLife.23235.001 PMID:28508747

Different spatial distributions of sodium channels in the slowly and rapidly adapting stretch receptor neuron of the crayfish.

PubMed

Lin, J H; Rydqvist, B

1999-06-05

Inward Na+ currents were studied, using a two-microelectrode intracellular voltage-clamp technique, in the slowly adapting (SA) and rapidly adapting (RA) stretch receptor neurons of the crayfish after the axons were cut at different distances from the soma. In the SA neuron, inward Na+ currents were recorded in the soma even when the axon was cut as close as 100 microm from the center of the soma, indicating the presence of Na+ channels in these parts. Also, two populations of Na+ channels seem to exist in the SA neuron. In the RA neuron, only minute Na+ currents were observed if the axon was shorter than 250 microm. The results strongly indicate that the voltage-gated Na+ channels in the SA and RA neurons have different distributions and that the difference in the spatial distribution of Na+ channel types may be important for the difference in firing properties in the two types of neurons. Copyright 1999 Elsevier Science B.V.

Amyotrophic lateral sclerosis immunoglobulins increase Ca2+ currents in a motoneuron cell line.

PubMed

Mosier, D R; Baldelli, P; Delbono, O; Smith, R G; Alexianu, M E; Appel, S H; Stefani, E

1995-01-01

The sporadic form of amyotrophic lateral sclerosis (ALS) is an idiopathic and eventually lethal disorder causing progressive degeneration of cortical and spinal motoneurons. Recent studies have shown that the majority of patients with sporadic ALS have serum antibodies that bind to purified L-type voltage-gated calcium channels and that antibody titer correlates with the rate of disease progression. Furthermore, antibodies purified from ALS patient sera have been found to alter the physiologic function of voltage-gated calcium channels in nonmotoneuron cell types. Using whole-cell patch-clamp techniques, immunoglobulins purified from sera of 5 of 6 patients with sporadic ALS are now shown to increase calcium currents in a hybrid motoneuron cell line, VSC4.1. These calcium currents are blocked by the polyamine funnel-web spider toxin FTX, which has previously been shown to block Ca2+ currents and evoked transmitter release at mammalian motoneuron terminals. These data provide additional evidence linking ALS to an autoimmune process and suggest that antibody-induced increases in calcium entry through voltage-gated calcium channels may occur in motoneurons in this disease, with possible deleterious effects in susceptible neurons.

Electrostatic actuation and electromechanical switching behavior of one-dimensional nanostructures.

PubMed

Subramanian, Arunkumar; Alt, Andreas R; Dong, Lixin; Kratochvil, Bradley E; Bolognesi, Colombo R; Nelson, Bradley J

2009-10-27

We report on the electromechanical actuation and switching performance of nanoconstructs involving doubly clamped, individual multiwalled carbon nanotubes. Batch-fabricated, three-state switches with low ON-state voltages (6.7 V average) are demonstrated. A nanoassembly architecture that permits individual probing of one device at a time without crosstalk from other nanotubes, which are originally assembled in parallel, is presented. Experimental investigations into device performance metrics such as hysteresis, repeatability and failure modes are presented. Furthermore, current-driven shell etching is demonstrated as a tool to tune the nanomechanical clamping configuration, stiffness, and actuation voltage of fabricated devices. Computational models, which take into account the nonlinearities induced by stress-stiffening of 1-D nanowires at large deformations, are presented. Apart from providing accurate estimates of device performance, these models provide new insights into the extension of stable travel range in electrostatically actuated nanowire-based constructs as compared to their microscale counterparts.

Mathematical modeling of electrical activity of uterine muscle cells.

PubMed

Rihana, Sandy; Terrien, Jeremy; Germain, Guy; Marque, Catherine

2009-06-01

The uterine electrical activity is an efficient parameter to study the uterine contractility. In order to understand the ionic mechanisms responsible for its generation, we aimed at building a mathematical model of the uterine cell electrical activity based upon the physiological mechanisms. First, based on the voltage clamp experiments found in the literature, we focus on the principal ionic channels and their cognate currents involved in the generation of this electrical activity. Second, we provide the methodology of formulations of uterine ionic currents derived from a wide range of electrophysiological data. The model is validated step by step by comparing simulated voltage-clamp results with the experimental ones. The model reproduces successfully the generation of single spikes or trains of action potentials that fit with the experimental data. It allows analyzing ionic channels implications. Likewise, the calcium-dependent conductance influences significantly the cellular oscillatory behavior.

How the early voltage clamp studies of José del Castillo inform "modern" neuroscience.

PubMed

Zottoli, Steven J

2012-10-01

The description of ionic currents that flow across the membrane of the squid giant axon during an action potential sparked an interest in determining whether there were similar currents in vertebrates. The preparation of choice was the node of Ranvier in single myelinated fibers in frog. José del Castillo spent 3 years on the United States mainland from 1956 to 1959. During that time, he collaborated with Jerome Y. Lettvin and John W. Moore. I discuss how these individuals met one another and some of their scientific discoveries using the voltage clamp to study squid giant axons and frog nodes. Much of this work was conducted at the Marine Biological Laboratory in Woods Hole, MA, and I attempt to convey a sense of the unique scientific "melting pot" that existed at the Marine Biological Laboratory and the broader effect that del Castillo had on "modern" neuroscience.

Voltage-gated currents in identified rat olfactory receptor neurons.

PubMed

Trombley, P Q; Westbrook, G L

1991-02-01

Whole-cell recording techniques were used to characterize voltage-gated membrane currents in neonatal rat olfactory receptor neurons (ORNs) in cell culture. Mature ORNs were identified in culture by their characteristic bipolar morphology, by retrograde labeling techniques, and by olfactory marker protein (OMP) immunoreactivity. ORNs did not have spontaneous activity, but fired action potentials to depolarizing current pulses. Action potentials were blocked by tetrodotoxin (TTX), which contrasts with the TTX-resistant action potentials in salamander olfactory receptor cells (e.g., Firestein and Werblin, 1987). Prolonged, suprathreshold current pulses evoked only a single action potential; however, repetitive firing up to 35 Hz could be elicited by a series of brief depolarizing pulses. Under voltage clamp, the TTX-sensitive sodium current had activation and inactivation properties similar to other excitable cells. In TTX and 20 mM barium, sustained inward current were evoked by voltage steps positive to -30 mV. This current was blocked by Cd (100 microM) and by nifedipine (IC50 = 368 nM) consistent with L-type calcium channels in other neurons. No T-type calcium current was observed. Voltage steps positive to -20 mV also evoked an outward current that did not inactivate during 100-msec depolarizations. Tail current analysis of this current was consistent with a selective potassium conductance. The outward current was blocked by external tetraethylammonium but was unaffected by Cd or 4-aminopyridine (4-AP) or by removal of external calcium. A transient outward current was not observed. The 3 voltage-dependent conductances in cultured rat ORNs appear to be sufficient for 2 essential functions: action potential generation and transmitter release. As a single odorant-activated channel can trigger an action potential (e.g., Lynch and Barry, 1989), the repetitive firing seen with brief depolarizing pulses suggests that ORNs do not integrate sensory input, but rather act as high-fidelity relays such that each opening of an odorant-activated channel reaches the olfactory bulb glomeruli as an action potential.

E-beam high voltage switching power supply

DOEpatents

Shimer, D.W.; Lange, A.C.

1996-10-15

A high-power power supply produces a controllable, constant high voltage output under varying and arcing loads. The power supply includes a voltage regulator, an inductor, an inverter for producing a high frequency square wave current of alternating polarity, an improved inverter voltage clamping circuit, a step up transformer, an output rectifier for producing a dc voltage at the output of each module, and a current sensor for sensing output current. The power supply also provides dynamic response to varying loads by controlling the voltage regulator duty cycle and circuitry is provided for sensing incipient arc currents at the output of the power supply to simultaneously decouple the power supply circuitry from the arcing load. The power supply includes a plurality of discrete switching type dc--dc converter modules. 5 figs.

E-beam high voltage switching power supply

DOEpatents

Shimer, Daniel W.; Lange, Arnold C.

1996-01-01

A high-power power supply produces a controllable, constant high voltage put under varying and arcing loads. The power supply includes a voltage regulator, an inductor, an inverter for producing a high frequency square wave current of alternating polarity, an improved inverter voltage clamping circuit, a step up transformer, an output rectifier for producing a dc voltage at the output of each module, and a current sensor for sensing output current. The power supply also provides dynamic response to varying loads by controlling the voltage regulator duty cycle and circuitry is provided for sensing incipient arc currents at the output of the power supply to simultaneously decouple the power supply circuitry from the arcing load. The power supply includes a plurality of discrete switching type dc--dc converter modules.

High voltage dc--dc converter with dynamic voltage regulation and decoupling during load-generated arcs

DOEpatents

Shimer, D.W.; Lange, A.C.

1995-05-23

A high-power power supply produces a controllable, constant high voltage output under varying and arcing loads. The power supply includes a voltage regulator, an inductor, an inverter for producing a high frequency square wave current of alternating polarity, an improved inverter voltage clamping circuit, a step up transformer, an output rectifier for producing a dc voltage at the output of each module, and a current sensor for sensing output current. The power supply also provides dynamic response to varying loads by controlling the voltage regulator duty cycle and circuitry is provided for sensing incipient arc currents at the output of the power supply to simultaneously decouple the power supply circuitry from the arcing load. The power supply includes a plurality of discrete switching type dc--dc converter modules. 5 Figs.

High voltage dc-dc converter with dynamic voltage regulation and decoupling during load-generated arcs

DOEpatents

Shimer, Daniel W.; Lange, Arnold C.

1995-01-01

A high-power power supply produces a controllable, constant high voltage output under varying and arcing loads. The power supply includes a voltage regulator, an inductor, an inverter for producing a high frequency square wave current of alternating polarity, an improved inverter voltage clamping circuit, a step up transformer, an output rectifier for producing a dc voltage at the output of each module, and a current sensor for sensing output current. The power supply also provides dynamic response to varying loads by controlling the voltage regulator duty cycle and circuitry is provided for sensing incipient arc currents at the output of the power supply to simultaneously decouple the power supply circuitry from the arcing load. The power supply includes a plurality of discrete switching type dc--dc converter modules.

Outside-out "sniffer-patch" clamp technique for in situ measures of neurotransmitter release.

PubMed

Muller-Chrétien, Emilie

2014-01-01

The mechanism underlying neurotransmitter release is a critical research domain for the understanding of neuronal network function; however, few techniques are available for the direct detection and measurement of neurotransmitter release. To date, the sniffer-patch clamp technique is mainly used to investigate these mechanisms from individual cultured cells. In this study, we propose to adapt the sniffer-patch clamp technique to in situ detection of neurosecretion. Using outside-out patches from donor cells as specific biosensors plunged in acute cerebral slices, this technique allows for proper detection and quantification of neurotransmitter release at the level of the neuronal network.

The transport systems of Ventricaria ventricosa: hypotonic and hypertonic turgor regulation.

PubMed

Bisson, M A; Beilby, M J

2002-11-01

The time course of hypertonic and hypotonic turgor regulation was studied in Ventricaria (Valonia) using pressure probe and I/V(current-voltage) analysis. Of 11 cells, 9 exhibited hypertonic turgor regulation, ranging from 100% regulation in 150 min to 14% regulation (14% recovery of the decrease in turgor) in 314 min. Some cells began regulating immediately, others took up to 90 min to begin. The resting PD (potential difference) became more positive in most cells. The I/V characteristics became more nonlinear with high resistance between -150 and -20 mV and negative conductance region near -70 mV. Prolonged (16 sec) voltage clamps to negative levels (-100 to -150 mV) showed progressively more rapid current turn-off, but subsequent I/V characteristics were not affected. Clamping to +150 mV, however, abolished the high conductance between -50 and +100 mV to yield a uniform high resistance I/V characteristic, similar to that in high [K+]o. Decreasing illumination from 2.02 micromol sec(-1) m(-2) to 0.5 micromol sec(-1)1 m(-2) had a similar effect. Two out of a total of three cells exhibited hypotonic turgor regulation. Both cells started regulating within minutes and achieved near 50% regulation within 50 min. The PD became more negative. The I/V curves exhibited high resistance between +50 and +150 mV. The characteristics were similar to those in cells exposed to low [K+]o. Prolonged voltage clamps to both negative and positive levels showed slow current increase. Decreased illumination increased the membrane resistance.

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse, a model of Huntington’s disease

PubMed Central

Braubach, Peter; Orynbayev, Murat; Andronache, Zoita; Hering, Tanja; Landwehrmeyer, Georg Bernhard; Lindenberg, Katrin S.

2014-01-01

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca2+ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca2+ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca2+ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca2+ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca2+ removal from the myoplasm and Ca2+ release flux from the sarcoplasmic reticulum were characterized using a Ca2+ binding and transport model, which indicated a significant reduction in slow Ca2+ removal activity and Ca2+ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca2+ channel conductance. These results indicate profound changes of Ca2+ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD. PMID:25348412

Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.

PubMed

Zahanich, Ihor; Graf, Eva M; Heubach, Jürgen F; Hempel, Ute; Boxberger, Sabine; Ravens, Ursula

2005-09-01

We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function. During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique. mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.

Activation of cannabinoid CB1 receptors modulates evoked action potentials in rat retinal ganglion cells.

PubMed

Jiang, Shu-Xia; Li, Qian; Wang, Xiao-Han; Li, Fang; Wang, Zhong-Feng

2013-08-25

Activation of cannabinoid CB1 receptors (CB1Rs) regulates a variety of physiological functions in the vertebrate retina through modulating various types of ion channels. The aim of the present study was to investigate the effects of this receptor on cell excitability of rat retinal ganglion cells (RGCs) in retinal slices using whole-cell patch-clamp techniques. The results showed that under current-clamped condition perfusing WIN55212-2 (WIN, 5 μmol/L), a CB1R agonist, did not significantly change the spontaneous firing frequency and resting membrane potential of RGCs. In the presence of cocktail synaptic blockers, including excitatory postsynaptic receptor blockers CNQX and D-APV, and inhibitory receptor blockers bicuculline and strychnine, perfusion of WIN (5 μmol/L) hardly changed the frequencies of evoked action potentials by a series of positive current injection (from +10 to +100 pA). Phase-plane plot analysis showed that both average threshold voltage for triggering action potential and delay time to reach threshold voltage were not affected by WIN. However, WIN significantly decreased +dV/dtmax and -dV/dtmax of action potentials, suggestive of reduced rising and descending velocities of action potentials. The effects of WIN were reversed by co-application of SR141716, a CB1R selective antagonist. Moreover, WIN did not influence resting membrane potential of RGCs with synaptic inputs being blocked. These results suggest that activation of CB1Rs may regulate intrinsic excitability of rat RGCs through modulating evoked action potentials.

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

PubMed Central

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

2016-01-01

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane. PMID:27330112

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid.

PubMed

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

2016-07-05

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

Variable-speed wind power system with improved energy capture via multilevel conversion

DOEpatents

Erickson, Robert W.; Al-Naseem, Osama A.; Fingersh, Lee Jay

2005-05-31

A system and method for efficiently capturing electrical energy from a variable-speed generator are disclosed. The system includes a matrix converter using full-bridge, multilevel switch cells, in which semiconductor devices are clamped to a known constant DC voltage of a capacitor. The multilevel matrix converter is capable of generating multilevel voltage wave waveform of arbitrary magnitude and frequencies. The matrix converter can be controlled by using space vector modulation.

Membrane voltage changes in passive dendritic trees: a tapering equivalent cylinder model.

PubMed

Poznański, R R

1988-01-01

An exponentially tapering equivalent cylinder model is employed in order to approximate the loss of the dendritic trunk parameter observed from anatomical data on apical and basilar dendrites of CA1 and CA3 hippocampal pyramidal neurons. This model allows dendritic trees with a relative paucity of branching to be treated. In particular, terminal branches are not required to end at the same electrotonic distance. The Laplace transform method is used to obtain analytic expressions for the Green's function corresponding to an instantaneous pulse of current injected at a single point along a tapering equivalent cylinder with sealed ends. The time course of the voltage in response to an arbitrary input is computed using the Green's function in a convolution integral. Examples of current input considered are (1) an infinitesimally brief (Dirac delta function) pulse and (2) a step pulse. It is demonstrated that inputs located on a tapering equivalent cylinder are more effective at the soma than identically placed inputs on a nontapering equivalent cylinder. Asymptotic solutions are derived to enable the voltage response behaviour over both relatively short and long time periods to be analysed. Semilogarithmic plots of these solutions provide a basis for estimating the membrane time constant tau m from experimental transients. Transient voltage decrement from a clamped soma reveals that tapering tends to reduce the error associated with inadequate voltage clamping of the dendritic membrane. A formula is derived which shows that tapering tends to increase the estimate of the electrotonic length parameter L.

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

PubMed Central

Martín, Pedro; Enrique, Nicolás; Palomo, Ana R. Roldán; Rebolledo, Alejandro; Milesi, Veronica

2012-01-01

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K+ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca2+-activated K+ channels (BKCa). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K+ currents carried by BKCa channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC50 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K+ currents (~45% blockage) in which, under our working conditions, BKCa is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BKCa channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account. PMID:22688134

Spontaneous activity of isolated dopaminergic periglomerular cells of the main olfactory bulb.

PubMed

Puopolo, Michelino; Bean, Bruce P; Raviola, Elio

2005-11-01

We examined the electrophysiological properties of a population of identified dopaminergic periglomerular cells of the main olfactory bulb using transgenic mice in which catecholaminergic neurons expressed human placental alkaline phosphatase (PLAP) on the outer surface of the plasma membrane. After acute dissociation, living dopaminergic periglomerular cells were identified by a fluorescently labeled monoclonal antibody to PLAP. In current-clamp mode, dopaminergic periglomerular cells spontaneously generated action potentials in a rhythmic fashion with an average frequency of 8 Hz. The hyperpolarization-activated cation current (Ih) did not seem important for pacemaking because blocking the current with ZD 7288 or Cs+ had little effect on spontaneous firing. To investigate what ionic currents do drive pacemaking, we performed action-potential-clamp experiments using records of pacemaking as voltage command in voltage-clamp experiments. We found that substantial TTX-sensitive Na+ current flows during the interspike depolarization. In addition, substantial Ca2+ current flowed during the interspike interval, and blocking Ca2+ current hyperpolarized the neurons and stopped spontaneous firing. These results show that dopaminergic periglomerular cells have intrinsic pacemaking activity, supporting the possibility that they can maintain a tonic release of dopamine to modulate the sensitivity of the olfactory system during odor detection. Calcium entry into these neurons provides electrical drive for pacemaking as well as triggering transmitter release.

Nonlinear effects of hyperpolarizing shifts in activation of mutant Nav1.7 channels on resting membrane potential

PubMed Central

Estacion, Mark

2017-01-01

The Nav1.7 sodium channel is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons. Gain-of-function mutations that cause the painful disorder inherited erythromelalgia (IEM) shift channel activation in a hyperpolarizing direction. When expressed within DRG neurons, these mutations produce a depolarization of resting membrane potential (RMP). The biophysical basis for the depolarized RMP has to date not been established. To explore the effect on RMP of the shift in activation associated with a prototypical IEM mutation (L858H), we used dynamic-clamp models that represent graded shifts that fractionate the effect of the mutation on activation voltage dependence. Dynamic-clamp recording from DRG neurons using a before-and-after protocol for each cell made it possible, even in the presence of cell-to-cell variation in starting RMP, to assess the effects of these graded mutant models. Our results demonstrate a nonlinear, progressively larger effect on RMP as the shift in activation voltage dependence becomes more hyperpolarized. The observed differences in RMP were predicted by the “late” current of each mutant model. Since the depolarization of RMP imposed by IEM mutant channels is known, in itself, to produce hyperexcitability of DRG neurons, the development of pharmacological agents that normalize or partially normalize activation voltage dependence of IEM mutant channels merits further study. NEW & NOTEWORTHY Inherited erythromelalgia (IEM), the first human pain disorder linked to a sodium channel, is widely regarded as a genetic model of neuropathic pain. IEM is produced by Nav1.7 mutations that hyperpolarize activation. These mutations produce a depolarization of resting membrane potential (RMP) in dorsal root ganglion neurons. Using dynamic clamp to explore the effect on RMP of the shift in activation, we demonstrate a nonlinear effect on RMP as the shift in activation voltage dependence becomes more hyperpolarized. PMID:28148645

Inhibitory actions of synthesised polyamine spider toxins and their analogues on Ca2+-activated Cl- currents recorded from cultured DRG neurones from neonatal rats.

PubMed

Sutton, K G; Stapleton, S R; Scott, R H

1998-07-24

The whole cell variant of the patch clamp technique was used to investigate the actions of polyamine spider toxins and their analogues on high voltage-activated Ca2+ currents and Ca2+-activated Cl- currents (I(Cl(Ca))). The actions of synthesised FTX (putative natural toxin from the American funnel web spider), sFTX-3.3, Orn-FTX-3.3, Lys-FTX-3.3, and argiotoxin-636 on cultured dorsal root ganglion neurones from neonatal rats were investigated. Synthesised FTX (1 microM) inhibited I(Cl(Ca)) but did not inhibit high voltage-activated Ca2+ currents. In contrast, sFTX-3.3 (10 microM) inhibited both high voltage-activated Ca2+ currents and the associated I(Cl(Ca)) in near equal proportions. Argiotoxin-636 (1-10 microM) inhibited I(Cl(Ca)) evoked by Ca2+ entry through voltage-activated channels and by intracellular photorelease of Ca2+ from a caged precursor DM-nitrophen. This data indicates that synthesised FTX and argiotoxin-636 directly inhibit Ca2+-activated Cl- channels. In conclusion, the potency of polyamines as non-selective inhibitors of Ca2+ channels and Ca2+-activated Cl- channels is in part determined by the presence of a terminal arginine and this may involve an interaction between terminal guanidino groups and Ca2+ binding sites.

The protein kinase C inhibitor, bisindolylmaleimide (I), inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells.

PubMed

Park, Won Sun; Son, Youn Kyoung; Ko, Eun A; Ko, Jae-Hong; Lee, Hyang Ae; Park, Kyoung Sun; Earm, Yung E

2005-06-17

We examined the effects of the protein kinase C (PKC) inhibitor, bisindolylmaleimide (BIM) (I), on voltage-dependent K+ (K(V)) channels in rabbit coronary arterial smooth muscle cells using whole-cell patch clamp technique. BIM (I) reversibly and dose-dependently inhibited the K(V) currents with an apparent Kd value of 0.27 microM. The inhibition of the K(V) current by BIM (I) was highly voltage-dependent between -30 and +10 mV (voltage range of channel activation), and the additive inhibition of the K(V) current by BIM (I) was voltage-dependence in the full activation voltage range. The rate constants of association and dissociation for BIM (I) were 18.4 microM(-1) s(-1) and 4.7 s(-1), respectively. BIM (I) had no effect on the steady-state activation and inactivation of K(V) channels. BIM (I) caused use-dependent inhibition of K(V) current, which was consistent with the slow recovery from inactivation in the presence of BIM (I) (recovery time constants were 856.95 +/- 282.6 ms for control, and 1806.38 +/- 110.0 ms for 300 nM BIM (I)). ATP-sensitive K+ (K(ATP)), inward rectifier K+ (K(IR)), Ca2+-activated K+ (BK(Ca)) channels, which regulate the membrane potential and arterial tone, were not affected by BIM (I). The PKC inhibitor, chelerythrine, and protein kinase A (PKA) inhibitor, PKA-IP, had little effect on the K(V) current and did not significantly alter the inhibitory effects of BIM (I) on the K(V) current. These results suggest that BIM (I) inhibits K(V) channels in a phosphorylation-independent, and voltage-, time- and use-dependent manner.

Phosphatase activity of the voltage-sensing phosphatase, VSP, shows graded dependence on the extent of activation of the voltage sensor

PubMed Central

Sakata, Souhei; Okamura, Yasushi

2014-01-01

The voltage-sensing phosphatase (VSP) consists of a voltage sensor and a cytoplasmic phosphatase region, and the movement of the voltage sensor is coupled to the phosphatase activity. However, its coupling mechanisms still remain unclear. One possible scenario is that the phosphatase is activated only when the voltage sensor is in a fully activated state. Alternatively, the enzymatic activity of single VSP proteins could be graded in distinct activated states of the voltage sensor, and partial activation of the voltage sensor could lead to partial activation of the phosphatase. To distinguish between these two possibilities, we studied a voltage sensor mutant of zebrafish VSP, where the voltage sensor moves in two steps as evidenced by analyses of charge movements of the voltage sensor and voltage clamp fluorometry. Measurements of the phosphatase activity toward phosphatidylinositol 4,5-bisphosphate revealed that both steps of voltage sensor activation are coupled to the tuning of phosphatase activities, consistent with the idea that the phosphatase activity is graded by the magnitude of the movement of the voltage sensor. PMID:24277865

Phosphatase activity of the voltage-sensing phosphatase, VSP, shows graded dependence on the extent of activation of the voltage sensor.

PubMed

Sakata, Souhei; Okamura, Yasushi

2014-03-01

The voltage-sensing phosphatase (VSP) consists of a voltage sensor and a cytoplasmic phosphatase region, and the movement of the voltage sensor is coupled to the phosphatase activity. However, its coupling mechanisms still remain unclear. One possible scenario is that the phosphatase is activated only when the voltage sensor is in a fully activated state. Alternatively, the enzymatic activity of single VSP proteins could be graded in distinct activated states of the voltage sensor, and partial activation of the voltage sensor could lead to partial activation of the phosphatase. To distinguish between these two possibilities, we studied a voltage sensor mutant of zebrafish VSP, where the voltage sensor moves in two steps as evidenced by analyses of charge movements of the voltage sensor and voltage clamp fluorometry. Measurements of the phosphatase activity toward phosphatidylinositol 4,5-bisphosphate revealed that both steps of voltage sensor activation are coupled to the tuning of phosphatase activities, consistent with the idea that the phosphatase activity is graded by the magnitude of the movement of the voltage sensor.

Dendritic calcium channels and their activation by synaptic signals in auditory coincidence detector neurons.

PubMed

Blackmer, Trillium; Kuo, Sidney P; Bender, Kevin J; Apostolides, Pierre F; Trussell, Laurence O

2009-08-01

The avian nucleus laminaris (NL) encodes the azimuthal location of low-frequency sound sources by detecting the coincidence of binaural signals. Accurate coincidence detection requires precise developmental regulation of the lengths of the fine, bitufted dendrites that characterize neurons in NL. Such regulation has been suggested to be driven by local, synaptically mediated, dendritic signals such as Ca(2+). We examined Ca(2+) signaling through patch clamp and ion imaging experiments in slices containing nucleus laminaris from embryonic chicks. Voltage-clamp recordings of neurons located in the NL showed the presence of large Ca(2+) currents of two types, a low voltage-activated, fast inactivating Ni(2+) sensitive channel resembling mammalian T-type channels, and a high voltage-activated, slowly inactivating Cd(2+) sensitive channel. Two-photon Ca(2+) imaging showed that both channel types were concentrated on dendrites, even at their distal tips. Single action potentials triggered synaptically or by somatic current injection immediately elevated Ca(2+) throughout the entire cell. Ca(2+) signals triggered by subthreshold synaptic activity were highly localized. Thus when electrical activity is suprathreshold, Ca(2+) channels ensure that Ca(2+) rises in all dendrites, even those that are synaptically inactive.

Molecular motions that shape the cardiac action potential: Insights from voltage clamp fluorometry.

PubMed

Zhu, Wandi; Varga, Zoltan; Silva, Jonathan R

2016-01-01

Very recently, voltage-clamp fluorometry (VCF) protocols have been developed to observe the membrane proteins responsible for carrying the ventricular ionic currents that form the action potential (AP), including those carried by the cardiac Na(+) channel, NaV1.5, the L-type Ca(2+) channel, CaV1.2, the Na(+)/K(+) ATPase, and the rapid and slow components of the delayed rectifier, KV11.1 and KV7.1. This development is significant, because VCF enables simultaneous observation of ionic current kinetics with conformational changes occurring within specific channel domains. The ability gained from VCF, to connect nanoscale molecular movement to ion channel function has revealed how the voltage-sensing domains (VSDs) control ion flux through channel pores, mechanisms of post-translational regulation and the molecular pathology of inherited mutations. In the future, we expect that this data will be of great use for the creation of multi-scale computational AP models that explicitly represent ion channel conformations, connecting molecular, cell and tissue electrophysiology. Here, we review the VCF protocol, recent results, and discuss potential future developments, including potential use of these experimental findings to create novel computational models. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sodium and potassium conductance changes during a membrane action potential

PubMed Central

Bezanilla, Francisco; Rojas, Eduardo; Taylor, Robert E.

1970-01-01

1. A method for turning a membrane potential control system on and off in less than 10 μsec is described. This method was used to record membrane currents in perfused giant axons from Dosidicus gigas and Loligo forbesi after turning on the voltage clamp system at various times during the course of a membrane action potential. 2. The membrane current measured just after the capacity charging transient was found to have an almost linear relation to the controlled membrane potential. 3. The total membrane conductance taken from these current—voltage curves was found to have a time course during the action potential similar to that found by Cole & Curtis (1939). 4. The instantaneous current voltage curves were linear enough to make it possible to obtain a good estimate of the individual sodium and potassium channel conductances, either algebraically or by clamping to the sodium, or potassium, reversal potentials. Good general agreement was obtained with the predictions of the Hodgkin—Huxley equations. 5. We consider these results to constitute the first direct experimental demonstration of the conductance changes to sodium and potassium during the course of an action potential. PMID:5505231

Flutter and divergence instability of supported piezoelectric nanotubes conveying fluid

NASA Astrophysics Data System (ADS)

Bahaadini, Reza; Hosseini, Mohammad; Jamali, Behnam

2018-01-01

In this paper, divergence and flutter instabilities of supported piezoelectric nanotubes containing flowing fluid are investigated. To take the size effects into account, the nonlocal elasticity theory is implemented in conjunction with the Euler-Bernoulli beam theory incorporating surface stress effects. The Knudsen number is applied to investigate the slip boundary conditions between the flow and wall of nanotube. The nonlocal governing equations of nanotube are obtained using Newtonian method, including the influence of piezoelectric voltage, surface effects, Knudsen number and nonlocal parameter. Applying Galerkin approach to transform resulting equations into a set of eigenvalue equations under the simple-simple (S-S) and clamped-clamped (C-C) boundary conditions. The effects of the piezoelectric voltage, surface effects, Knudsen number, nonlocal parameter and boundary conditions on the divergence and flutter boundaries of nanotubes are discussed. It is observed that the fluid-conveying nanotubes with both ends supported lose their stability by divergence first and then by flutter with increase in fluid velocity. Results indicate the importance of using piezoelectric voltage, nonlocal parameter and Knudsen number in decrease of critical flow velocities of system. Moreover, the surface effects have a significant role on the eigenfrequencies and critical fluid velocity.

A Calmodulin C-Lobe Ca2+-Dependent Switch Governs Kv7 Channel Function.

PubMed

Chang, Aram; Abderemane-Ali, Fayal; Hura, Greg L; Rossen, Nathan D; Gate, Rachel E; Minor, Daniel L

2018-02-21

Kv7 (KCNQ) voltage-gated potassium channels control excitability in the brain, heart, and ear. Calmodulin (CaM) is crucial for Kv7 function, but how this calcium sensor affects activity has remained unclear. Here, we present X-ray crystallographic analysis of CaM:Kv7.4 and CaM:Kv7.5 AB domain complexes that reveal an Apo/CaM clamp conformation and calcium binding preferences. These structures, combined with small-angle X-ray scattering, biochemical, and functional studies, establish a regulatory mechanism for Kv7 CaM modulation based on a common architecture in which a CaM C-lobe calcium-dependent switch releases a shared Apo/CaM clamp conformation. This C-lobe switch inhibits voltage-dependent activation of Kv7.4 and Kv7.5 but facilitates Kv7.1, demonstrating that mechanism is shared by Kv7 isoforms despite the different directions of CaM modulation. Our findings provide a unified framework for understanding how CaM controls different Kv7 isoforms and highlight the role of membrane proximal domains for controlling voltage-gated channel function. VIDEO ABSTRACT. Copyright © 2018 Elsevier Inc. All rights reserved.

Laminar Instability and Transition on the X-51A

DTIC Science & Technology

2009-08-01

AIM15 controller. Surface thermocouples are affixed to the contraction by hose clamps and are used by the controllers to determine when each heater...installed in the model. One 20- gauge, braided , high-voltage wire is soldered to the inner electrode. This wire then passes through the model, angle...and another 20-gauge, braided , high-voltage wire leading from the sting to the ground of the glow electronics. From the back of the sting to the glow

A new pH-sensitive rectifying potassium channel in mitochondria from the embryonic rat hippocampus.

PubMed

Kajma, Anna; Szewczyk, Adam

2012-10-01

Patch-clamp single-channel studies on mitochondria isolated from embryonic rat hippocampus revealed the presence of two different potassium ion channels: a large-conductance (288±4pS) calcium-activated potassium channel and second potassium channel with outwardly rectifying activity under symmetric conditions (150/150mM KCl). At positive voltages, this channel displayed a conductance of 67.84pS and a strong voltage dependence at holding potentials from -80mV to +80mV. The open probability was higher at positive than at negative voltages. Patch-clamp studies at the mitoplast-attached mode showed that the channel was not sensitive to activators and inhibitors of mitochondrial potassium channels but was regulated by pH. Moreover, we demonstrated that the channel activity was not affected by the application of lidocaine, an inhibitor of two-pore domain potassium channels, or by tertiapin, an inhibitor of inwardly rectifying potassium channels. In summary, based on the single-channel recordings, we characterised for the first time mitochondrial pH-sensitive ion channel that is selective for cations, permeable to potassium ions, displays voltage sensitivity and does not correspond to any previously described potassium ion channels in the inner mitochondrial membrane. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). Copyright © 2012 Elsevier B.V. All rights reserved.

Computing rates of Markov models of voltage-gated ion channels by inverting partial differential equations governing the probability density functions of the conducting and non-conducting states.

PubMed

Tveito, Aslak; Lines, Glenn T; Edwards, Andrew G; McCulloch, Andrew

2016-07-01

Markov models are ubiquitously used to represent the function of single ion channels. However, solving the inverse problem to construct a Markov model of single channel dynamics from bilayer or patch-clamp recordings remains challenging, particularly for channels involving complex gating processes. Methods for solving the inverse problem are generally based on data from voltage clamp measurements. Here, we describe an alternative approach to this problem based on measurements of voltage traces. The voltage traces define probability density functions of the functional states of an ion channel. These probability density functions can also be computed by solving a deterministic system of partial differential equations. The inversion is based on tuning the rates of the Markov models used in the deterministic system of partial differential equations such that the solution mimics the properties of the probability density function gathered from (pseudo) experimental data as well as possible. The optimization is done by defining a cost function to measure the difference between the deterministic solution and the solution based on experimental data. By evoking the properties of this function, it is possible to infer whether the rates of the Markov model are identifiable by our method. We present applications to Markov model well-known from the literature. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Intracellularly applied anti-P70 antibody blocks the induction of abnormal membrane properties by pentylenetetrazole in identified Euhadra neurons.

PubMed

Onozuka, M; Watanabe, K

1996-04-15

Using the voltage-clamp technique combined with pressure injection, we have studied the action of pentylenetetrazole (PTZ) on identified Euhadra neurons by examining how the PTZ-induced changes in membrane properties are affected by an antibody against P70, a protein found in the experimentally-induced epileptogenic cortex of rats. Intracellular injection of anti-P70 antibody blocked the induction by PTZ; bursting activity with both of development of negative slope resistance region in the steady state 1-V curve and a reduction in the delayed outward potassium current. These results suggest a novel mechanism of action for PTZ, involving intracellular protein(s) which react with anti-P70 antibody.

Nerve membrane ion channels as the target site of environmental toxicants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Narahashi, T.

1987-04-01

There are many environmentally important chemicals which exhibit potent effects on the nervous system. Since nerve excitation takes place in a fraction of a second, electrophysiological methods provide the authors with the most straightforward approach to the study of the mechanisms of action of environmental toxicants on the nervous system. Aquatic animals such as crayfish, lobster, squid, and marine snails represent extremely useful materials for such electrophysiological studies, because much of the authors knowledge of nerve excitation is derived from those animals. Nerve excitation takes place as a result of opening and closing of ion channels of the membrane. Thesemore » functions are independent of metabolic energy, and can be measured most effectively by voltage clamp techniques as applied to the giant axons of the crayfish and the squid. Patch clamp techniques developed during the past 10 years have added a new dimension to the electrophysiological investigation. These techniques allow them to measure the activity of individual ion channels, thereby making it possible to analyze the interaction of toxic molecules directly with single ion channels. Examples are given summarizing electrophysiological studies of environmental neurotoxicants. The abdominal nerve cords and neuromuscular preparations isolated from the crayfish are convenient materials for bioassay of certain environmental toxicants such as pyrethroids, chlorinated hydrocarbons, and other insecticides. Only a small fraction of the flux through the sodium channel, less than 1%, must be modified by pyrethroids for the animal to develop symptoms of poisoning. Such a toxicological application from channel to animal is important is understanding the potent toxic effect.« less

Contamination of current-clamp measurement of neuron capacitance by voltage-dependent phenomena

PubMed Central

White, William E.

2013-01-01

Measuring neuron capacitance is important for morphological description, conductance characterization, and neuron modeling. One method to estimate capacitance is to inject current pulses into a neuron and fit the resulting changes in membrane potential with multiple exponentials; if the neuron is purely passive, the amplitude and time constant of the slowest exponential give neuron capacitance (Major G, Evans JD, Jack JJ. Biophys J 65: 423–449, 1993). Golowasch et al. (Golowasch J, Thomas G, Taylor AL, Patel A, Pineda A, Khalil C, Nadim F. J Neurophysiol 102: 2161–2175, 2009) have shown that this is the best method for measuring the capacitance of nonisopotential (i.e., most) neurons. However, prior work has not tested for, or examined how much error would be introduced by, slow voltage-dependent phenomena possibly present at the membrane potentials typically used in such work. We investigated this issue in lobster (Panulirus interruptus) stomatogastric neurons by performing current clamp-based capacitance measurements at multiple membrane potentials. A slow, voltage-dependent phenomenon consistent with residual voltage-dependent conductances was present at all tested membrane potentials (−95 to −35 mV). This phenomenon was the slowest component of the neuron's voltage response, and failure to recognize and exclude it would lead to capacitance overestimates of several hundredfold. Most methods of estimating capacitance depend on the absence of voltage-dependent phenomena. Our demonstration that such phenomena make nonnegligible contributions to neuron responses even at well-hyperpolarized membrane potentials highlights the critical importance of checking for such phenomena in all work measuring neuron capacitance. We show here how to identify such phenomena and minimize their contaminating influence. PMID:23576698

Dynamic, nonlinear feedback regulation of slow pacemaking by A-type potassium current in ventral tegmental area neurons.

PubMed

Khaliq, Zayd M; Bean, Bruce P

2008-10-22

We analyzed ionic currents that regulate pacemaking in dopaminergic neurons of the mouse ventral tegmental area by comparing voltage trajectories during spontaneous firing with ramp-evoked currents in voltage clamp. Most recordings were made in brain slice, with key experiments repeated using acutely dissociated neurons, which gave identical results. During spontaneous firing, net ionic current flowing between spikes was calculated from the time derivative of voltage multiplied by cell capacitance, signal-averaged over many firing cycles to enhance resolution. Net inward interspike current had a distinctive nonmonotonic shape, reaching a minimum (generally <1 pA) between -60 and -55 mV. Under voltage clamp, ramps over subthreshold voltages elicited a time- and voltage-dependent outward current that peaked near -55 mV. This current was undetectable with 5 mV/s ramps and increased steeply with depolarization rate over the range (10-50 mV/s) typical of natural pacemaking. Ramp-evoked subthreshold current was resistant to alpha-dendrotoxin, paxilline, apamin, and tetraethylammonium but sensitive to 4-aminopyridine and 0.5 mM Ba2+, consistent with A-type potassium current (I(A)). Same-cell comparison of currents elicited by various ramp speeds with natural spontaneous depolarization showed how the steep dependence of I(A) on depolarization rate results in small net inward currents during pacemaking. These results reveal a mechanism in which subthreshold I(A) is near zero at steady state, but is engaged at depolarization rates >10 mV/s to act as a powerful, supralinear feedback element. This feedback mechanism explains how net ionic current can be constrained to <1-2 pA but reliably inward, thus enabling slow, regular firing.

A new balancing three level three dimensional space vector modulation strategy for three level neutral point clamped four leg inverter based shunt active power filter controlling by nonlinear back stepping controllers.

PubMed

Chebabhi, Ali; Fellah, Mohammed Karim; Kessal, Abdelhalim; Benkhoris, Mohamed F

2016-07-01

In this paper is proposed a new balancing three-level three dimensional space vector modulation (B3L-3DSVM) strategy which uses a redundant voltage vectors to realize precise control and high-performance for a three phase three-level four-leg neutral point clamped (NPC) inverter based Shunt Active Power Filter (SAPF) for eliminate the source currents harmonics, reduce the magnitude of neutral wire current (eliminate the zero-sequence current produced by single-phase nonlinear loads), and to compensate the reactive power in the three-phase four-wire electrical networks. This strategy is proposed in order to gate switching pulses generation, dc bus voltage capacitors balancing (conserve equal voltage of the two dc bus capacitors), and to switching frequency reduced and fixed of inverter switches in same times. A Nonlinear Back Stepping Controllers (NBSC) are used for regulated the dc bus voltage capacitors and the SAPF injected currents to robustness, stabilizing the system and to improve the response and to eliminate the overshoot and undershoot of traditional PI (Proportional-Integral). Conventional three-level three dimensional space vector modulation (C3L-3DSVM) and B3L-3DSVM are calculated and compared in terms of error between the two dc bus voltage capacitors, SAPF output voltages and THDv, THDi of source currents, magnitude of source neutral wire current, and the reactive power compensation under unbalanced single phase nonlinear loads. The success, robustness, and the effectiveness of the proposed control strategies are demonstrated through simulation using Sim Power Systems and S-Function of MATLAB/SIMULINK. Copyright © 2016 ISA. Published by Elsevier Ltd. All rights reserved.

Mechanism of block by tedisamil of transient outward current in human ventricular subepicardial myocytes

PubMed Central

Wettwer, Erich; Himmel, Herbert M; Amos, Gregory J; Li, Qi; Metzger, Franz; Ravens, Ursula

1998-01-01

Tedisamil is a new antiarrhythmic drug with predominant class III action. The aim of the present study was to investigate the blocking pattern of the compound on the transient outward current (Ito) in human subepicardial myocytes isolated from explanted left ventricles. Using the single electrode whole cell voltage clamp technique, Ito was analysed after appropriate voltage inactivation of sodium current and block of calcium current.Tedisamil reduced the amplitude of peak Ito, but did not affect the amplitude of non-inactivating outward current. The drug accelerated the apparent rate of Ito inactivation. The reduction in time constant of Ito inactivation depended on drug concentration, the apparent IC50 value was 4.4 μM.Tedisamil affected Ito amplitude in a use-dependent manner. After 2 min at −80 mV, maximum block of Ito was reached after 4–5 clamp steps either at the frequency of 0.2 or 2 Hz, indicating that the block was not frequency-dependent in an experimentally relevant range. Recovery from block was very slow and proceeded with a time constant of 12.1±1.8 s. Also in the presence of drug, a fraction of channels recovered from inactivation with a similar time constant as in control myocytes (i.e. 81±40 ms and 51±8 ms, respectively, n.s.).From the onset of fractional block of Ito by tedisamil during the initial 60 ms of a clamp step, we calculated k1=9×106 mol−1 s−1 for the association rate constant, and k2=23 s−1 for the dissociation rate constant. The resulting apparent KD was 2.6 μM and is similar to the IC50 value.The effects of tedisamil on Ito could be simulated by assuming a four state channel model where the drug binds to the channel in an open (activated) conformation. It is concluded that in human subepicardial myocytes tedisamil is an open channel blocker of Ito and that this effect probably contributes to the antiarrhythmic potential of this drug. PMID:9831899

Prevention of Ca(2+)-mediated action potentials in GABAergic local circuit neurones of rat thalamus by a transient K+ current.

PubMed Central

Pape, H C; Budde, T; Mager, R; Kisvárday, Z F

1994-01-01

1. Neurones enzymatically dissociated from the rat dorsal lateral geniculate nucleus (LGN) were identified as GABAergic local circuit interneurones and geniculocortical relay cells, based upon quantitative analysis of soma profiles, immunohistochemical detection of GABA or glutamic acid decarboxylase, and basic electrogenic behaviour. 2. During whole-cell current-clamp recording, isolated LGN neurones generated firing patterns resembling those in intact tissue, with the most striking difference relating to the presence in relay cells of a Ca2+ action potential with a low threshold of activation, capable of triggering fast spikes, and the absence of a regenerative Ca2+ response with a low threshold of activation in local circuit cells. 3. Whole-cell voltage-clamp experiments demonstrated that both classes of LGN neurones possess at least two voltage-dependent membrane currents which operate in a range of membrane potentials negative to the threshold for generation of Na(+)-K(+)-mediated spikes: the T-type Ca2+ current (IT) and an A-type K+ current (IA). Taking into account the differences in membrane surface area, the average size of IT was similar in the two types of neurones, and interneurones possessed a slightly larger A-conductance. 4. In local circuit neurones, the ranges of steady-state inactivation and activation of IT and IA were largely overlapping (VH = 81.1 vs. -82.8 mV), both currents activated at around -70 mV, and they rapidly increased in amplitude with further depolarization. In relay cells, the inactivation curve of IT was negatively shifted along the voltage axis by about 20 mV compared with that of IA (Vh = -86.1 vs. -69.2 mV), and the activation threshold for IT (at -80 mV) was 20 mV more negative than that for IA. In interneurones, the activation range of IT was shifted to values more positive than that in relay cells (Vh = -54.9 vs. -64.5 mV), whereas the activation range of IA was more negative (Vh = -25.2 vs. -14.5 mV). 5. Under whole-cell voltage-clamp conditions that allowed the combined activation of Ca2+ and K+ currents, depolarizing voltage steps from -110 mV evoked inward currents resembling IT in relay cells and small outward currents indicative of IA in local circuit neurones. After blockade of IA with 4-aminopyridine (4-AP), the same pulse protocol produced IT in both types of neurones. Under current clamp, 4-AP unmasked a regenerative membrane depolarization with a low threshold of activation capable of triggering fast spikes in local circuit neurones.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 1 PMID:7965855

Cellular defibrillation: interaction of micro-scale electric fields with voltage-gated ion channels.

PubMed

Kargol, Armin; Malkinski, Leszek; Eskandari, Rahmatollah; Carter, Maya; Livingston, Daniel

2015-09-01

We study the effect of micro-scale electric fields on voltage-gated ion channels in mammalian cell membranes. Such micro- and nano-scale electric fields mimic the effects of multiferroic nanoparticles that were recently proposed [1] as a novel way of controlling the function of voltage-sensing biomolecules such as ion channels. This article describes experimental procedures and initial results that reveal the effect of the electric field, in close proximity of cells, on the ion transport through voltage-gated ion channels. We present two configurations of the whole-cell patch-clamping apparatus that were used to detect the effect of external stimulation on ionic currents and discuss preliminary results that indicate modulation of the ionic currents consistent with the applied stimulus.

Novel switching method for single-phase NPC three-level inverter with neutral-point voltage control

NASA Astrophysics Data System (ADS)

Lee, June-Seok; Lee, Seung-Joo; Lee, Kyo-Beum

2018-02-01

This paper proposes a novel switching method with the neutral-point voltage control in a single-phase neutral-point-clamped three-level inverter (SP-NPCI) used in photovoltaic systems. A proposed novel switching method for the SP-NPCI improves the efficiency. The main concept is to fix the switching state of one leg. As a result, the switching loss decreases and the total efficiency is improved. In addition, it enables the maximum power-point-tracking operation to be performed by applying the proposed neutral-point voltage control algorithm. This control is implemented by modifying the reference signal. Simulation and experimental results provide verification of the performance of a novel switching method with the neutral-point voltage control.

Automatic parameter estimation of multicompartmental neuron models via minimization of trace error with control adjustment.

PubMed

Brookings, Ted; Goeritz, Marie L; Marder, Eve

2014-11-01

We describe a new technique to fit conductance-based neuron models to intracellular voltage traces from isolated biological neurons. The biological neurons are recorded in current-clamp with pink (1/f) noise injected to perturb the activity of the neuron. The new algorithm finds a set of parameters that allows a multicompartmental model neuron to match the recorded voltage trace. Attempting to match a recorded voltage trace directly has a well-known problem: mismatch in the timing of action potentials between biological and model neuron is inevitable and results in poor phenomenological match between the model and data. Our approach avoids this by applying a weak control adjustment to the model to promote alignment during the fitting procedure. This approach is closely related to the control theoretic concept of a Luenberger observer. We tested this approach on synthetic data and on data recorded from an anterior gastric receptor neuron from the stomatogastric ganglion of the crab Cancer borealis. To test the flexibility of this approach, the synthetic data were constructed with conductance models that were different from the ones used in the fitting model. For both synthetic and biological data, the resultant models had good spike-timing accuracy. Copyright © 2014 the American Physiological Society.

Effects of van der Waals Force and Thermal Stresses on Pull-in Instability of Clamped Rectangular Microplates

PubMed Central

Batra, Romesh C.; Porfiri, Maurizio; Spinello, Davide

2008-01-01

We study the influence of von Kármán nonlinearity, van der Waals force, and thermal stresses on pull-in instability and small vibrations of electrostatically actuated microplates. We use the Galerkin method to develop a tractable reduced-order model for electrostatically actuated clamped rectangular microplates in the presence of van der Waals forces and thermal stresses. More specifically, we reduce the governing two-dimensional nonlinear transient boundary-value problem to a single nonlinear ordinary differential equation. For the static problem, the pull-in voltage and the pull-in displacement are determined by solving a pair of nonlinear algebraic equations. The fundamental vibration frequency corresponding to a deflected configuration of the microplate is determined by solving a linear algebraic equation. The proposed reduced-order model allows for accurately estimating the combined effects of van der Waals force and thermal stresses on the pull-in voltage and the pull-in deflection profile with an extremely limited computational effort. PMID:27879752

Effects of van der Waals Force and Thermal Stresses on Pull-in Instability of Clamped Rectangular Microplates.

PubMed

Batra, Romesh C; Porfiri, Maurizio; Spinello, Davide

2008-02-15

We study the influence of von Karman nonlinearity, van der Waals force, and a athermal stresses on pull-in instability and small vibrations of electrostatically actuated mi-croplates. We use the Galerkin method to develop a tractable reduced-order model for elec-trostatically actuated clamped rectangular microplates in the presence of van der Waals forcesand thermal stresses. More specifically, we reduce the governing two-dimensional nonlineartransient boundary-value problem to a single nonlinear ordinary differential equation. For thestatic problem, the pull-in voltage and the pull-in displacement are determined by solving apair of nonlinear algebraic equations. The fundamental vibration frequency corresponding toa deflected configuration of the microplate is determined by solving a linear algebraic equa-tion. The proposed reduced-order model allows for accurately estimating the combined effectsof van der Waals force and thermal stresses on the pull-in voltage and the pull-in deflectionprofile with an extremely limited computational effort.

The ac power line protection for an IEEE 587 Class B environment

NASA Technical Reports Server (NTRS)

Roehr, W. D.; Clark, O. M.

1984-01-01

The 587B series of protectors are unique, low clamping voltage transient suppressors to protect ac-powered equipment from the 6000V peak open-circuit voltage and 3000A short circuit current as defined in IEEE standard 587 for Category B transients. The devices, which incorporate multiple-stage solid-state protector components, were specifically designed to operate under multiple exposures to maximum threat levels in this severe environment. The output voltage peaks are limited to 350V under maximum threat conditions for a 120V ac power line, thus providing adequate protection to vulnerable electronic equipment. The principle of operation and test performance data is discussed.

Piezoresistive cantilever force-clamp system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Sung-Jin; Petzold, Bryan C.; Pruitt, Beth L.

2011-04-15

We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or ''clamps'' the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to amore » sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of {mu}N force and nm up to tens of {mu}m displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode.« less

Antagonism of Lidocaine Inhibition by Open-Channel Blockers That Generate Resurgent Na Current

PubMed Central

Bant, Jason S.; Aman, Teresa K.; Raman, Indira M.

2013-01-01

Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVβ4 (the β4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the β4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 μM lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the β4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs. PMID:23486968

Role of action potential configuration and the contribution of Ca2+ and K+ currents to isoprenaline-induced changes in canine ventricular cells

PubMed Central

Szentandrássy, N; Farkas, V; Bárándi, L; Hegyi, B; Ruzsnavszky, F; Horváth, B; Bányász, T; Magyar, J; Márton, I; Nánási, PP

2012-01-01

BACKGROUND AND PURPOSE Although isoprenaline (ISO) is known to activate several ion currents in mammalian myocardium, little is known about the role of action potential morphology in the ISO-induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca2+ current (ICa), slow delayed rectifier K+ current (IKs) and fast delayed rectifier K+ current (IKr) were studied and compared in a frequency-dependent manner using canine isolated ventricular myocytes from various transmural locations. EXPERIMENTAL APPROACH Action potentials were recorded with conventional sharp microelectrodes; ion currents were measured using conventional and action potential voltage clamp techniques. KEY RESULTS In myocytes displaying a spike-and-dome action potential configuration (epicardial and midmyocardial cells), ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. ISO-induced action potential shortening was absent in endocardial cells and in myocytes pretreated with 4-aminopyridine. Application of the IKr blocker E-4031 failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of the IKs blocker HMR-1556. Both action potential shortening and elevation of the plateau were prevented by pretreatment with the ICa blocker nisoldipine. Action potential voltage clamp experiments revealed a prominent slowly inactivating ICa followed by a rise in IKs, both currents increased with increasing the cycle length. CONCLUSIONS AND IMPLICATIONS The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of IKs– but not IKr– may be responsible for the observed shortening of action potentials. PMID:22563726

Role of action potential configuration and the contribution of C²⁺a and K⁺ currents to isoprenaline-induced changes in canine ventricular cells.

PubMed

Szentandrássy, N; Farkas, V; Bárándi, L; Hegyi, B; Ruzsnavszky, F; Horváth, B; Bányász, T; Magyar, J; Márton, I; Nánási, P P

2012-10-01

Although isoprenaline (ISO) is known to activate several ion currents in mammalian myocardium, little is known about the role of action potential morphology in the ISO-induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca²⁺ current (I(Ca)), slow delayed rectifier K⁺ current (I(Ks)) and fast delayed rectifier K⁺ current (I(Kr)) were studied and compared in a frequency-dependent manner using canine isolated ventricular myocytes from various transmural locations. Action potentials were recorded with conventional sharp microelectrodes; ion currents were measured using conventional and action potential voltage clamp techniques. In myocytes displaying a spike-and-dome action potential configuration (epicardial and midmyocardial cells), ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. ISO-induced action potential shortening was absent in endocardial cells and in myocytes pretreated with 4-aminopyridine. Application of the I(Kr) blocker E-4031 failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of the I(Ks) blocker HMR-1556. Both action potential shortening and elevation of the plateau were prevented by pretreatment with the I(Ca) blocker nisoldipine. Action potential voltage clamp experiments revealed a prominent slowly inactivating I(Ca) followed by a rise in I(Ks) , both currents increased with increasing the cycle length. The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of I(Ks) - but not I(Kr) - may be responsible for the observed shortening of action potentials. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Effect of ethanol at clinically relevant concentrations on atrial inward rectifier potassium current sensitive to acetylcholine.

PubMed

Bébarová, Markéta; Matejovič, Peter; Pásek, Michal; Hořáková, Zuzana; Hošek, Jan; Šimurdová, Milena; Šimurda, Jiří

2016-10-01

Alcohol intoxication tends to induce arrhythmias, most often the atrial fibrillation. To elucidate arrhythmogenic mechanisms related to alcohol consumption, the effect of ethanol on main components of the ionic membrane current is investigated step by step. Considering limited knowledge, we aimed to examine the effect of clinically relevant concentrations of ethanol (0.8-80 mM) on acetylcholine-sensitive inward rectifier potassium current I K(Ach). Experiments were performed by the whole-cell patch clamp technique at 23 ± 1 °C on isolated rat and guinea-pig atrial myocytes, and on expressed human Kir3.1/3.4 channels. Ethanol induced changes of I K(Ach) in the whole range of concentrations applied; the effect was not voltage dependent. The constitutively active component of I K(Ach) was significantly increased by ethanol with the maximum effect (an increase by ∼100 %) between 8 and 20 mM. The changes were comparable in rat and guinea-pig atrial myocytes and also in expressed human Kir3.1/3.4 channels (i.e., structural correlate of I K(Ach)). In the case of the acetylcholine-induced component of I K(Ach), a dual ethanol effect was apparent with a striking heterogeneity of changes in individual cells. The effect correlated with the current magnitude in control: the current was increased by eth-anol in the cells showing small current in control and vice versa. The average effect peaked at 20 mM ethanol (an increase of the current by ∼20 %). Observed changes of action potential duration agreed well with the voltage clamp data. Ethanol significantly affected both components of I K(Ach) even in concentrations corresponding to light alcohol consumption.

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats.

PubMed

Zhang, Man; Zang, Kai-Hong; Luo, Jia-Lie; Leung, Fung-Ping; Huang, Yu; Lin, Cheng-Yuan; Yang, Zhi-Jun; Lu, Ai-Ping; Tang, Xu-Dong; Xu, Hong-Xi; Sung, Joseph Jao-yiu; Bian, Zhao-Xiang

2013-11-15

This study aimed to investigate the effect of magnolol (5,5'-diallyl-2,2'-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca(2+) currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3-100 μM). In the presence of Bay K8644 (100 nM), magnolol (10-100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and Nώ-nitro-L-arginine methyl ester (L-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3-100 μM) inhibited the L-type Ca(2+) currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca(2+) channel activity. Copyright © 2013 Elsevier GmbH. All rights reserved.

Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

NASA Astrophysics Data System (ADS)

Brew, Helen; Attwell, David

1987-06-01

Glutamate is taken up avidly by glial cells in the central nervous system1. Glutamate uptake may terminate the transmitter action of glutamate released from neurons1, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique2. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

Chloride channel blockers activate an endogenous cationic current in oocytes of Bufo arenarum.

PubMed

Cavarra, M S; del Mónaco, S M; Kotsias, B A

2004-07-01

A two-electrode, voltage-clamp technique was used to measure the effect of the Cl(-) channel blockers, 9-anthracene carboxylic acid and niflumic acid, upon the ionic currents of oocytes of the South American toad Bufo arenarum. The main results were: (1) both blockers produced a reversible increase of the outward currents on a dose-dependent manner; (2) the activated outward current was voltage dependent; (3) the 9-anthracene carboxylic acid-sensitive current was blocked with barium; and (4) the effect of 9-anthracene carboxylic acid was more pronounced in a zero-K(+) solution than in standard (2 mmol l(-1)) or high (20 mmol l(-1)) K(+) solutions, indicating that a K(+) conductance is activated. The effect of the Cl(-) channel blockers could be due to a direct interaction with endogenous cationic channels. Another possible explanation is that Cl(-) that enter the cell during depolarizing steps in control solution inhibit this cationic conductance; thus, the blockade of Cl(-) channels by 9-anthracene carboxylic acid and niflumic acid would remove this inhibition, allowing the cationic current to flow freely.

Effects of cevimeline on excitability of parasympathetic preganglionic neurons in the superior salivatory nucleus of rats.

PubMed

Mitoh, Yoshihiro; Ueda, Hirotaka; Ichikawa, Hiroyuki; Fujita, Masako; Kobashi, Motoi; Matsuo, Ryuji

2017-09-01

The superior salivatory nucleus (SSN) contains parasympathetic preganglionic neurons innervating the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, is a sialogogue that possibly stimulates SSN neurons in addition to the salivary glands themselves because it can cross the blood-brain barrier (BBB). In the present study, we examined immunoreactivities for mAChR subtypes in SSN neurons retrogradely labeled with a fluorescent tracer in neonatal rats. Additionally, we examined the effects of cevimeline in labeled SSN neurons of brainstem slices using a whole-cell patch-clamp technique. Mainly M1 and M3 receptors were detected by immunohistochemical staining, with low-level detection of M4 and M5 receptors and absence of M2 receptors. Most (110 of 129) SSN neurons exhibited excitatory responses to application of cevimeline. In responding neurons, voltage-clamp recordings showed that 84% (101/120) of the neurons exhibited inward currents. In the neurons displaying inward currents, the effects of the mAChR antagonists were examined. A mixture of M1 and M3 receptor antagonists most effectively reduced the peak amplitude of inward currents, suggesting that the excitatory effects of cevimeline on SSN neurons were mainly mediated by M1 and M3 receptors. Current-clamp recordings showed that application of cevimeline induced membrane depolarization (9/9 neurons). These results suggest that most SSN neurons are excited by cevimeline via M1 and M3 muscarinic receptors. Copyright © 2017 Elsevier B.V. All rights reserved.

Randomized clinical trial of stapler versus clamp-crushing transection in elective liver resection.

PubMed

Rahbari, N N; Elbers, H; Koch, M; Vogler, P; Striebel, F; Bruckner, T; Mehrabi, A; Schemmer, P; Büchler, M W; Weitz, J

2014-02-01

Various devices have been developed to facilitate liver transection and reduce blood loss in liver resections. None of these has proven superiority compared with the classical clamp-crushing technique. This randomized clinical trial compared the effectiveness and safety of stapler transection with that of clamp-crushing during open liver resection. Patients admitted for elective open liver resection between January 2010 and October 2011 were assigned randomly to stapler transection or the clamp-crushing technique. The primary endpoint was the total amount of intraoperative blood loss. Secondary endpoints included transection time, duration of operation, complication rates and resection margins. A total of 130 patients were enrolled, 65 to clamp-crushing and 65 to stapler transection. There was no difference between groups in total intraoperative blood loss: median (i.q.r.) 1050 (525-1650) versus 925 (450-1425) ml respectively (P = 0·279). The difference in total intraoperative blood loss normalized to the transection surface area was not statistically significant (P = 0·092). Blood loss during parenchymal transection was significantly lower in the stapler transection group (P = 0·002), as were the parenchymal transection time (mean(s.d.) 30(21) versus 9(7) min for clamp-crushing and stapler transection groups respectively; P < 0·001) and total duration of operation (mean(s.d.) 221(86) versus 190(85) min; P = 0·047). There were no significant differences in postoperative morbidity (P = 0·863) or mortality (P = 0·684) between groups. Stapler transection is a safe technique but does not reduce intraoperative blood loss in elective liver resection compared with the clamp-crushing technique. NCT01049607 (http://www.clinicaltrials.gov). © 2014 BJS Society Ltd. Published by John Wiley & Sons Ltd.

Fast calcium transients translate the distribution and conduction of neural activity in different regions of a single sensory neuron.

PubMed

Purali, Nuhan

2017-09-01

In the present study, cytosolic calcium concentration changes were recorded in response to various forms of excitations, using the fluorescent calcium indicator dye OG-BAPTA1 together with the current or voltage clamp methods in stretch receptor neurons of crayfish. A single action potential evoked a rise in the resting calcium level in the axon and axonal hillock, whereas an impulse train or a large saturating current injection would be required to evoke an equivalent response in the dendrite region. Under voltage clamp conditions, amplitude differences between axon and dendrite responses vanished completely. The fast activation time and the modulation of the response by extracellular calcium concentration changes indicated that the evoked calcium transients might be mediated by calcium entry into the cytosol through a voltage-gated calcium channel. The decay of the responses was slow and sensitive to extracellular sodium and calcium concentrations as well as exposure to 1-10 mM NiCl 2 and 10-500 µM lanthanum. Thus, a sodium calcium exchanger and a calcium ATPase might be responsible for calcium extrusion from the cytosol. Present results indicate that the calcium indicator OG-BAPTA1 might be an efficient but indirect way of monitoring regional membrane potential differences in a single neuron.

The Touch and Zap method for in vivo whole-cell patch recording of intrinsic and visual responses of cortical neurons and glial cells.

PubMed

Schramm, Adrien E; Marinazzo, Daniele; Gener, Thomas; Graham, Lyle J

2014-01-01

Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe "Touch and Zap", an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the "Touch". By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or "Zap", as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi-automatically, this approach is more reproducible and less dependent on experimenter technique.

The Touch and Zap Method for In Vivo Whole-Cell Patch Recording of Intrinsic and Visual Responses of Cortical Neurons and Glial Cells

PubMed Central

Schramm, Adrien E.; Marinazzo, Daniele; Gener, Thomas; Graham, Lyle J.

2014-01-01

Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe “Touch and Zap”, an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the “Touch”. By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or “Zap”, as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi-automatically, this approach is more reproducible and less dependent on experimenter technique. PMID:24875855

Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

PubMed

Rayaprolu, Vamseedhar; Royal, Perrine; Stengel, Karen; Sandoz, Guillaume; Kohout, Susy C

2018-05-07

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P 3 or PI(3,4)P 2 Our results indicate that dimerization plays a significant role in Ci-VSP function. © 2018 Rayaprolu et al.

Scale-Up Synthesis and Identification of GLYX-13, a NMDAR Glycine-Site Partial Agonist for the Treatment of Major Depressive Disorder.

PubMed

Li, Wenchao; Liu, Jingjian; Fan, Minghua; Li, Zhongtang; Chen, Yin; Zhang, Guisen; Huang, Zhuo; Zhang, Liangren

2018-04-24

GLYX-13, a NMDAR glycine-site partial agonist, was discovered as a promising antidepressant with rapidly acting effects but no ketamine-like side effects. However, the reported synthetic process route had deficiencies of low yield and the use of unfriendly reagents. Here, we report a scaled-up synthesis of GLYX-13 with an overall yield of 30% on the hectogram scale with a column chromatography-free strategy, where the coupling and deprotection reaction conditions were systematically optimized. Meanwhile, the absolute configuration of precursor compound of GLYX-13 was identified by X-ray single crystal diffraction. Finally, the activity of GLYX-13 was verified in the cortical neurons of mice through whole-cell voltage-clamp technique.

Pore forming properties of cecropin-melittin hybrid peptide in a natural membrane.

PubMed

Milani, Alberto; Benedusi, Mascia; Aquila, Marco; Rispoli, Giorgio

2009-12-11

The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH(2); CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in approximately 50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was >or=1 G Omega, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15.

Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown.

PubMed

Shalygin, A V; Vigont, V A; Glushankova, L N; Zimina, O A; Kolesnikov, D O; Skopin, A Yu; Kaznacheeva, E V

2017-07-01

An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

Open channel noise. I. Noise in acetylcholine receptor currents suggests conformational fluctuations.

PubMed

Sigworth, F J

1985-05-01

The random passage of ions through an open channel is expected to result in shot noise fluctuations in the channel current. The patch-clamp technique now allows fluctuations of this size to be observed in single-channel currents. In the experiments reported here the acetylcholine-induced currents in cultured rat muscle cells were analyzed; fluctuations were found that were considerably larger than expected for shot noise. A low-frequency component, which was fitted with a Lorentzian, was examined in detail; it appears to arise from fluctuations in channel conductance of approximately 3% on a time scale of 1 ms. The characteristic relaxation time is voltage dependent and temperature dependent (Q10 approximately equal to 3) suggesting that the fluctuations arise from conformational fluctuations in the channel protein.

The discovery of the sub-threshold currents M and Q/H in central neurons.

PubMed

Adams, Paul

2016-08-15

The history, content and consequences of the highly-cited 1982 Brain Research paper by Halliwell and Adams are summarized. The paper pioneered the use of the single-electrode voltage clamp in mammalian brain slices, described 2 novel sub-threshold voltage-dependent ionic currents, IM and IQ/H, and suggested that cholinergic inputs "enabled" pyramidal cell firing in response to conventional synaptic input, the first example of central neuromodulation. The paper, published in Brain Research to give the first author appropriate importance, heralded an ongoing tidal wave of quantitative electrophysiology in mammalian central neurons. Voltage-clamp analysis of muscarinic excitation in hippocampal neurons Pyramidal cells in the CA1 field of guinea pig hippocampal slices were voltage-clamped using a single microelectrode, at 23-30°C. Small inwardly relaxing currents triggered by step hyperpolarizations from holding potentials of -80 to -40mV were investigated. Inward relaxations occurring for negative steps between -40mV and -70mV resembled M-currents of sympathetic ganglion cells: they were abolished by addition of carbachol, muscarine or bethanechol, as well as by 1mM barium; the relaxations appeared to invert at around -80mV; they became faster at more negative potentials; and the inversion potential was shifted positively by raising external K(+) concentration. Inward relaxations triggered by steps negative to -80mV, in contrast, appeared to reflect passage of another current species, which has been labeled IQ.Thus IQ did not invert negative to -80mV, it was insensitive to muscarinic agonizts or to barium, and it was blocked by 0.5-3mM cesium (which does not block IM). Turn-on of IQ causes the well known droop in the hyperpolarizing electrotonic potential in these cells. The combined effects of IQ and IM make the steady-state current-voltage relation of CA1 cells slightly sigmoidal around rest potential. It is suggested that activation of cholinergic septal inputs to the hippocampus facilitates repetitive firing off pyramidal cells by turning off the M-conductance, without much change in the resting potential of the cell. © 1982. This article is part of a Special Issue entitled SI:50th Anniversary Issue. Copyright © 2016. Published by Elsevier B.V.

QPatch: the past, present and future of automated patch clamp.

PubMed

Mathes, Chris

2006-04-01

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC(50) values. The microfluidic pathways also allow for very small amounts of compound used for each experiment ( approximately 5 microl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel

PubMed Central

Goldschen-Ohm, Marcel P.; Capes, Deborah L.; Oelstrom, Kevin M.; Chanda, Baron

2013-01-01

Voltage-dependent Na+ channels are crucial for electrical signalling in excitable cells. Membrane depolarization initiates asynchronous movements in four non-identical voltage-sensing domains of the Na+ channel. It remains unclear to what extent this structural asymmetry influences pore gating as compared with outwardly rectifying K+ channels, where channel opening results from a final concerted transition of symmetric pore gates. Here we combine single channel recordings, cysteine accessibility and voltage clamp fluorimetry to probe the relationships between voltage sensors and pore conformations in an inactivation deficient Nav1.4 channel. We observe three distinct conductance levels such that DI-III voltage sensor activation is kinetically correlated with formation of a fully open pore, whereas DIV voltage sensor movement underlies formation of a distinct subconducting pore conformation preceding inactivation in wild-type channels. Our experiments reveal that pore gating in sodium channels involves multiple transitions driven by asynchronous movements of voltage sensors. These findings shed new light on the mechanism of coupling between activation and fast inactivation in voltage-gated sodium channels. PMID:23322038

Rediscovering sperm ion channels with the patch-clamp technique

PubMed Central

Kirichok, Yuriy; Lishko, Polina V.

2011-01-01

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca2+ in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca2+ and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (Hv1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible. PMID:21642646

Prolonged post-inhibitory rebound firing in the cerebellar nuclei mediated by group I mGluR potentiation of L-type Ca currents

PubMed Central

Zheng, Nan; Raman, Indira M.

2011-01-01

Neurons in the cerebellar nuclei fire at accelerated rates for prolonged periods after trains of synaptic inhibition that interrupt spontaneous firing. Both in vitro and in vivo, however, this prolonged rebound firing is favored by strong stimulation of afferents, suggesting that neurotransmitters other than GABA may contribute to the increased firing rates. Here, we tested whether metabotropic glutamate receptors modulate excitability of nuclear cells in cerebellar slices from mouse. In current clamp, the prolonged rebound firing rate after high-frequency synaptic stimulation was reduced by a variety of group I mGluR antagonists, including CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester), JNJ16259685 ((3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone)+MPEP, or 3-MATIDA (α-amino-5-carboxy-3-methyl-2-thiopheneacetic acid) +MPEP, as long as both mGluR1 and mGluR5 were blocked. This mGluR-dependent acceleration of firing was reduced but still evident when IPSPs were prevented by GABAA receptor antagonists. In voltage clamp, voltage ramps revealed a non-inactivating, low-voltage-activated, nimodipine-sensitive current that was enhanced by the selective group I mGluR agonist s-DHPG ((S)-3,5-dihydroxyphenylglycine). This putative L-type current also increased when mGluRs were activated by trains of evoked synaptic currents instead of direct application of agonist. In current clamp, blocking L-type Ca channels with the specific blocker nifedipine greatly reduced prolonged post-stimulus firing and occluded the effect of adding group I mGluR antagonists. Thus, potentiation of a low-voltage-activated L-type current by synaptically released glutamate accounted nearly fully for the mGluR-dependent acceleration of firing. Together, these data suggest that prolonged rebound firing in the cerebellar nuclei in vivo is most likely to occur when GABAA and mGluRs are simultaneously activated by concurrent excitation and inhibition. PMID:21753005

Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

PubMed Central

Zhang, J; Loew, L M; Davidson, R M

1996-01-01

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells. PMID:8913589

Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

PubMed

Zhang, J; Loew, L M; Davidson, R M

1996-11-01

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells.

Clamp-Crushing versus stapler hepatectomy for transection of the parenchyma in elective hepatic resection (CRUNSH) - A randomized controlled trial (NCT01049607)

PubMed Central

2011-01-01

Background Hepatic resection is still associated with significant morbidity. Although the period of parenchymal transection presents a crucial step during the operation, uncertainty persists regarding the optimal technique of transection. It was the aim of the present randomized controlled trial to evaluate the efficacy and safety of hepatic resection using the technique of stapler hepatectomy compared to the simple clamp-crushing technique. Methods/Design The CRUNSH Trial is a prospective randomized controlled single-center trial with a two-group parallel design. Patients scheduled for elective hepatic resection without extrahepatic resection at the Department of General-, Visceral- and Transplantation Surgery, University of Heidelberg are enrolled into the trial and randomized intraoperatively to hepatic resection by the clamp-crushing technique and stapler hepatectomy, respectively. The primary endpoint is total intraoperative blood loss. A set of general and surgical variables are documented as secondary endpoints. Patients and outcome-assessors are blinded for the treatment intervention. Discussion The CRUNSH Trial is the first randomized controlled trial to evaluate efficacy and safety of stapler hepatectomy compared to the clamp-crushing technique for parenchymal transection during elective hepatic resection. Trial Registration ClinicalTrials.gov: NCT01049607 PMID:21888669

Clamp-crushing vs. radiofrequency-assisted liver resection:changes in liver function tests.

PubMed

Palibrk, Ivan; Milicic, Biljana; Stojiljkovic, Ljuba; Manojlovic, Nebojsa; Dugalic, Vladimir; Bumbasirevic, Vesna; Kalezic, Nevena; Zuvela, Marinko; Milicevic, Miroslav

2012-05-01

Liver resection is the gold standard in managing patients with metastatic or primary liver cancer. The aim of our study was to compare the traditional clamp-crushing technique to the radiofrequency- assisted liver resection technique in terms of postoperative liver function. Liver function was evaluated preoperatively and on postoperative days 3 and 7. Liver synthetic function parameters (serum albumin level, prothrombin time and international normalized ratio), markers of hepatic injury and necrosis (serum alanine aminotransferase, aspartate aminotransferase and total bilirubin level) and microsomal activity (quantitative lidocaine test) were compared. Forty three patients completed the study (14 had clamp-crushing and 29 had radiofrequency assisted liver resection). The groups did not differ in demographic characteristics, pre-operative liver function, operative time and perioperative transfusion rate. In postoperative period, there were similar changes in monitored parameters in both groups except albumin levels, that were higher in radiofrequency-assisted liver resection group (p=0.047). Both, traditional clamp-crushing technique and radiofrequency assisted liver resection technique, result in similar postoperative changes of most monitored liver function parameters.

Closed-Loop, Multichannel Experimentation Using the Open-Source NeuroRighter Electrophysiology Platform

PubMed Central

Newman, Jonathan P.; Zeller-Townson, Riley; Fong, Ming-Fai; Arcot Desai, Sharanya; Gross, Robert E.; Potter, Steve M.

2013-01-01

Single neuron feedback control techniques, such as voltage clamp and dynamic clamp, have enabled numerous advances in our understanding of ion channels, electrochemical signaling, and neural dynamics. Although commercially available multichannel recording and stimulation systems are commonly used for studying neural processing at the network level, they provide little native support for real-time feedback. We developed the open-source NeuroRighter multichannel electrophysiology hardware and software platform for closed-loop multichannel control with a focus on accessibility and low cost. NeuroRighter allows 64 channels of stimulation and recording for around US $10,000, along with the ability to integrate with other software and hardware. Here, we present substantial enhancements to the NeuroRighter platform, including a redesigned desktop application, a new stimulation subsystem allowing arbitrary stimulation patterns, low-latency data servers for accessing data streams, and a new application programming interface (API) for creating closed-loop protocols that can be inserted into NeuroRighter as plugin programs. This greatly simplifies the design of sophisticated real-time experiments without sacrificing the power and speed of a compiled programming language. Here we present a detailed description of NeuroRighter as a stand-alone application, its plugin API, and an extensive set of case studies that highlight the system’s abilities for conducting closed-loop, multichannel interfacing experiments. PMID:23346047

Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal neurons.

PubMed

Borde, M; Bonansco, C; Fernández de Sevilla, D; Le Ray, D; Buño, W

2000-01-01

Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca2+-activated K+ current (sl(AHP)) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sl(AHP) using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sl(AHP). Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sl(AHP) remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca2+ current by nifedipine (20 microM) markedly reduced the sl(AHP) (79%) and its potentiation (55%). Ryanodine (20 microM) that blocks the release of intracellular Ca2+ also reduced sl(AHP) (29%) and its potentiation (25%). The potentiation of the sl(AHP) induced a marked and prolonged (>50%; approximately equals 8 min) decrease in excitability. The results suggest that sl(AHP) is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage-gated L-type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+-binding capacity of the channels mediating the sl(AHP). This potentiation of the sl(AHP) could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca2+ to toxic levels when intense synaptic activation occurs.

Electroporation of DC-3F cells is a dual process.

PubMed

Wegner, Lars H; Frey, Wolfgang; Silve, Aude

2015-04-07

Treatment of biological material by pulsed electric fields is a versatile technique in biotechnology and biomedicine used, for example, in delivering DNA into cells (transfection), ablation of tumors, and food processing. Field exposure is associated with a membrane permeability increase usually ascribed to electroporation, i.e., formation of aqueous membrane pores. Knowledge of the underlying processes at the membrane level is predominantly built on theoretical considerations and molecular dynamics (MD) simulations. However, experimental data needed to monitor these processes with sufficient temporal resolution are scarce. The whole-cell patch-clamp technique was employed to investigate the effect of millisecond pulsed electric fields on DC-3F cells. Cellular membrane permeabilization was monitored by a conductance increase. For the first time, to our knowledge, it could be established experimentally that electroporation consists of two clearly separate processes: a rapid membrane poration (transient electroporation) that occurs while the membrane is depolarized or hyperpolarized to voltages beyond so-called threshold potentials (here, +201 mV and -231 mV, respectively) and is reversible within ∼100 ms after the pulse, and a long-term, or persistent, permeabilization covering the whole voltage range. The latter prevailed after the pulse for at least 40 min, the postpulse time span tested experimentally. With mildly depolarizing or hyperpolarizing pulses just above threshold potentials, the two processes could be separated, since persistent (but not transient) permeabilization required repetitive pulse exposure. Conductance increased stepwise and gradually with depolarizing and hyperpolarizing pulses, respectively. Persistent permeabilization could also be elicited by single depolarizing/hyperpolarizing pulses of very high field strength. Experimental measurements of propidium iodide uptake provided evidence of a real membrane phenomenon, rather than a mere patch-clamp artifact. In short, the response of DC-3F cells to strong pulsed electric fields was separated into a transient electroporation and a persistent permeabilization. The latter dominates postpulse membrane properties but to date has not been addressed by electroporation theory or MD simulations. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Allosteric substrate switching in a voltage-sensing lipid phosphatase.

PubMed

Grimm, Sasha S; Isacoff, Ehud Y

2016-04-01

Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis.

Allosteric substrate switching in a voltage sensing lipid phosphatase

PubMed Central

Grimm, Sasha S.; Isacoff, Ehud Y.

2016-01-01

Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis. PMID:26878552

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

PubMed

Thuleau, P; Ward, J M; Ranjeva, R; Schroeder, J I

1994-07-01

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.

Role of the Local Anesthetic Receptor in the State-Dependent Inhibition of Voltage-Gated Sodium Channels by the Insecticide Metaflumizone

PubMed Central

von Stein, Richard T.

2012-01-01

Sodium channel inhibitor (SCI) insecticides selectively target voltage-gated sodium (Nav) channels in the slow-inactivated state by binding at or near the local anesthetic receptor within the sodium channel pore. Metaflumizone is a new insecticide for the treatment of fleas on domesticated pets and has recently been reported to block insect sodium channels in the slow-inactivated state, thereby implying that it is also a member of the SCI class. Using the two-electrode voltage-clamp technique, we examined metaflumizone inhibition of rat Nav1.4 sodium channels expressed in Xenopus laevis oocytes. Metaflumizone selectively inhibited Nav1.4 channels at potentials that promoted slow inactivation and shifted the voltage dependence of slow inactivation in the direction of hyperpolarization. Metaflumizone perfusion at a hyperpolarized holding potential also shifted the conductance-voltage curve for activation in the direction of depolarization and antagonized use-dependent lidocaine inhibition of fast-inactivated sodium channels, actions not previously observed with other SCI insecticides. We expressed mutated Nav1.4/F1579A and Nav1.4/Y1586A channels to investigate whether metaflumizone shares the domain IV segment S6 (DIV-S6) binding determinants identified for other SCI insecticides. Consistent with previous investigations of SCI insecticides on rat Nav1.4 channels, the F1579A mutation reduced sensitivity to block by metaflumizone, whereas the Y1586A mutation paradoxically increased the sensitivity to metaflumizone. We conclude that metaflumizone selectively inhibits slow-inactivated Nav1.4 channels and shares DIV-S6 binding determinants with other SCI insecticides and therapeutic drugs. However, our results suggest that metaflumizone interacts with resting and fast-inactivated channels in a manner that is distinct from other compounds in this insecticide class. PMID:22127519

Design and testing of a novel piezoelectric micro-motor actuated by asymmetrical inertial impact driving principle.

PubMed

Zeng, Ping; Sun, Shujie; Li, Li'an; Xu, Feng; Cheng, Guangming

2014-03-01

In this paper, an asymmetrical inertial impact driving principle is first proposed, and accordingly a novel piezoelectrically actuated linear micro-motor is developed. It is driven by the inertial impact force generated by piezoelectric smart cantilever (PSC) with asymmetrical clamping locations during a driving cycle. When the PSC is excited by typical harmonic voltage signals, different equivalent stiffness will be induced due to its asymmetrical clamping locations when it is vibrating back and forth, leading to a tiny displacement difference on the two opposite directions in a cycle, and then the accumulation of tiny displacement difference will allow directional movements. A prototype of the proposed motor has been developed and investigated by means of experimental tests. The motion and dynamics characteristics of the prototype are well studied. The experimental results demonstrate that the resolution of the micro-motor is 0.02 μm, the maximum velocity is 16.87 mm/s, and the maximum loading capacity can reach up to 1 kg with a voltage of 100 V and 35 Hz.

Room temperature, very sensitive thermometer using a doubly clamped microelectromechanical beam resonator for bolometer applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Y., E-mail: zhangya@iis.u-tokyo.ac.jp; Watanabe, Y.; Hosono, S.

We propose a room temperature, all electrical driving and detecting, very sensitive thermometer structure using a microelectromechanical (MEMS) resonator for bolometer applications. We have fabricated a GaAs doubly clamped MEMS beam resonator whose oscillation can be excited and detected by the piezoelectric effect. When a heating power is applied to a NiCr film deposited on the MEMS beam surface, internal thermal stress is generated in the beam, leading to a reduction in the resonance frequency. The present device detects the shift in the resonance frequency caused by heating and works as a very sensitive thermometer. When the resonator was drivenmore » by a voltage slightly below the threshold for the nonlinear, hysteretic oscillation, the thermometer showed a voltage responsivity of about 3300 V/W, while keeping a low noise spectral density of about 60 nV/Hz{sup 1/2}, demonstrating a noise equivalent power of <20 pW/Hz{sup 1/2} even at room temperature. The observed effect can be used for realizing high-sensitivity terahertz bolometers for room-temperature operation.« less

Ionic currents and charge movements in organ-cultured rat skeletal muscle.

PubMed

Hollingworth, S; Marshall, M W; Robson, E

1984-12-01

The middle of the fibre voltage-clamp technique was used to measure ionic currents and non-linear charge movements in intact, organ-cultured (in vitro denervated) mammalian fast-twitch (rat extensor digitorum longus) muscle fibres. Muscle fibres organ cultured for 4 days can be used as electrophysiological and morphological models for muscles in vivo denervated for the same length of time. Sodium currents in organ-cultured muscle fibres are similar to innervated fibres except that in the temperature range 0-20 degrees C (a) in the steady state, the voltage distribution of inactivation in cultured fibres is shifted negatively some 20 mV; (b) at the same temperature and membrane potential, the time constant of inactivation in cultured fibres is about twice that of innervated fibres. Potassium currents in innervated and cultured fibres at 15 degrees C can be fitted with the Hodgkin-Huxley n variable raised to the second power. Despite the large range we would estimate that the maximum value of the steady-state potassium conductance of cultured fibres is about one-half that of innervated fibres. The estimated maximum amount of charge moved in cultured fibre is about one-third that in innervated fibres. Compared to innervated fibres, culturing doubles the kinetics of the decay phase of charge movement. The possibility of a negative shift of the voltage distribution of charge movements in cultured fibres is discussed.

Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation

PubMed Central

Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

2010-01-01

Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (−8 mV by manual profiling, −11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (∼20% manual, ∼40% robotic), and enhances slow inactivation (hyperpolarizing shift −15 mV by human, −13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (∼2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations. PMID:20123784

Localized accumulation of cytosolic calcium near the fused sperm is associated with the calcium- and voltage-dependent block of sperm entry in the sea urchin egg.

PubMed

Ivonnet, Pedro I; Mohri, Tatsuma; McCulloh, David H

2017-10-01

Interaction of the sperm and egg depolarizes the egg membrane, allowing the sperm to enter; however, if the egg membrane is not allowed to depolarize from its resting potential (e.g., by voltage-clamp), the sperm will not enter. Previous studies demonstrated that sperm entry into sea urchin eggs that are voltage-clamped at negative membrane potentials is regulated both by the egg's membrane potential and a voltage-dependent influx of calcium into the egg. In these cases, electrical or cytoplasmic continuity (sperm-egg membrane fusion) occurs at negative membrane potentials, but subsequent loss of cytoplasmic continuity results in failure of sperm entry (unfusion). The work presented herein examined where, in relation to the sperm, and when, in relation to the sperm-induced electrophysiological events, the egg's calcium influx occurs, and how these events relate to successful or failed sperm entry. When sperm entered the egg, elevation of intracellular calcium concentration ([Ca 2+ ] i ) began near the fused sperm on average 5.9 s after sperm-egg membrane fusion. Conversely, when sperm failed to enter the egg, [Ca 2+ ] i elevated near the site of sperm-egg fusion on average 0.7 s after sperm-egg membrane fusion, which is significantly earlier than in eggs for which sperm entered. Therefore, the accumulation of calcium near the site of sperm-egg fusion is spatially and temporally consistent with the mechanism that may be responsible for loss of cytoplasmic continuity and failure of sperm entry. © 2017 Wiley Periodicals, Inc.

External protons destabilize the activated voltage sensor in hERG channels.

PubMed

Shi, Yu Patrick; Cheng, Yen May; Van Slyke, Aaron C; Claydon, Tom W

2014-03-01

Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca(2+) and Cd(2+), mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd(2+) (0.1 mM), suggesting a common binding site for the Cd(2+) and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.

A proposed route to independent measurements of tight junction conductance at discrete cell junctions

PubMed Central

Zhou, Lushan; Zeng, Yuhan; Baker, Lane A; Hou, Jianghui

2015-01-01

Direct recording of tight junction permeability is of pivotal importance to many biologic fields. Previous approaches bear an intrinsic disadvantage due to the difficulty of separating tight junction conductance from nearby membrane conductance. Here, we propose the design of Double whole-cell Voltage Clamp - Ion Conductance Microscopy (DVC-ICM) based on previously demonstrated potentiometric scanning of local conductive pathways. As proposed, DVC-ICM utilizes two coordinated whole-cell patch-clamps to neutralize the apical membrane current during potentiometric scanning, which in models described here will profoundly enhance the specificity of tight junction recording. Several potential pitfalls are considered, evaluated and addressed with alternative countermeasures. PMID:26716077

Ion channel electrophysiology via integrated planar patch-clamp chip with on-demand drug exchange.

PubMed

Chen, Chang-Yu; Tu, Ting-Yuan; Jong, De-Shien; Wo, Andrew M

2011-06-01

Planar patch clamp has revolutionized characterization of ion channel behavior in drug discovery primarily via advancement in high throughput. Lab use of planar technology, however, addresses different requirements and suffers from inflexibility to enable wide range of interrogation via a single cell. This work presents integration of planar patch clamp with microfluidics, achieving multiple solution exchanges for tailor-specific measurement and allowing rapid replacement of the cell-contacting aperture. Studies via endogenously expressed ion channels in HEK 293T cells were commenced to characterize the device. Results reveal the microfluidic concentration generator produces distinct solution/drug combination/concentrations on-demand. Volume-regulated chloride channel and voltage-gated potassium channels in HEK 293T cells immersed in generated solutions under various osmolarities or drug concentrations show unique channel signature under specific condition. Excitation and blockage of ion channels in a single cell was demonstrated via serial solution exchange. Robustness of the reversible bonding and ease of glass substrate replacement were proven via repeated usage of the integrated device. The present approach reveals the capability and flexibility of integrated microfluidic planar patch-clamp system for ion channel assays. Copyright © 2011 Wiley Periodicals, Inc.

Kv2 Channel Regulation of Action Potential Repolarization and Firing Patterns in Superior Cervical Ganglion Neurons and Hippocampal CA1 Pyramidal Neurons

PubMed Central

Liu, Pin W.

2014-01-01

Kv2 family “delayed-rectifier” potassium channels are widely expressed in mammalian neurons. Kv2 channels activate relatively slowly and their contribution to action potential repolarization under physiological conditions has been unclear. We explored the function of Kv2 channels using a Kv2-selective blocker, Guangxitoxin-1E (GxTX-1E). Using acutely isolated neurons, mixed voltage-clamp and current-clamp experiments were done at 37°C to study the physiological kinetics of channel gating and action potentials. In both rat superior cervical ganglion (SCG) neurons and mouse hippocampal CA1 pyramidal neurons, 100 nm GxTX-1E produced near-saturating block of a component of current typically constituting ∼60–80% of the total delayed-rectifier current. GxTX-1E also reduced A-type potassium current (IA), but much more weakly. In SCG neurons, 100 nm GxTX-1E broadened spikes and voltage clamp experiments using action potential waveforms showed that Kv2 channels carry ∼55% of the total outward current during action potential repolarization despite activating relatively late in the spike. In CA1 neurons, 100 nm GxTX-1E broadened spikes evoked from −70 mV, but not −80 mV, likely reflecting a greater role of Kv2 when other potassium channels were partially inactivated at −70 mV. In both CA1 and SCG neurons, inhibition of Kv2 channels produced dramatic depolarization of interspike voltages during repetitive firing. In CA1 neurons and some SCG neurons, this was associated with increased initial firing frequency. In all neurons, inhibition of Kv2 channels depressed maintained firing because neurons entered depolarization block more readily. Therefore, Kv2 channels can either decrease or increase neuronal excitability depending on the time scale of excitation. PMID:24695716

Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

PubMed

Pohl, P; Saparov, S M; Borgnia, M J; Agre, P

2001-08-14

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.

Effect of extracellular ATP on contraction, cytosolic calcium activity, membrane voltage and ion currents of rat mesangial cells in primary culture.

PubMed Central

Pavenstädt, H.; Gloy, J.; Leipziger, J.; Klär, B.; Pfeilschifter, J.; Schollmeyer, P.; Greger, R.

1993-01-01

1. The effects of extracellular ATP on contraction, membrane voltage (Vm), ion currents and intracellular calcium activity [Ca2+]i were studied in rat mesangial cells (MC) in primary culture. 2. Addition of extracellular ATP (10(-5) and 10(-4) M) to MC led to a cell contraction which was independent of extracellular calcium. 3. Membrane voltage (Vm) and ion currents were measured with the nystatin patch clamp technique. ATP induced a concentration-dependent transient depolarization of Vm (ED50: 2 x 10(-6) M). During the transient depolarization ion currents were monitored simultaneously and showed an increase of the inward- and outward current. 4. In a buffer with a reduced extracellular chloride concentration (from 145 to 30 mM) ATP induced a depolarization augmented to -4 +/- 4 mV. 5. ATP-gamma-S and 2-methylthio-ATP depolarized Vm to the same extent as ATP, whereas alpha,beta-methylene-ATP (all 10(-5) M) had no effect on Vm. 6. The Ca2+ ionophore, A23187, depolarized Vm transiently from -51 +/- 2 to -28 +/- 4 mV and caused an increase of the inward current. 7. The intracellular calcium activity [Ca2+]i was measured with the fura-2 technique. ATP stimulated a concentration-dependent increase of [Ca2+]i (ED50: 5 x 10(-6) M). The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. 8. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free buffer, whereas the plateau was abolished. Verapamil (10(-4) M) did not inhibit the [Ca2+]i increase induced by ATP.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 PMID:7691366

Piezoresistive cantilever force-clamp system

PubMed Central

Park, Sung-Jin; Petzold, Bryan C.; Goodman, Miriam B.; Pruitt, Beth L.

2011-01-01

We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or “clamps” the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to a sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of μN force and nm up to tens of μm displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode. PMID:21529009

Gating by Cyclic Gmp and Voltage in the α Subunit of the Cyclic Gmp–Gated Channel from Rod Photoreceptors

PubMed Central

Benndorf, Klaus; Koopmann, Rolf; Eismann, Elisabeth; Kaupp, U. Benjamin

1999-01-01

Gating by cGMP and voltage of the α subunit of the cGMP-gated channel from rod photoreceptor was examined with a patch-clamp technique. The channels were expressed in Xenopus oocytes. At low [cGMP] (<20 μM), the current displayed strong outward rectification. At low and high (700 μM) [cGMP], the channel activity was dominated by only one conductance level. Therefore, the outward rectification at low [cGMP] results solely from an increase in the open probability, P o. Kinetic analysis of single-channel openings revealed two exponential distributions. At low [cGMP], the larger P o at positive voltages with respect to negative voltages is caused by an increased frequency of openings in both components of the open-time distribution. In macroscopic currents, depolarizing voltage steps, starting from −100 mV, generated a time-dependent current that increased with the step size (activation). At low [cGMP] (20 μM), the degree of activation was large and the time course was slow, whereas at saturating [cGMP] (7 mM) the respective changes were small and fast. The dose–response relation at −100 mV was shifted to the right and saturated at significantly lower P o values with respect to that at +100 mV (0.77 vs. 0.96). P o was determined as function of the [cGMP] (at +100 and −100 mV) and voltage (at 20, 70, and 700 μM, and 7 mM cGMP). Both relations could be fitted with an allosteric state model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric opening reaction. At saturating [cGMP] (7 mM), the activation time course was monoexponential, which allowed us to determine the individual rate constants for the allosteric reaction. For the rapid rate constants of cGMP binding and unbinding, lower limits are determined. It is concluded that an allosteric model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric reaction, describes the cGMP- and voltage-dependent gating of cGMP-gated channels adequately. PMID:10498668

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons.

PubMed

Mafra, R A; Leão, R M; Beirão, P S L; Cruz, J S

2003-07-01

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 M and a maximum increase of 51.2 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

PubMed Central

Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K.; Craik, David J.; Kent, Stephen B. H.; French, Robert J.; Bezanilla, Francisco; Correa, Ana M.

2017-01-01

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating. PMID:28202723

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

PubMed

Kubota, Tomoya; Durek, Thomas; Dang, Bobo; Finol-Urdaneta, Rocio K; Craik, David J; Kent, Stephen B H; French, Robert J; Bezanilla, Francisco; Correa, Ana M

2017-03-07

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Micromachined patch-clamp apparatus

DOEpatents

Okandan, Murat

2012-12-04

A micromachined patch-clamp apparatus is disclosed for holding one or more cells and providing electrical, chemical, or mechanical stimulation to the cells during analysis with the patch-clamp technique for studying ion channels in cell membranes. The apparatus formed on a silicon substrate utilizes a lower chamber formed from silicon nitride using surface micromachining and an upper chamber formed from a molded polymer material. An opening in a common wall between the chambers is used to trap and hold a cell for analysis using the patch-clamp technique with sensing electrodes on each side of the cell. Some embodiments of the present invention utilize one or more electrostatic actuators formed on the substrate to provide mechanical stimulation to the cell being analyzed, or to provide information about mechanical movement of the cell in response to electrical or chemical stimulation.

Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells.

PubMed Central

Fleischmann, B K; Washabau, R J; Kotlikoff, M I

1993-01-01

1. In order to determine the physiological role of specific potassium currents in airway smooth muscle, potassium currents were measured in freshly dissociated ferret trachealis cells using the nystatin-permeabilized, whole-cell method, at 35 degrees C. 2. The magnitude of the outward currents was markedly increased as bath temperature was increased from 22 to 35 degrees C. This increase was primarily due to the increase in maximum potassium conductance (gK,max), although there was also a small leftward shift in the relationship between gK and voltage at higher temperatures. The maximum conductance and the kinetics of current activation and inactivation were also temperature dependent. At 35 degrees C, gating of the current was steeply voltage dependent between -40 and 0 mV. Current activation was well fitted by fourth-order kinetics; the mean time constants of activation (30 mV clamp step) were 1.09 +/- 0.17 and 1.96 +/- 0.27 ms at 35 and 22 degrees C, respectively. 3. Outward currents using the nystatin method were qualitatively similar to delayed rectifier currents recorded in dialysed cells with high calcium buffering capacity solutions. 4-Aminopyridine (4-AP; 2 mM), a specific blocker of delayed rectifier potassium channels in this tissue, inhibited over 80% of the outward current evoked by voltage-clamp steps to between -10 and +20 mV (n = 6). Less than 5% of the outward current was blocked over the same voltage range by charybdotoxin (100 nM; n = 15), a specific antagonist of large-conductance, calcium-activated potassium channels in this tissue. 4. The degree to which delayed rectifier and calcium-activated potassium conductances control resting membrane potential was examined in current-clamp experiments. The resting membrane potential of current clamped cells was -33.6 +/- 1.0 mV (n = 62). Application of 4-AP (2 mM) resulted in a 14.4 +/- 1.0 mV depolarization (n = 8) and an increase in input resistance. Charybdotoxin (100 nM) had no effect on resting membrane potential (n = 6). 5. Force measurements were made in isolated strips of trachealis muscle to determine the effect of pharmacological blockade of individual potassium conductances on resting tone. In the presence of tetrodotoxin (1 microM) and atropine (1 microM), 4-AP increased baseline tension in a dose-dependent manner, with an EC50 of 1.8 mM (n = 13); application of 5 mM 4-AP increased tone to 86.8 +/- 8.1% of that produced by 1 microM methacholine, and this tone was almost completely inhibited by nifedipine (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8271220

A comparative study of charge movement in rat and frog skeletal muscle fibres.

PubMed

Hollingworth, S; Marshall, M W

1981-12-01

1. The middle of the fibre voltage--clamp technique (Adrian & Marshall, 1977), modified where necessary for electrically short muscle fibres, has been used to measure non-linear charge movements in mammalian fast twitch (rat extensor digitorum longus), mammalian slow twitch (rat soleus) and frog (sartorius) muscles. 2. The maximum amount of charge moved in mammalian fast twitch muscle at 2 degrees C in hypertonic solution, was 3--5 times greater than in slow twitch muscle. The voltage distribution of fast twitch charge was 10--15 mV more positive when compared to slow twitch. 3. In both mammalian muscle types hypertonic Ringer solution negatively shifted the voltage distribution of charge some 6 mV. The steepness of charge moved around mechanical threshold was unaffected by hypertonicity. 4. The amount of charge in frog sartorius fibres at 2 degrees C in hypertonic solution was about half of that in rat fast twitch muscle; the voltage distribution of the frog charge was similar to rat soleus muscle. 5. Warming between 2 and 15 degrees C had no effect on either the amount of steady-state distribution of charge in mammalian or frog muscles. 6. At 2 degrees C, the kinetics of charge movement in fast and slow twitch mammalian muscles were similar and 2--3 times faster than frog muscle at the same temperature. In fast and slow mammalian fibres at 2 degrees C similar times were taken to shift the same fractions of the total amount of charge. The Q10 of charge movement kinetics was between 1.2 and 2.0 in the three muscles studied.

If and SR Ca2+ release both contribute to pacemaker activity in canine sinoatrial node cells

PubMed Central

Gao, Zhan; Chen, Biyi; Joiner, Mei-ling A.; Wu, Yuejin; Guan, Xiaoqun; Koval, Olha M.; Chaudhary, Ashok K.; Cunha, Shane R.; Mohler, Peter J.; Martins, James B.; Song, Long-Sheng; Anderson, Mark E.

2010-01-01

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a ‘voltage clock’ and a Ca2+ dependent process, or ‘Ca2+ clock.’ The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (If) is thought to be particularly important. A Ca2+ dependent process triggers APs when sarcoplasmic reticulum (SR) Ca2+ release activates inward current carried by the forward mode of the electrogenic Na+/Ca2+ exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca2+ clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective If antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest (∼14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca2+ release, but did not affect basal or isoproterenol-enhanced If. Taken together, these results indicate that voltage and Ca2+ dependent automaticity mechanisms coexist in canine SAN cells, and suggest If and SR Ca2+ release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells. PMID:20380837

Electromagnetic fields (UHF) increase voltage sensitivity of membrane ion channels; possible indication of cell phone effect on living cells.

PubMed

Ketabi, N; Mobasheri, H; Faraji-Dana, R

2015-03-01

The effects of ultra high frequency (UHF) nonionizing electromagnetic fields (EMF) on the channel activities of nanopore forming protein, OmpF porin, were investigated. The voltage clamp technique was used to study the single channel activity of the pore in an artificial bilayer in the presence and absence of the electromagnetic fields at 910 to 990 MHz in real time. Channel activity patterns were used to address the effect of EMF on the dynamic, arrangement and dielectric properties of water molecules, as well as on the hydration state and arrangements of side chains lining the channel barrel. Based on the varied voltage sensitivity of the channel at different temperatures in the presence and absence of EMF, the amount of energy transferred to nano-environments of accessible groups was estimated to address the possible thermal effects of EMF. Our results show that the effects of EMF on channel activities are frequency dependent, with a maximum effect at 930 MHz. The frequency of channel gating and the voltage sensitivity is increased when the channel is exposed to EMF, while its conductance remains unchanged at all frequencies applied. We have not identified any changes in the capacitance and permeability of membrane in the presence of EMF. The effect of the EMF irradiated by cell phones is measured by Specific Absorption Rate (SAR) in artificial model of human head, Phantom. Thus, current approach applied to biological molecules and electrolytes might be considered as complement to evaluate safety of irradiating sources on biological matter at molecular level.

Protein kinase C enhances the swelling-induced chloride current in human atrial myocytes.

PubMed

Li, Ye-Tao; Du, Xin-Ling

2016-06-01

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Development of Ca2+ hotspots between Lymnaea neurons during synaptogenesis

PubMed Central

Feng, Zhong-Ping; Grigoriev, Nikita; Munno, David; Lukowiak, Ken; MacVicar, Brian A; Goldberg, Jeffrey I; Syed, Naweed I

2002-01-01

Calcium (Ca2+) channel clustering at specific presynaptic sites is a hallmark of mature synapses. However, the spatial distribution patterns of Ca2+ channels at newly formed synapses have not yet been demonstrated. Similarly, it is unclear whether Ca2+ ‘hotspots’ often observed at the presynaptic sites are indeed target cell contact specific and represent a specialized mechanism by which Ca2+ channels are targeted to select synaptic sites. Utilizing both soma–soma paired (synapsed) and single neurons from the mollusk Lymnaea, we have tested the hypothesis that differential gradients of voltage-dependent Ca2+ signals develop in presynaptic neuron at its contact point with the postsynaptic neuron; and that these Ca2+ hotspots are target cell contact specific. Fura-2 imaging, or two-photon laser scanning microscopy of Calcium Green, was coupled with electrophysiological techniques to demonstrate that voltage-induced Ca2+ gradients (hotspots) develop in the presynaptic cell at its contact point with the postsynaptic neuron, but not in unpaired single cells. The incidence of Ca2+ hotspots coincided with the appearance of synaptic transmission between the paired cells, and these gradients were target cell contact specific. In contrast, the voltage-induced Ca2+ signal in unpaired neurons was uniformly distributed throughout the somata; a similar pattern of Ca2+ gradient was observed in the presynaptic neuron when it was soma–soma paired with a non-synaptic partner cell. Moreover, voltage clamp recording techniques, in conjunction with a fast, optical differential perfusion system, were used to demonstrate that the total whole-cell Ca2+ (or Ba2+) current density in single and paired cells was not significantly different. However, the amplitude of Ba2+ current was significantly higher in the presynaptic cell at its contact side with the postsynaptic neurons, compared with non-contacted regions. In summary, this study demonstrates that voltage-induced Ca2+ hotspots develop in the presynaptic cell, concomitant with the appearance of synaptic transmission between the soma–soma paired cells. The appearance of Ca2+ gradients in presynaptic neurons is target cell contact specific and is probably due to a spatial redistribution of existing channels during synaptogenesis. PMID:11850501

Propylparaben reduces the excitability of hippocampal neurons by blocking sodium channels.

PubMed

Lara-Valderrábano, Leonardo; Rocha, Luisa; Galván, Emilio J

2016-12-01

Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (I Na ) amplitude, causing a hyperpolarizing shift in the inactivation curve of the I Na, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (I NaP ). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner. Copyright © 2016 Elsevier B.V. All rights reserved.

Calcium current in type I hair cells isolated from the semicircular canal crista ampullaris of the rat.

PubMed

Almanza, Angélica; Vega, Rosario; Soto, Enrique

2003-12-24

The low voltage gain in type I hair cells implies that neurotransmitter release at their afferent synapse should be mediated by low voltage activated calcium channels, or that some peculiar mechanism should be operating in this synapse. With the patch clamp technique, we studied the characteristics of the Ca(2+) current in type I hair cells enzymatically dissociated from rat semicircular canal crista ampullaris. Calcium current in type I hair cells exhibited a slow inactivation (during 2-s depolarizing steps), was sensitive to nimodipine and was blocked by Cd(2+) and Ni(2+). This current was activated at potentials above -60 mV, had a mean half maximal activation of -36 mV, and exhibited no steady-state inactivation at holding potentials between -100 and -60 mV. This data led us to conclude that hair cell Ca(2+) current is most likely of the L type. Thus, other mechanisms participating in neurotransmitter release such as K(+) accumulation in the synaptic cleft, modulation of K(+) currents by nitric oxide, participation of a Na(+) current and possible metabotropic cascades activated by depolarization should be considered.

Inhibitory effects of antihistamines, diphenhydramine and chlorpheniramine, on proton currents in BV2 microglial cells.

PubMed

Kim, Jiwon; Song, Jin-Ho

2017-03-05

Microglial NADPH oxidase is a major source of toxic reactive oxygen species produced during chronic neuroinflammation. Voltage-gated proton channel (H V 1) functions to maintain the intense activity of NADPH oxidase, and channel inhibition alleviates the pathology of neurodegenerative diseases such as ischemic stroke and multiple sclerosis associated with oxidative neuroinflammation. Antagonists of histamine H 1 receptors have beneficial effects against microglia-mediated oxidative stress and neurotoxicity. We examined the effects of the H 1 antihistamines, diphenhydramine and chlorpheniramine, on proton currents in BV2 microglial cells recorded using the whole-cell patch clamp technique. Diphenhydramine and chlorpheniramine reduced the proton currents with almost the same potency, yielding IC 50 values of 42 and 43μM, respectively. Histamine did not affect proton currents, excluding the involvement of histamine receptors in their action. Neither drug shifted the voltage-dependence of activation or the reversal potential of the proton currents, even though diphenhydramine slowed the activation and deactivation kinetics. The inhibitory effects of the two antihistamines on proton currents could be utilized to develop therapeutic agents for neurodegenerative diseases and other diseases associated with H V 1 proton channel abnormalities. Copyright © 2017 Elsevier B.V. All rights reserved.

Subdiaphragmatic vagotomy increases the sensitivity of lumbar Aδ primary afferent neurons along with voltage-dependent potassium channels in rats.

PubMed

Furuta, Sadayoshi; Watanabe, Lisa; Doi, Seira; Horiuchi, Hiroshi; Matsumoto, Kenjiro; Kuzumaki, Naoko; Suzuki, Tsutomu; Narita, Minoru

2012-02-01

Subdiaphragmatic vagal dysfunction causes chronic pain. To verify whether this chronic pain is accompanied by enhanced peripheral nociceptive sensitivity, we evaluated primary afferent neuronal excitability in subdiaphragmatic vagotomized (SDV) rats. SDV rats showed a decrease in the electrical stimuli-induced hind limb-flexion threshold at 250 Hz, but showed no similar effect at 5 or 2000 Hz, which indicated that lumbar primary afferent Aδ sensitivity was enhanced in SDV rats. The whole-cell patch-clamp technique also revealed the hyper-excitability of acutely dissociated medium-sized lumbar dorsal root ganglion (DRG) neurons isolated from SDV rats. The contribution of changes in voltage-dependent potassium (Kv) channels was assessed, and transient A-type K(+) (I(A) ) current density was apparently decreased. Moreover, Kv4.3 immunoreactivity in medium-sized DRG neurons was significantly reduced in SDV rats compared to sham. These results indicate that SDV causes hyper-excitability of lumbar primary Aδ afferent neurons, which may be induced along with suppressing I(A) currents via the decreased expression of Kv4.3. Thus, peripheral Aδ neuroplasticity may contribute to the chronic lower limb pain caused by SDV. Copyright © 2011 Wiley Periodicals, Inc.

Inhibitory Effects of Honokiol on the Voltage-Gated Potassium Channels in Freshly Isolated Mouse Dorsal Root Ganglion Neurons.

PubMed

Sheng, Anqi; Zhang, Yan; Li, Guang; Zhang, Guangqin

2018-02-01

Voltage-gated potassium (K V ) currents, subdivided into rapidly inactivating A-type currents (I A ) and slowly inactivating delayed rectifier currents (I K ), play a fundamental role in modulating pain by controlling neuronal excitability. The effects of Honokiol (Hon), a natural biphenolic compound derived from Magnolia officinalis, on K V currents were investigated in freshly isolated mouse dorsal root ganglion neurons using the whole-cell patch clamp technique. Results showed that Hon inhibited I A and I K in concentration-dependent manner. The IC 50 values for block of I A and I K were 30.5 and 25.7 µM, respectively. Hon (30 µM) shifted the steady-state activation curves of I A and I K to positive potentials by 17.6 and 16.7 mV, whereas inactivation and recovery from the inactivated state of I A were unaffected. These results suggest that Hon preferentially interacts with the active states of the I A and I K channels, and has no effect on the resting state and inactivated state of the I A channel. Blockade on K + channels by Hon may contribute to its antinociceptive effect, especially anti-inflammatory pain.

A nitric oxide concentration clamp.

PubMed

Zhelyaskov, V R; Godwin, D W

1999-10-01

We report a new method of generating nitric oxide (NO) that possesses several advantages for experimental use. This method consists of a photolysis chamber where NO is released by illuminating photolabile NO donors with light from a xenon lamp, in conjunction with feedback control. Control of the photolysis light was achieved by selectively gating light projected through a shutter before the light was launched into a light guide that conveyed the light to the photolysis chamber. By gating the light in proportion to a sensor that reported nearly instantaneous concentration from the photolysis chamber, a criterion NO concentration could be achieved, which could be easily adjusted to higher or lower criterion levels. To denote the similarity of this process with the electrophysiological process of voltage clamp, we term this process a concentration "clamp." This development enhances the use of the fiber-optic-based system for NO delivery and should enable the execution of experiments where the in situ concentration of NO is particularly critical, such as in biological preparations. Copyright 1999 Academic Press.

Resonant-type Smooth Impact Drive Mechanism (SIDM) actuator using a bolt-clamped Langevin transducer.

PubMed

Nishimura, Takuma; Hosaka, Hiroshi; Morita, Takeshi

2012-01-01

The Smooth Impact Drive Mechanism (SIDM) is a linear piezoelectric actuator that has seen practically applied to camera lens modules. Although previous SIDM actuators are easily miniaturized and enable accurate positioning, these actuators cannot actuate at high speed and cannot provide powerful driving because they are driven at an off-resonant frequency using a soft-type PZT. In the present study, we propose a resonant-type SIDM using a bolt-clamped Langevin transducer (BLT) with a hard-type PZT. The resonant-type SIDM overcomes the above-mentioned problems and high-power operation becomes possible with a very simple structure. As a result, we confirmed the operation of resonant-type SIDM by designing a bolt-clamped Langevin transducer. The properties of no-load maximum speed was 0.28m/s at driving voltages of 80V(p-p) for 44.9kHz and 48V(p-p) for 22.45kHz with a pre-load of 3.1N. Copyright © 2011 Elsevier B.V. All rights reserved.

Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance.

PubMed

Veenstra, Richard D

2016-01-01

The development of the patch clamp technique has enabled investigators to directly measure gap junction conductance between isolated pairs of small cells with resolution to the single channel level. The dual patch clamp recording technique requires specialized equipment and the acquired skill to reliably establish gigaohm seals and the whole cell recording configuration with high efficiency. This chapter describes the equipment needed and methods required to achieve accurate measurement of macroscopic and single gap junction channel conductances. Inherent limitations with the dual whole cell recording technique and methods to correct for series access resistance errors are defined as well as basic procedures to determine the essential electrical parameters necessary to evaluate the accuracy of gap junction conductance measurements using this approach.

Dynamic Action Potential Restitution Contributes to Mechanical Restitution in Right Ventricular Myocytes From Pulmonary Hypertensive Rats.

PubMed

Hardy, Matthew E L; Pervolaraki, Eleftheria; Bernus, Olivier; White, Ed

2018-01-01

We investigated the steepened dynamic action potential duration (APD) restitution of rats with pulmonary artery hypertension (PAH) and right ventricular (RV) failure and tested whether the observed APD restitution properties were responsible for negative mechanical restitution in these myocytes. PAH and RV failure were provoked in male Wistar rats by a single injection of monocrotaline (MCT) and compared with saline-injected animals (CON). Action potentials were recorded from isolated RV myocytes at stimulation frequencies between 1 and 9 Hz. Action potential waveforms recorded at 1 Hz were used as voltage clamp profiles (action potential clamp) at stimulation frequencies between 1 and 7 Hz to evoke rate-dependent currents. Voltage clamp profiles mimicking typical CON and MCT APD restitution were applied and cell shortening simultaneously monitored. Compared with CON myocytes, MCT myocytes were hypertrophied; had less polarized diastolic membrane potentials; had action potentials that were triggered by decreased positive current density and shortened by decreased negative current density; APD was longer and APD restitution steeper. APD90 restitution was unchanged by exposure to the late Na + -channel blocker (5 μM) ranolazine or the intracellular Ca 2+ buffer BAPTA. Under AP clamp, stimulation frequency-dependent inward currents were smaller in MCT myocytes and were abolished by BAPTA. In MCT myocytes, increasing stimulation frequency decreased contraction amplitude when depolarization duration was shortened, to mimic APD restitution, but not when depolarization duration was maintained. We present new evidence that the membrane potential of PAH myocytes is less stable than normal myocytes, being more easily perturbed by external currents. These observations can explain increased susceptibility to arrhythmias. We also present novel evidence that negative APD restitution is at least in part responsible for the negative mechanical restitution in PAH myocytes. Thus, our study links electrical restitution remodeling to a defining mechanical characteristic of heart failure, the reduced ability to respond to an increase in demand.

Biological Activity of Bacillus thuringiensis and Associated Toxins against the Asian Longhorned Beetle (Coleoptera: Cerambycidae)

Treesearch

Vincent D' amico; John D. Podgwaite; Sara Duke; Sara Duke

2004-01-01

Bacillus thuringiensis Berliner var. tenebrionis and B. thuringiensis toxins were assayed against larval and adult Asian longhorned beetles, Anoplophora glabripennis (A. glabripennis). Preliminary in vitro assays showed some toxins to be active on whole midgut preparations in voltage clamp assays and in assays on brush border membrane vesicles formed from midgut...

Easily disassembled electrical connector for high voltage, high frequency connections

DOEpatents

Milner, Joseph R.

1994-01-01

An easily accessible electrical connector capable of rapid assembly and disassembly wherein a wide metal conductor sheet may be evenly contacted over the entire width of the conductor sheet by opposing surfaces on the connector which provide an even clamping pressure against opposite surfaces of the metal conductor sheet using a single threaded actuating screw.

Lightweight In-Plane Actuated Deformable Mirrors for Space Telescopes

DTIC Science & Technology

2006-09-01

dimensional beam-string and axisymmetric plate-membrane. The beam-string (a clamped beam simultaneously under an axial load ) is an important...Tensile load versus radius. . . . . . . . . . . . . . . . . . . . . . 175 7.4. Actuation voltage functions. . . . . . . . . . . . . . . . . . . . 179...membrane Asymptotic finite element Flint and De- noyer [45] 2003 In-plane Circular membrane Numerical least squares fit Actuators modelled as line loads

30 CFR 90.204 - Approved sampling devices; maintenance and calibration.

Code of Federal Regulations, 2013 CFR

2013-07-01

... performed to assure that the sampling devices are clean and in proper working condition by a person... voltage per cell value; (2) Examination of all components of the cyclone to assure that they are clean and... sampling device to assure that it is clean and free of leaks; and (5) Examination of the clamping and...

30 CFR 90.204 - Approved sampling devices; maintenance and calibration.

Code of Federal Regulations, 2014 CFR

2014-07-01

... performed to assure that the sampling devices are clean and in proper working condition by a person... voltage per cell value; (2) Examination of all components of the cyclone to assure that they are clean and... sampling device to assure that it is clean and free of leaks; and (5) Examination of the clamping and...

30 CFR 90.204 - Approved sampling devices; maintenance and calibration.

Code of Federal Regulations, 2012 CFR

2012-07-01

... performed to assure that the sampling devices are clean and in proper working condition by a person... voltage per cell value; (2) Examination of all components of the cyclone to assure that they are clean and... sampling device to assure that it is clean and free of leaks; and (5) Examination of the clamping and...

A grid-connected single-phase photovoltaic micro inverter

NASA Astrophysics Data System (ADS)

Wen, X. Y.; Lin, P. J.; Chen, Z. C.; Wu, L. J.; Cheng, S. Y.

2017-11-01

In this paper, the topology of a single-phase grid-connected photovoltaic (PV) micro-inverter is proposed. The PV micro-inverter consists of DC-DC stage with high voltage gain boost and DC-AC conversion stage. In the first stage, we apply the active clamp circuit and two voltage multipliers to achieve soft switching technology and high voltage gain. In addition, the flower pollination algorithm (FPA) is employed for the maximum power point tracking (MPPT) in the PV module in this stage. The second stage cascades a H-bridge inverter and LCL filter. To feed high quality sinusoidal power into the grid, the software phase lock, outer voltage loop and inner current loop control method are adopted as the control strategy. The performance of the proposed topology is tested by Matlab/Simulink. A PV module with maximum power 300W and maximum power point voltage 40V is applied as the input source. The simulation results indicate that the proposed topology and the control strategy are feasible.

Patch-clamp, ion-sensing, and glutamate-sensing techniques to study glutamate transport in isolated retinal glial cells.

PubMed

Billups, B; Szatkowski, M; Rossi, D; Attwell, D

1998-01-01

We have described how a combination of electrical, ion-sensing, and glutamate-sensing techniques has advanced our understanding of glutamate uptake into isolated salamander retinal glial cells. The next steps in understanding glutamate transport will inevitably depend strongly on molecular biological methods, as described elsewhere in this book, but will also require more detailed study of transporters in their normal environment, perhaps by using patch-clamping or imaging techniques to study cells in situ.

Direct block by bisindolylmaleimide of rat Kv1.5 expressed in Chinese hamster ovary cells.

PubMed

Choi, B H; Choi, J S; Jeong, S W; Hahn, S J; Yoon, S H; Jo, Y H; Kim, M S

2000-05-01

The interaction of bisindolylmaleimide (BIM), widely used as a specific protein kinase C (PKC) inhibitor, with rat brain Kv1.5 (rKv1.5) channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. BIM (I) and its inactive analog, BIM (V), inhibited rKv1.5 currents at +50 mV in a reversible concentration-dependent manner with an apparent K(d) value of 0.38 and 1.70 microM, respectively. BIM (I) accelerated the decay rate of inactivation of rKv1.5 currents but did not significantly modify the kinetics of current activation. Other specific PKC inhibitors, chelerythrine and PKC 19-36, had no effect on rKv1.5 and did not prevent the inhibitory effect of BIM (I). The inhibition of rKv1.5 by BIM (I) and BIM (V) was highly voltage-dependent between -30 and 0 mV (voltage range of channel opening), suggesting that both drugs interact preferentially with the open state of the channel. The additional inhibition by BIM (I) displayed a voltage dependence (delta = 0.19) in the full activation voltage range positive to 0 mV, but was not shown in BIM (V) (delta = 0). The rate constants of association and dissociation for BIM (I) were 9.63 microM(-1) s(-1) and 5.82 s(-1), respectively. BIM (I) increased the time constant of deactivation of tail currents from 26. 35 to 45.79 ms, resulting in tail crossover phenomenon. BIM (I) had no effect on the voltage dependence of steady-state inactivation. BIM (I) produced use-dependent inhibition of rKv1.5, which was consistent with the slow recovery from inactivation in the presence of drug. These results suggest that BIM (I) directly inhibits rKv1.5 channels in a phosphorylation-independent, and state-, voltage-, time-, and use-dependent manner.

Ph-Dependent Inhibition of Voltage-Gated H+ Currents in Rat Alveolar Epithelial Cells by Zn2+ and Other Divalent Cations

PubMed Central

Cherny, Vladimir V.; DeCoursey, Thomas E.

1999-01-01

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl2 reduced the H+ current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H+ current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn2+ were inconsistent with classical voltage-dependent block in which Zn2+ binds within the membrane voltage field. Instead, Zn2+ binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn2+ were strongly pHo dependent but were insensitive to pHi, suggesting that protons and Zn2+ compete for external sites on H+ channels. The apparent potency of Zn2+ in slowing activation was ∼10× greater at pHo 7 than at pHo 6, and ∼100× greater at pHo 6 than at pHo 5. The pHo dependence suggests that Zn2+, not ZnOH+, is the active species. Evidently, the Zn2+ receptor is formed by multiple groups, protonation of any of which inhibits Zn2+ binding. The external receptor bound H+ and Zn2+ with pK a 6.2–6.6 and pK M 6.5, as described by several models. Zn2+ effects on the proton chord conductance–voltage (g H–V) relationship indicated higher affinities, pK a 7 and pK M 8. CdCl2 had similar effects as ZnCl2 and competed with H+, but had lower affinity. Zn2+ applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H+ currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn2+ receptor may be the same modulatory protonation site(s) at which pHo regulates H+ channel gating. PMID:10578017

HTS techniques for patch clamp-based ion channel screening - advances and economy.

PubMed

Farre, Cecilia; Fertig, Niels

2012-06-01

Ten years ago, the first publication appeared showing patch clamp recordings performed on a planar glass chip instead of using a conventional patch clamp pipette. "Going planar" proved to revolutionize ion channel drug screening as we know it, by allowing high quality measurements of ion channels and their effectors at a higher throughput and at the same time de-skilling the highly laborious technique. Over the years, platforms evolved in response to user requirements regarding experimental features, data handling plus storage, and suitable target diversity. This article gives a snapshot image of patch clamp-based ion channel screening with focus on platforms developed to meet requirements of high-throughput screening environments. The commercially available platforms are described, along with their benefits and drawbacks in ion channel drug screening. Automated patch clamp (APC) platforms allow faster investigation of a larger number of ion channel active compounds or cell clones than previously possible. Since patch clamp is the only method allowing direct, real-time measurements of ion channel activity, APC holds the promise of picking up high quality leads, where they otherwise would have been overseen using indirect methods. In addition, drug candidate safety profiling can be performed earlier in the drug discovery process, avoiding late-phase compound withdrawal due to safety liability issues, which is highly costly and inefficient.

Unitaxial constant velocity microactuator

DOEpatents

McIntyre, Timothy J.

1994-01-01

A uniaxial drive system or microactuator capable of operating in an ultra-high vacuum environment. The mechanism includes a flexible coupling having a bore therethrough, and two clamp/pusher assemblies mounted in axial ends of the coupling. The clamp/pusher assemblies are energized by voltage-operated piezoelectrics therewithin to operatively engage the shaft and coupling causing the shaft to move along its rotational axis through the bore. The microactuator is capable of repeatably positioning to sub-manometer accuracy while affording a scan range in excess of 5 centimeters. Moreover, the microactuator generates smooth, constant velocity motion profiles while producing a drive thrust of greater than 10 pounds. The system is remotely controlled and piezoelectrically driven, hence minimal thermal loading, vibrational excitation, or outgassing is introduced to the operating environment.

Simultaneous recording of the action potential and its whole-cell associated ion current on NG108-15 cells cultured over a MWCNT electrode

NASA Astrophysics Data System (ADS)

Morales-Reyes, I.; Seseña-Rubfiaro, A.; Acosta-García, M. C.; Batina, N.; Godínez-Fernández, R.

2016-08-01

It is well known that, in excitable cells, the dynamics of the ion currents (I i) is extremely important to determine both the magnitude and time course of an action potential (A p). To observe these two processes simultaneously, we cultured NG108-15 cells over a multi-walled carbon nanotubes electrode (MWCNTe) surface and arranged a two independent Patch Clamp system configuration (Bi-Patch Clamp). The first system was used in the voltage or current clamp mode, using a glass micropipette as an electrode. The second system was modified to connect the MWCNTe to virtual ground. While the A p was recorded through the micropipette electrode, the MWCNTe was used to measure the underlying whole-cell current. This configuration allowed us to record both the membrane voltage (V m) and the current changes simultaneously. Images acquired by atomic force microscopy (AFM) and scanning electron microscopy (SEM) indicate that cultured cells developed a complex network of neurites, which served to establish the necessary close contact and strong adhesion to the MWCNTe surface. These features were a key factor to obtain the recording of the whole-cell I i with a high signal to noise ratio (SNR). The experimental results were satisfactorily reproduced by a theoretical model developed to simulate the proposed system. Besides the contribution to a better understanding of the fundamental mechanisms involved in cell communication, the developed method could be useful in cell physiology studies, pharmacology and diseases diagnosis.

Nonlinear analysis of thermally and electrically actuated functionally graded material microbeam.

PubMed

Li, Yingli; Meguid, S A; Fu, Yiming; Xu, Daolin

2014-02-08

In this paper, we provide a unified and self-consistent treatment of a functionally graded material (FGM) microbeam with varying thermal conductivity subjected to non-uniform or uniform temperature field. Specifically, it is our objective to determine the effect of the microscopic size of the beam, the electrostatic gap, the temperature field and material property on the pull-in voltage of the microbeam under different boundary conditions. The non-uniform temperature field is obtained by integrating the steady-state heat conduction equation. The governing equations account for the microbeam size by introducing an internal material length-scale parameter that is based on the modified couple stress theory. Furthermore, it takes into account Casimir and van der Waals forces, and the associated electrostatic force with the first-order fringing field effects. The resulting nonlinear differential equations were converted to a coupled system of algebraic equations using the differential quadrature method. The outcome of our work shows the dramatic effect and dependence of the pull-in voltage of the FGM microbeam upon the temperature field, its gradient for a given boundary condition. Specifically, both uniform and non-uniform thermal loading can actuate the FGM microbeam even without an applied voltage. Our work also reveals that the non-uniform temperature field is more effective than the uniform temperature field in actuating a FGM cantilever-type microbeam. For the clamped-clamped case, care must be taken to account for the effective use of thermal loading in the design of microbeams. It is also observed that uniform thermal loading will lead to a reduction in the pull-in voltage of a FGM microbeam for all the three boundary conditions considered.

Timing and efficacy of Ca2+ channel activation in hippocampal mossy fiber boutons.

PubMed

Bischofberger, Josef; Geiger, Jörg R P; Jonas, Peter

2002-12-15

The presynaptic Ca2+ signal is a key determinant of transmitter release at chemical synapses. In cortical synaptic terminals, however, little is known about the kinetic properties of the presynaptic Ca2+ channels. To investigate the timing and magnitude of the presynaptic Ca2+ inflow, we performed whole-cell patch-clamp recordings from mossy fiber boutons (MFBs) in rat hippocampus. MFBs showed large high-voltage-activated Ca(2+) currents, with a maximal amplitude of approximately 100 pA at a membrane potential of 0 mV. Both activation and deactivation were fast, with time constants in the submillisecond range at a temperature of approximately 23 degrees C. An MFB action potential (AP) applied as a voltage-clamp command evoked a transient Ca2+ current with an average amplitude of approximately 170 pA and a half-duration of 580 microsec. A prepulse to +40 mV had only minimal effects on the AP-evoked Ca2+ current, indicating that presynaptic APs open the voltage-gated Ca2+ channels very effectively. On the basis of the experimental data, we developed a kinetic model with four closed states and one open state, linked by voltage-dependent rate constants. Simulations of the Ca2+ current could reproduce the experimental data, including the large amplitude and rapid time course of the current evoked by MFB APs. Furthermore, the simulations indicate that the shape of the presynaptic AP and the gating kinetics of the Ca2+ channels are tuned to produce a maximal Ca2+ influx during a minimal period of time. The precise timing and high efficacy of Ca2+ channel activation at this cortical glutamatergic synapse may be important for synchronous transmitter release and temporal information processing.

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na+ channel inactivation.

PubMed

Holtkamp, Dominik; Opitz, Thoralf; Niespodziany, Isabelle; Wolff, Christian; Beck, Heinz

2017-01-01

In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na + currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Core-coat conductor of lipid bilayer and micromachined silicon.

PubMed

Fromherz, P; Klingler, J

1991-02-11

We have etched a groove into a (110) plane of silicon and have covered it with a bilayer of glycerol monooleate. We have varied the depth of the groove, the concentration of salt in the electrolyte and the density of gramicidin in the membrane. We have clamped one end of the groove at a constant voltage with respect to the bath keeping the other end sealed or electrically open with respect to the bath. We have measured (i) the voltage at the center of the groove and at the sealed distal end and (ii) the current through the system in sealed and open configuration. We have found that the spread of voltage is in quantitative agreement with the stationary solutions of Kelvin's equation for a homogeneous cable.

Design of a Miniaturized RAD Hard Point-of-Load Converter

NASA Astrophysics Data System (ADS)

Lofgren, Henrik; Landstrom, Sven; Gunnarsson, Marcus; Hagstrom, Maria

2014-08-01

As an ARTES 5.2 activity, a miniaturized radiation hardened Point-Of-Load converter (uPOL) has been developed. Several different design options have been evaluated before the final system level design was selected. The selected topology is a buck regulator with synchronous rectification utilizing peak current mode control. The PWM logic is designed using discrete electronics. Inside the POL converter package, an independent latching current limiter and clamping over- voltage protection are included as protection devices. The converter has an input voltage range of 4.8-6.2V, output voltage range of 1.2-3.5V and an output current of 0-3.5A. The final converter will be a metal packaged hybrid built on LTCC technology with an operating case temperature range of -40 to +85 °C.

Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison.

PubMed Central

Llinás, R; Sugimori, M; Lin, J W; Cherksey, B

1989-01-01

A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the "P" channel, as it was first described in Purkinje cells. Images PMID:2537980

Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison.

PubMed

Llinás, R; Sugimori, M; Lin, J W; Cherksey, B

1989-03-01

A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the "P" channel, as it was first described in Purkinje cells.

Synaptic control of the shape of the motoneuron pool input-output function

PubMed Central

Heckman, Charles J.

2017-01-01

Although motoneurons have often been considered to be fairly linear transducers of synaptic input, recent evidence suggests that strong persistent inward currents (PICs) in motoneurons allow neuromodulatory and inhibitory synaptic inputs to induce large nonlinearities in the relation between the level of excitatory input and motor output. To try to estimate the possible extent of this nonlinearity, we developed a pool of model motoneurons designed to replicate the characteristics of motoneuron input-output properties measured in medial gastrocnemius motoneurons in the decerebrate cat with voltage-clamp and current-clamp techniques. We drove the model pool with a range of synaptic inputs consisting of various mixtures of excitation, inhibition, and neuromodulation. We then looked at the relation between excitatory drive and total pool output. Our results revealed that the PICs not only enhance gain but also induce a strong nonlinearity in the relation between the average firing rate of the motoneuron pool and the level of excitatory input. The relation between the total simulated force output and input was somewhat more linear because of higher force outputs in later-recruited units. We also found that the nonlinearity can be increased by increasing neuromodulatory input and/or balanced inhibitory input and minimized by a reciprocal, push-pull pattern of inhibition. We consider the possibility that a flexible input-output function may allow motor output to be tuned to match the widely varying demands of the normal motor repertoire. NEW & NOTEWORTHY Motoneuron activity is generally considered to reflect the level of excitatory drive. However, the activation of voltage-dependent intrinsic conductances can distort the relation between excitatory drive and the total output of a pool of motoneurons. Using a pool of realistic motoneuron models, we show that pool output can be a highly nonlinear function of synaptic input but linearity can be achieved through adjusting the time course of excitatory and inhibitory synaptic inputs. PMID:28053245

Relaxin suppresses atrial fibrillation in aged rats by reversing fibrosis and upregulating Na+ channels.

PubMed

Henry, Brian L; Gabris, Beth; Li, Qiao; Martin, Brian; Giannini, Marianna; Parikh, Ashish; Patel, Divyang; Haney, Jamie; Schwartzman, David S; Shroff, Sanjeev G; Salama, Guy

2016-04-01

Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly patients and has been correlated with enhanced age-dependent atrial fibrosis. Reversal of atrial fibrosis has been proposed as therapeutic strategy to suppress AF. To test the ability of relaxin to reverse age-dependent atrial fibrosis and suppress AF. Aged F-344 rats (24 months old) were treated with subcutaneous infusion of vehicle or relaxin (0.4 mg/kg/day) for 2 weeks. Rat hearts were excised, perfused on a Langendorff apparatus, and stained with voltage and Ca(2+) indicator dyes. Optical mapping and programmed electrical stimulation was used to test arrhythmia vulnerability and changes in electrophysiological characteristics. Changes in protein expression and Na(+) current density (INa) were measured by tissue immunofluorescence and whole-cell patch clamp technique. In aged rats, sustained AF was readily induced with a premature pulse (n = 7/8) and relaxin treatment suppressed sustained AF by a premature impulse or burst pacing (n = 1/6) (P < .01). Relaxin significantly increased atrial action potential conduction velocity and decreased atrial fibrosis. Relaxin treatment increased Nav1.5 expression (n = 6; 36% ± 10%) and decreased total collagen and collagen I (n = 5-6; 55%-66% ± 15%) in aged atria (P < .05) and decreased collagen I and III and TGF-β1 mRNA (P < .05). Voltage-clamp experiments demonstrated that relaxin treatment (100 nM for 2 days) increased atrial INa by 46% ± 4% (n = 12-13/group, P < .02). Relaxin suppresses AF through an increase in atrial conduction velocity by decreasing atrial fibrosis and increasing INa. These data provide compelling evidence that relaxin may serve as an effective therapy to manage AF in geriatric patients by reversing fibrosis and modulating cardiac ionic currents. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane

NASA Technical Reports Server (NTRS)

Miedema, H.; Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1999-01-01

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

CaV3.1 isoform of T-type calcium channels supports excitability of rat and mouse ventral tegmental area neurons.

PubMed

Tracy, Matthew E; Tesic, Vesna; Stamenic, Tamara Timic; Joksimovic, Srdjan M; Busquet, Nicolas; Jevtovic-Todorovic, Vesna; Todorovic, Slobodan M

2018-03-23

Recent data have implicated voltage-gated calcium channels in the regulation of the excitability of neurons within the mesolimbic reward system. While the attention of most research has centered on high voltage L-type calcium channel activity, the presence and role of the low voltage-gated T-type calcium channel (T-channels) has not been well explored. Hence, we investigated T-channel properties in the neurons of the ventral tegmental area (VTA) utilizing wild-type (WT) rats and mice, Ca V 3.1 knock-out (KO) mice, and TH-eGFP knock-in (KI) rats in acute horizontal brain slices of adolescent animals. In voltage-clamp experiments, we first assessed T-channel activity in WT rats with characteristic properties of voltage-dependent activation and inactivation, as well as characteristic crisscrossing patterns of macroscopic current kinetics. T-current kinetics were similar in WT mice and WT rats but T-currents were abolished in Ca V 3.1 KO mice. In ensuing current-clamp experiments, we observed the presence of hyperpolarization-induced rebound burst firing in a subset of neurons in WT rats, as well as dopaminergic and non-dopaminergic neurons in TH-eGFP KI rats. Following the application of a pan-selective T-channel blocker TTA-P2, rebound bursting was significantly inhibited in all tested cells. In a behavioral assessment, the acute locomotor increase induced by a MK-801 (Dizocilpine) injection in WT mice was abolished in Ca V 3.1 KO mice, suggesting a tangible role for 3.1 T-type channels in drug response. We conclude that pharmacological targeting of Ca V 3.1 isoform of T-channels may be a novel approach for the treatment of disorders of mesolimbic reward system. Copyright © 2018. Published by Elsevier Ltd.

A linkage analysis toolkit for studying allosteric networks in ion channels

PubMed Central

2013-01-01

A thermodynamic approach to studying allosterically regulated ion channels such as the large-conductance voltage- and Ca2+-dependent (BK) channel is presented, drawing from principles originally introduced to describe linkage phenomena in hemoglobin. In this paper, linkage between a principal channel component and secondary elements is derived from a four-state thermodynamic cycle. One set of parallel legs in the cycle describes the “work function,” or the free energy required to activate the principal component. The second are “lever operations” activating linked elements. The experimental embodiment of this linkage cycle is a plot of work function versus secondary force, whose asymptotes are a function of the parameters (displacements and interaction energies) of an allosteric network. Two essential work functions play a role in evaluating data from voltage-clamp experiments. The first is the conductance Hill energy WH[g], which is a “local” work function for pore activation, and is defined as kT times the Hill transform of the conductance (G-V) curve. The second is the electrical capacitance energy WC[q], representing “global” gating charge displacement, and is equal to the product of total gating charge per channel times the first moment (VM) of normalized capacitance (slope of Q-V curve). Plots of WH[g] and WC[q] versus voltage and Ca2+ potential can be used to measure thermodynamic parameters in a model-independent fashion of the core gating constituents (pore, voltage-sensor, and Ca2+-binding domain) of BK channel. The method is easily generalized for use in studying other allosterically regulated ion channels. The feasibility of performing linkage analysis from patch-clamp data were explored by simulating gating and ionic currents of a 17-particle model BK channel in response to a slow voltage ramp, which yielded interaction energies deviating from their given values in the range of 1.3 to 7.2%. PMID:23250867

Effect of an N-terminus deletion on voltage-dependent gating of the ClC-2 chloride channel

PubMed Central

Varela, Diego; Niemeyer, María Isabel; Cid, L Pablo; Sepúlveda, Francisco V

2002-01-01

ClC-2, a chloride channel widely expressed in mammalian tissues, is activated by hyperpolarisation and extracellular acidification. Deletion of amino acids 16–61 in rat ClC-2 abolishes voltage and pH dependence in two-electrode voltage-clamp experiments in amphibian oocytes. These results have been interpreted in terms of a ball-and-chain type of mechanism in which the N-terminus would behave as a ball that is removed from an inactivating site upon hyperpolarisation. We now report whole-cell patch-clamp measurements in mammalian cells showing hyperpolarization-activation of rClC-2Δ16–61 differing only in presenting faster opening and closing kinetics than rClC-2. The lack of time and voltage dependence observed previously was reproduced, however, in nystatin-perforated patch experiments. The behaviour of wild-type rClC-2 did not differ between conventional and nystatin-perforated patches. Similar results were obtained with ClC-2 from guinea-pig. One possible explanation of the results is that some diffusible component is able to lock the channel in an open state but does so only to the mutated channel. Alternative explanations involving the osmotic state of the cell and cytoskeleton structure are also considered. Low extracellular pH activates the wild-type channel but not rClC-2Δ16–61 when expressed in oocytes, a result that had been interpreted to suggest that protons affect the ball-and-chain mechanism. In our experiments no difference was seen in the effect of extracellular pH upon rClC-2 and rClC-2Δ16–61 in either recording configuration, suggesting that protons act independently from possible effects of the N-terminus on gating. Our observations of voltage-dependent gating of the N-terminal deleted ClC-2 are an argument against a ball-and-chain mechanism for this channel. PMID:12381811

Vertebrate rod photoreceptors express both BK and IK calcium-activated potassium channels, but only BK channels are involved in receptor potential regulation.

PubMed

Pelucchi, Bruna; Grimaldi, Annalisa; Moriondo, Andrea

2008-01-01

In salamander rods, Ca(2+)-activated K(+) current (I(KCa)) provides an effective "clamp" of the dark membrane potential to its normal resting level. By a combination of electrophysiological, pharmacological, and immunohistochemical approaches, we show that salamander rods functionally express large-conductance Ca(2+)- and voltage-dependent potassium (BK) channel and intermediate-conductance Ca(2+)-dependent potassium (IK) channel, but not small-conductance Ca(2+)-dependent potassium channel (SK) subtypes. Application of 100 nM iberiotoxin and 100 nM clotrimazole reduced net I(KCa) to 36% and 63%, respectively, whereas the current was unaffected by application of 1 microM apamin. Consistently, anti- SK1, -SK2, and -SK3 antibodies were unable to stain rod photoreceptors, whereas both anti-BK and -SK4/ IK1 antibodies heavily stained the ellipsoid region of the inner segments of the rods. Moreover, by using current-clamp experiments, it was clearly seen that the strong clamping effect of the total I(KCa) was lost when IbTx, but not CLTZ, was applied to the bath. This behavior strongly suggests that of BK and IK channels, only the former are responsible for the clamping effect on the photoreceptor membrane potential.

Easily disassembled electrical connector for high voltage, high frequency connections

DOEpatents

Milner, J.R.

1994-05-10

An easily accessible electrical connector capable of rapid assembly and disassembly is described wherein a wide metal conductor sheet may be evenly contacted over the entire width of the conductor sheet by opposing surfaces on the connector which provide an even clamping pressure against opposite surfaces of the metal conductor sheet using a single threaded actuating screw. 13 figures.

Role of the pH in state-dependent blockade of hERG currents

NASA Astrophysics Data System (ADS)

Wang, Yibo; Guo, Jiqing; Perissinotti, Laura L.; Lees-Miller, James; Teng, Guoqi; Durdagi, Serdar; Duff, Henry J.; Noskov, Sergei Yu.

2016-10-01

Mutations that reduce inactivation of the voltage-gated Kv11.1 potassium channel (hERG) reduce binding for a number of blockers. State specific block of the inactivated state of hERG block may increase risks of drug-induced Torsade de pointes. In this study, molecular simulations of dofetilide binding to the previously developed and experimentally validated models of the hERG channel in open and open-inactivated states were combined with voltage-clamp experiments to unravel the mechanism(s) of state-dependent blockade. The computations of the free energy profiles associated with the drug block to its binding pocket in the intra-cavitary site display startling differences in the open and open-inactivated states of the channel. It was also found that drug ionization may play a crucial role in preferential targeting to the open-inactivated state of the pore domain. pH-dependent hERG blockade by dofetilie was studied with patch-clamp recordings. The results show that low pH increases the extent and speed of drug-induced block. Both experimental and computational findings indicate that binding to the open-inactivated state is of key importance to our understanding of the dofetilide’s mode of action.

MEMS earthworm: a thermally actuated peristaltic linear micromotor

NASA Astrophysics Data System (ADS)

Arthur, Craig; Ellerington, Neil; Hubbard, Ted; Kujath, Marek

2011-03-01

This paper examines the design, fabrication and testing of a bio-mimetic MEMS (micro-electro mechanical systems) earthworm motor with external actuators. The motor consists of a passive mobile shuttle with two flexible diamond-shaped segments; each segment is independently squeezed by a pair of stationary chevron-shaped thermal actuators. Applying a specific sequence of squeezes to the earthworm segments, the shuttle can be driven backward or forward. Unlike existing inchworm drives that use clamping and thrusting actuators, the earthworm actuators apply only clamping forces to the shuttle, and lateral thrust is produced by the shuttle's compliant geometry. The earthworm assembly is fabricated using the PolyMUMPs process with planar dimensions of 400 µm width by 800 µm length. The stationary actuators operate within the range of 4-9 V and provide a maximum shuttle range of motion of 350 µm (approximately half its size), a maximum shuttle speed of 17 mm s-1 at 10 kHz, and a maximum dc shuttle force of 80 µN. The shuttle speed was found to vary linearly with both input voltage and input frequency. The shuttle force was found to vary linearly with the actuator voltage.

Single Active Switch PV Inverter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramanan, V. R.; Pan, Zhiguo

This report presents a new PV inverter topology that uses only one active switch instead of 7 active switches in a conventional PV inverter. It has a buck boost converter and operates at discontinuous current control mode, which can reduce the output stage from an active switch bridge to a thyristor bridge. This concept, if successfully demonstrated, may have great cost and size/weight benefits over conventional solutions. Since the proposed topology is completely different from the traditional boost converter plus voltage source inverter approach, there is no existing control/modulation scheme available. A new modulation scheme for both the main switchmore » and the thyristors has been developed. An active clamping circuit has also been proposed to reduce switching losses and voltage spike during the switching transient. A simulation model has been set up to validate the control algorithm and clamping circuit. Simulated results show that a proposed 10 kW PV inverter can reach 5% total harmonic distortion (THD), 98.8% peak efficiency with only one main active switch, and an inductor weighing less than 3 kg. Based on that, a 10 kW prototype converter has been designed and built.« less

Mechanism of pain relief by low-power infrared irradiation: ATP is an IR-target molecule in nociceptive neurons.

PubMed

Yachnev, Igor L; Plakhova, Vera B; Podzorova, Svetlana A; Shelykh, Tatiana N; Rogachevsky, Ilya V; Krylov, Boris V

2012-01-01

Effects of infrared (IR) radiation generated by a low-power CO2-laser on the membrane of cultured dissociated nociceptive neurons of newborn rat spinal ganglia were investigated using the whole-cell patch-clamp method. Low-power IR radiation diminished the voltage sensitivity of activation gating machinery of slow sodium channels (Na(v)1.8). Ouabain known to block both transducer and pumping functions of Na+,K+-ATPase eliminated IR irradiation effects. The molecular mechanism of interaction of CO2-laser radiation with sensory membrane was proposed. The primary event of this interaction is the process of energy absorption by ATP molecules. The transfer of vibrational energy from Na+,K+- ATPase-bound and vibrationally excited ATP molecules to Na+,K+-ATPase activates this enzyme and converts it into a signal transducer. This effect leads to a decrease in the voltage sensitivity of Na(v)1.8 channels. The effect of IR-radiation was elucidated by the combined application of a very sensitive patch-clamp method and an optical facility with a controlled CO2-laser. As a result, the mechanism of interaction of non-thermal low-power IR radiation with the nociceptive neuron membrane is suggested.

Design and experimental verification of an improved magnetostrictive energy harvester

NASA Astrophysics Data System (ADS)

Germer, M.; Marschner, U.; Flatau, A. B.

2017-04-01

This paper summarizes and extends the modeling state of the art of magnetostrictive energy harvesters with a focus on the pick-up coil design. The harvester is a one-sided clamped galfenol unimorph loaded with two brass pieces each containing a permanent magnet to create a biased magnetic field. Measurements on different pick-up coils were conducted and compared with results from an analytic model. Resistance, mass and inductance were formulated and proved by measurements. Both the length for a constant number of turns and the number of turns for a constant coil length were also modeled and varied. The results confirm that the output voltage depends on the coil length for a constant number of turns and is higher for smaller coils. In contrast to a uniform magnetic field, the maximal output voltage is gained if the coil is placed not directly at but near the fixation. Two effects explain this behavior: Due to the permanent magnet next to the fixation, the magnetic force is higher and orientates the magnetic domains stronger. The clamping locally increases the stress and forces the magnetic domains to orientate, too. For that reason the material is stiffer and therefore the strain smaller. The tradeoff between a higher induced voltage in the coil and an increasing inductance and resistance for every additional turn are presented together with an experimental validation of the models. Based on the results guidelines are given to design an optimal coil which maximizes the output power for a given unimorph.

Sensory Transduction and Electrical Signaling in Guard Cells

PubMed Central

Serrano, Elba E.; Zeiger, Eduardo

1989-01-01

Guard cells are a valuable model system for the study of photoreception, ion transport, and osmoregulation in plant cells. Changes in stomatal apertures occur when sensing mechanisms within the guard cells transduce environmental stimull into the ion fluxes and biosynthesis of organic solutes that regulate turgor. The electrical events mediating sensory transduction in guard cells can be characterized with a variety of electrophysiological recording techniques. Recent experiments applying the patch clamp method to guard cell protoplasts have demonstrated activation of electrogenic pumps by blue and red light as well as the presence of potassium channels in guard cell plasmalemma. Light activation of electrogenic proton pumping and the ensuing gating of voltage-dependent ion channels appear to be components of sensory transduction of the stomatal response to light. Mechanisms underlying stomatal control by environmental signals can be understood by studying electrical events associated with ion transport. PMID:16667138

Membrane currents in the oocyte of the toad Bufo arenarum.

PubMed

Kotsias, Basilio A; Damiano, Alicia E; Godoy, Sebastian; Assef, Yanina; Ibarra, Cristina; Cantiello, Horacio F

2002-03-01

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes. Copyright 2002 Wiley-Liss, Inc.

Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

PubMed Central

Andersen, O S; Feldberg, S; Nakadomari, H; Levy, S; McLaughlin, S

1978-01-01

We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes. PMID:620077

[Effects of dauricine on action potentials and slow inward currents of guinea pig ventricular papillary muscles].

PubMed

Li, S N; Zhang, K Y

1992-11-01

Effects of dauricine (Dau) on the action potentials (AP), the slow action potentials (SAP), and the slow inward currents (Isi) of guinea pig ventricular papillary muscles were observed by means of intracellular microelectrode and single sucrose gap voltage clamp technique. In the early stage, Dau shortened action potential duration 100 (APD100) and effective refractory period (ERP) (ERP/APD < 1; P < 0.01), but did not affect APD20 and other parameters. In the late stage, Dau prolonged APD100, ERP, and APD20, significantly decreased action potential amplitude (APA), maximum velocity (Vmax), and overshot (OS) (ERP/APD > 1; P < 0.01), greatly diminished APA and OS of SAP induced by isoprenaline (P < 0.01), and remarkably inhibited Isi (P < 0.01). The results suggested that Dau exerted an inhibitory effect on Na+, Ca2+, and K+ channels.

The delayed rectifier, IKI, is the major conductance in type I vestibular hair cells across vestibular end organs

NASA Technical Reports Server (NTRS)

Ricci, A. J.; Rennie, K. J.; Correia, M. J.

1996-01-01

Hair cells were dissociated from the semicircular canal, utricle, lagena and saccule of white king pigeons. Type I hair cells were identified morphologically based on the ratios of neck width to cuticular plate width (NPR < 0.72) as well as neck width to cell body width (NBR < 0.64). The perforated patch variant of the whole-cell recording technique was used to measure electrical properties from type I hair cells. In voltage-clamp, the membrane properties of all identified type I cells were dominated by a predominantly outward potassium current, previously characterized in semicircular canal as IKI. Zero-current potential, activation, deactivation, slope conductance, pharmacologic and steady-state properties of the complex currents were not statistically different between type I hair cells of different vestibular end organs. The voltage dependence causes a significant proportion of this conductance to be active about the cell's zero-current potential. The first report of the whole-cell activation kinetics of the conductance is presented, showing a voltage dependence that could be best fit by an equation for a single exponential. Results presented here are the first data from pigeon dissociated type I hair cells from utricle, saccule and lagena suggesting that the basolateral conductances of a morphologically identified population of type I hair cells are conserved between functionally different vestibular end organs; the major conductance being a delayed rectifier characterized previously in semicircular canal hair cells as IKI.

The human ether-a-go-go-related gene (hERG) current inhibition selectively prolongs action potential of midmyocardial cells to augment transmural dispersion.

PubMed

Yasuda, C; Yasuda, S; Yamashita, H; Okada, J; Hisada, T; Sugiura, S

2015-08-01

The majority of drug induced arrhythmias are related to the prolongation of action potential duration following inhibition of rapidly activating delayed rectifier potassium current (I(Kr)) mediated by the hERG channel. However, for arrhythmias to develop and be sustained, not only the prolongation of action potential duration but also its transmural dispersion are required. Herein, we evaluated the effect of hERG inhibition on transmural dispersion of action potential duration using the action potential clamp technique that combined an in silico myocyte model with the actual I(Kr) measurement. Whole cell I(Kr) current was measured in Chinese hamster ovary cells stably expressing the hERG channel. The measured current was coupled with models of ventricular endocardial, M-, and epicardial cells to calculate the action potentials. Action potentials were evaluated under control condition and in the presence of 1, 10, or 100 μM disopyramide, an hERG inhibitor. Disopyramide dose-dependently increased the action potential durations of the three cell types. However, action potential duration of M-cells increased disproportionately at higher doses, and was significantly different from that of epicardial and endocardial cells (dispersion of repolarization). By contrast, the effects of disopyramide on peak I(Kr) and instantaneous current-voltage relation were similar in all cell types. Simulation study suggested that the reduced repolarization reserve of M-cell with smaller amount of slowly activating delayed rectifier potassium current levels off at longer action potential duration to make such differences. The action potential clamp technique is useful for studying the mechanism of arrhythmogenesis by hERG inhibition through the transmural dispersion of repolarization.

Voltage-driven reversible insertion into and leaving from a lipid bilayer: tuning transmembrane transport of artificial channels.

PubMed

Si, Wen; Li, Zhan-Ting; Hou, Jun-Li

2014-04-25

Three new artificial transmembrane channel molecules have been designed and synthesized by attaching positively charged Arg-incorporated tripeptide chains to pillar[5]arene. Fluorescent and patch-clamp experiments revealed that voltage can drive the molecules to insert into and leave from a lipid bilayer and thus switch on and off the transport of K(+) ions. One of the molecules was found to display antimicrobial activity toward Bacillus subtilis with half maximal inhibitory concentration (IC50 ) of 10 μM which is comparable to that of natural channel-forming peptide alamethicin. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Voltage-clamp analysis of membrane currents and excitation-contraction coupling in a crustacean muscle.

PubMed

Weiss, T; Erxleben, C; Rathmayer, W

2001-01-01

A single fibre preparation from the extensor muscle of a marine isopod crustacean is described which allows the analysis of membrane currents and simultaneously recorded contractions under two-electrode voltage-clamp conditions. We show that there are three main depolarisation-gated currents, two are outward and carried by K+, the third is an inward Ca2+ current, I(Ca). Normally, the K+ currents which can be isolated by using K+ channel blockers, mask I(Ca). I(Ca) activates at potentials more positive than -40 mV, is maximal around 0 mV, and shows strong inactivation at higher depolarisation. Inactivation depends on current rather than voltage. Ba2+, Sr2+ and Mg2+ can substitute for Ca2+. Ba2+ currents are about 80% larger than Ca2+ currents and inactivate little. The properties of I(Ca) characterise it as a high threshold L-type current. The outward current consists primarily of a fast, transient A current, I(K(A)) and a maintained, delayed rectifier current, I(K(V)). In some fibres, a small Ca2+-dependent K+ current is also present. I(K(A)) activates fast at depolarisation above -45 mV, shows pronounced inactivation and is almost completely inactivated at holding potentials more positive than -40 mV. I(K(A)) is half-maximally blocked by 70 microM 4-aminopyridine (4-AP), and 70 mM tetraethylammonium (TEA). I(K(V)) activates more slowly, at about -30 mV, and shows no inactivation. It is half-maximally blocked by 2 mM TEA but rather insensitive to 4-AP. Physiologically, the two K+ currents prevent all-or-nothing action potentials and determine the graded amplitude of active electrical responses and associated contractions. Tension development depends on and is correlated with depolarisation-induced Ca2+ influx mediated by I(Ca). The voltage dependence of peak tension corresponds directly to the voltage dependence of the integrated I(Ca). The threshold potential for contraction is at about -38 mV. Peak tension increases with increasing voltage steps, reaches maximum at around 0 mV, and declines with further depolarisation.

Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study

PubMed Central

Magistretti, Jacopo; Castelli, Loretta; Forti, Lia; D'Angelo, Egidio

2006-01-01

Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage-dependent Na+ currents. In this study we analysed voltage-dependent Na+ currents of rat cerebellar granule cells (GCs) by performing whole-cell, patch-clamp experiments in acute rat cerebellar slices. A transient Na+ current (INaT) was always present and had the properties of a typical fast-activating/inactivating Na+ current. In addition to INaT, robust persistent (INaP) and resurgent (INaR) Na+ currents were observed. INaP peaked at ∼−40 mV, showed half-maximal activation at ∼−55 mV, and its maximal amplitude was about 1.5% of that of INaT. INaR was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate INaT, and showed voltage- and time-dependent activation and voltage-dependent decay kinetics. The conductance underlying INaR showed a bell-shaped voltage dependence, with peak at −35 mV. A significant correlation was found between GC INaR and INaT peak amplitudes; however, GCs expressing INaT of similar size showed marked variability in terms of INaR amplitude, and in a fraction of cells INaR was undetectable. INaT, INaP and INaR could be accounted for by a 13-state kinetic scheme comprising closed, open, inactivated and blocked states. Current-clamp experiments carried out to identify possible functional correlates of INaP and/or INaR revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with INaR playing a role in their generation. Computer modelling showed that INaR promotes DAP generation and enhances high-frequency firing, whereas INaP boosts near-threshold firing activity. Our findings suggest that special properties of voltage-dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially important consequences for cerebellar information transfer and computation. PMID:16527854

Modulation of voltage-gated Na+ and K+ channels by pumiliotoxin 251D: a "joint venture" alkaloid from arthropods and amphibians.

PubMed

Vandendriessche, Thomas; Abdel-Mottaleb, Yousra; Maertens, Chantal; Cuypers, Eva; Sudau, Alexander; Nubbemeyer, Udo; Mebs, Dietrich; Tytgat, Jan

2008-03-01

Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown. In this work, we searched for the possible target of PTX 251D. The toxin was therefore made synthetically and tested on four VGSCs (mammalian rNa(v)1.2/beta(1), rNa(v)1.4/beta(1), hNa(v)1.5/beta(1) and insect Para/tipE) and five voltage-gated potassium channels (VGPCs) (mammalian rK(v)1.1-1.2, hK(v)1.3, hK(v)11.1 (hERG) and insect Shaker IR) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage clamp technique. PTX 251D not only inhibited the Na(+) influx through the mammalian VGSCs but also affected the steady-state activation and inactivation. Interestingly, in the insect ortholog, the inactivation process was dramatically affected. Additionally, PTX 251D inhibited the K(+) efflux through all five tested VGPCs and slowed down the deactivation kinetics of the mammalian VGPCs. hK(v)1.3 was the most sensitive channel, with an IC(50) value 10.8+/-0.5 microM. To the best of our knowledge this is the first report of a PTX affecting VGPCs.

OmpF, a nucleotide-sensing nanoprobe, computational evaluation of single channel activities

NASA Astrophysics Data System (ADS)

Abdolvahab, R. H.; Mobasheri, H.; Nikouee, A.; Ejtehadi, M. R.

2016-09-01

The results of highthroughput practical single channel experiments should be formulated and validated by signal analysis approaches to increase the recognition precision of translocating molecules. For this purpose, the activities of the single nano-pore forming protein, OmpF, in the presence of nucleotides were recorded in real time by the voltage clamp technique and used as a means for nucleotide recognition. The results were analyzed based on the permutation entropy of current Time Series (TS), fractality, autocorrelation, structure function, spectral density, and peak fraction to recognize each nucleotide, based on its signature effect on the conductance, gating frequency and voltage sensitivity of channel at different concentrations and membrane potentials. The amplitude and frequency of ion current fluctuation increased in the presence of Adenine more than Cytosine and Thymine in milli-molar (0.5 mM) concentrations. The variance of the current TS at various applied voltages showed a non-monotonic trend whose initial increasing slope in the presence of Thymine changed to a decreasing one in the second phase and was different from that of Adenine and Cytosine; e.g., by increasing the voltage from 40 to 140 mV in the 0.5 mM concentration of Adenine or Cytosine, the variance decreased by one third while for the case of Thymine it was doubled. Moreover, according to the structure function of TS, the fractality of current TS differed as a function of varying membrane potentials (pd) and nucleotide concentrations. Accordingly, the calculated permutation entropy of the TS, validated the biophysical approach defined for the recognition of different nucleotides at various concentrations, pd's and polarities. Thus, the promising outcomes of the combined experimental and theoretical methodologies presented here can be implemented as a complementary means in pore-based nucleotide recognition approaches.

Eslicarbazepine and the enhancement of slow inactivation of voltage-gated sodium channels: a comparison with carbamazepine, oxcarbazepine and lacosamide.

PubMed

Hebeisen, Simon; Pires, Nuno; Loureiro, Ana I; Bonifácio, Maria João; Palma, Nuno; Whyment, Andrew; Spanswick, David; Soares-da-Silva, Patrício

2015-02-01

This study aimed at evaluating the effects of eslicarbazepine, carbamazepine (CBZ), oxcarbazepine (OXC) and lacosamide (LCM) on the fast and slow inactivated states of voltage-gated sodium channels (VGSC). The anti-epileptiform activity was evaluated in mouse isolated hippocampal slices. The anticonvulsant effects were evaluated in MES and the 6-Hz psychomotor tests. The whole-cell patch-clamp technique was used to investigate the effects of eslicarbazepine, CBZ, OXC and LCM on sodium channels endogenously expressed in N1E-115 mouse neuroblastoma cells. CBZ and eslicarbazepine exhibit similar concentration dependent suppression of epileptiform activity in hippocampal slices. In N1E-115 mouse neuroblastoma cells, at a concentration of 250 μM, the voltage dependence of the fast inactivation was not influenced by eslicarbazepine, whereas LCM, CBZ and OXC shifted the V0.5 value (mV) by -4.8, -12.0 and -16.6, respectively. Eslicarbazepine- and LCM-treated fast-inactivated channels recovered similarly to control conditions, whereas CBZ- and OXC-treated channels required longer pulses to recover. CBZ, eslicarbazepine and LCM shifted the voltage dependence of the slow inactivation (V0.5, mV) by -4.6, -31.2 and -53.3, respectively. For eslicarbazepine, LCM, CBZ and OXC, the affinity to the slow inactivated state was 5.9, 10.4, 1.7 and 1.8 times higher than to the channels in the resting state, respectively. In conclusion, eslicarbazepine did not share with CBZ and OXC the ability to alter fast inactivation of VGSC. Both eslicarbazepine and LCM reduce VGSC availability through enhancement of slow inactivation, but LCM demonstrated higher interaction with VGSC in the resting state and with fast inactivation gating. Copyright © 2014 Elsevier Ltd. All rights reserved.

mRNAs coding for neurotransmitter receptors and voltage-gated sodium channels in the adult rabbit visual cortex after monocular deafferentiation

PubMed Central

Nguyen, Quoc-Thang; Matute, Carlos; Miledi, Ricardo

1998-01-01

It has been postulated that, in the adult visual cortex, visual inputs modulate levels of mRNAs coding for neurotransmitter receptors in an activity-dependent manner. To investigate this possibility, we performed a monocular enucleation in adult rabbits and, 15 days later, collected their left and right visual cortices. Levels of mRNAs coding for voltage-activated sodium channels, and for receptors for kainate/α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-d-aspartate (NMDA), γ-aminobutyric acid (GABA), and glycine were semiquantitatively estimated in the visual cortices ipsilateral and contralateral to the lesion by the Xenopus oocyte/voltage-clamp expression system. This technique also allowed us to study some of the pharmacological and physiological properties of the channels and receptors expressed in the oocytes. In cells injected with mRNA from left or right cortices of monocularly enucleated and control animals, the amplitudes of currents elicited by kainate or AMPA, which reflect the abundance of mRNAs coding for kainate and AMPA receptors, were similar. There was no difference in the sensitivity to kainate and in the voltage dependence of the kainate response. Responses mediated by NMDA, GABA, and glycine were unaffected by monocular enucleation. Sodium channel peak currents, activation, steady-state inactivation, and sensitivity to tetrodotoxin also remained unchanged after the enucleation. Our data show that mRNAs for major neurotransmitter receptors and ion channels in the adult rabbit visual cortex are not obviously modified by monocular deafferentiation. Thus, our results do not support the idea of a widespread dynamic modulation of mRNAs coding for receptors and ion channels by visual activity in the rabbit visual system. PMID:9501250

Comparison of detection limit in fiber-based conventional, amplified, and gain-clamped cavity ring-down techniques

NASA Astrophysics Data System (ADS)

Sharma, K.; Abdul Khudus, M. I. M.; Alam, S. U.; Bhattacharya, S.; Venkitesh, D.; Brambilla, G.

2018-01-01

Relative performance and detection limit of conventional, amplified, and gain-clamped cavity ring-down techniques (CRDT) in all-fiber configurations are compared experimentally for the first time. Refractive index measurement using evanescent field in tapered fibers is used as a benchmark for the comparison. The systematic optimization of a nested-loop configuration in gain-clamped CRDT is also discussed, which is crucial for achieving a constant gain in a CRDT experiment. It is found that even though conventional CRDT has the lowest standard error in ring-down time (Δτ), the value of ring-down time (τ) is very small, thus leading to poor detection limit. Amplified CRDT provides an improvement in τ, albeit with two orders of magnitude higher Δτ due to amplifier noise. The nested-loop configuration in gain-clamped CRDT helps in reducing Δτ by an order of magnitude as compared to amplified CRDT whilst retaining the improvement in τ. A detection limit of 1 . 03 × 10-4 RIU at refractive index of 1.322 with a 3 mm long and 4.5 μm diameter tapered fiber is demonstrated with the gain-clamped CRDT.

Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

PubMed Central

Yue, Jin-feng; Qiao, Guan-hua; Liu, Ni; Nan, Fa-jun; Gao, Zhao-bing

2016-01-01

Aim: To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators. Methods: KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique. Results: From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively. Conclusion: The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators. PMID:26725738

Involvement of mitochondrial Na+–Ca2+ exchange in intestinal pacemaking activity

PubMed Central

Kim, Byung Joo; Jun, Jae Yeoul; So, Insuk; Kim, Ki Whan

2006-01-01

AIM: Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. We have aimed to investigate the involvement of mitochondrial Na+-Ca2+ exchange in intestinal pacemaking activity in cultured interstitial cells of Cajal. METHODS: Enzymatic digestions were used to dissociate ICCs from the small intestine of a mouse. The whole-cell patch-clamp configuration was used to record membrane currents (voltage clamp) and potentials (current clamp) from cultured ICCs. RESULTS: Clonazepam and CGP37157 inhibited the pacemaking activity of ICCs in a dose-dependent manner. Clonazepam from 20 to 60 µmol/L and CGP37157 from 10 to 30 µmol/L effectively inhibited Ca2+ efflux from mitochondria in pacemaking activity of ICCs. The IC50s of clonazepam and CGP37157 were 37.1 and 18.2 µmol/L, respectively. The addition of 20 µmol/L NiCl2 to the internal solution caused a “wax and wane” phenomenon of pacemaking activity of ICCs. CONCLUSION: These results suggest that mitochondrial Na+-Ca2+ exchange has an important role in intestinal pacemaking activity. PMID:16521198

Rapid Changes of Potassium Concentration at the Outer Surface of Exposed Single Neurons during Membrane Current Flow

PubMed Central

Neher, E.; Lux, H. D.

1973-01-01

K+-sensitive liquid ion-exchanger microelectrodes are shown to be capable of measuring concentration changes which occur on a millisecond time scale. However, some quaternary ammonium ions, such as tetraethylammonium and acetylcholine, are able to block electrode function when present in concentrations as low as 10-4 to 10-3 M. Changes in extracellular potassium concentration caused by spike activity or voltage clamp pulses of exposed single neurons of the snail Helix pomatia may be measured by these electrodes. Quantitative analysis shows that the total amount of excess potassium found in the vicinity of the cell a short time after a clamp pulse, is in relatively good agreement with the amount of potassium carried by the membrane current. PMID:4689624

Progress Toward a Bulk Micromachined Tunneling Tip Microaccelerometer

NASA Technical Reports Server (NTRS)

Frank T. Hartley, Ben Dolgen, Paul M. Zavracky

1995-01-01

Ultrasensitive accelerometers are needed for microgravity measurement of orbital drag and active isolation systems. We have designed an accelerometer capable of measuring accelerations of the order of 10(i) g. A tunneling tip sensor can be used as a position sensor with a potential performance advantage of two orders of magnitude over capacitive sensors. In this paper, we disclose our progress in the fabrication and measurement of a bulk microaccelerometer which employs a tunneling tip. Fully assembled accelerometers consisting of four separate die have been fabricated. The device employs a unique folded spring system with a low spring constant. To protect the tunneling tip, we have employed electrostatic clamping. Stiction has not been observed, but the required clamping voltage is greater than expected. We have developed a simple model to analyze our results.

Octopamine increases the excitability of neurons in the snail feeding system by modulation of inward sodium current but not outward potassium currents.

PubMed

Vehovszky, Agnes; Szabó, Henriette; Elliott, Christopher J H

2005-12-06

Although octopamine has long been known to have major roles as both transmitter and modulator in arthropods, it has only recently been shown to be functionally important in molluscs, playing a role as a neurotransmitter in the feeding network of the snail Lymnaea stagnalis. The synaptic potentials cannot explain all the effects of octopamine-containing neurons on the feeding network, and here we test the hypothesis that octopamine is also a neuromodulator. The excitability of the B1 and B4 motoneurons in the buccal ganglia to depolarising current clamp pulses is significantly (P < < 0.05) increased by (10 microM) octopamine, whereas the B2 motoneuron becomes significantly less excitable. The ionic currents evoked by voltage steps were recorded using 2-electrode voltage clamp. The outward current of B1, B2 and B4 motoneurons had two components, a transient IA current and a sustained IK delayed-rectifier current, but neither was modulated by octopamine in any of these three buccal neurons. The fast inward current was eliminated in sodium-free saline and so is likely to be carried by sodium ions. 10 microM octopamine enhanced this current by 33 and 45% in the B1 and B4 motoneurons respectively (P < < 0.05), but a small reduction was seen in the B2 neuron. A Hodgkin-Huxley style simulation of the B1 motoneuron confirms that a 33% increase in the fast inward current by octopamine increases the excitability markedly. We conclude that octopamine is also a neuromodulator in snails, changing the excitability of the buccal neurons. This is supported by the close relationship from the voltage clamp data, through the quantitative simulation, to the action potential threshold, changing the properties of neurons in a rhythmic network. The increase in inward sodium current provides an explanation for the polycyclic modulation of the feeding system by the octopamine-containing interneurons, making feeding easier to initiate and making the feeding bursts more intense.

Quantitative analysis of the Ca2+ -dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes.

PubMed

Bartos, Daniel C; Morotti, Stefano; Ginsburg, Kenneth S; Grandi, Eleonora; Bers, Donald M

2017-04-01

[Ca 2+ ] i enhanced rabbit ventricular slowly activating delayed rectifier K + current (I Ks ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K + current (I Kr ) amplitude and voltage dependence were unaffected by high [Ca 2+ ] i . When measuring or simulating I Ks during an action potential, I Ks was not different during a physiological Ca 2+ transient or when [Ca 2+ ] i was buffered to 500 nm. The slowly activating delayed rectifier K + current (I Ks ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca 2+ ([Ca 2+ ] i ) and β-adrenergic receptor (β-AR) stimulation modulate I Ks amplitude and kinetics, but details of these important I Ks regulators and their interaction are limited. We assessed the [Ca 2+ ] i dependence of I Ks in steady-state conditions and with dynamically changing membrane potential and [Ca 2+ ] i during an AP. I Ks was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca 2+ ] i , we found that raising [Ca 2+ ] i from 100 to 600 nm produced similar increases in I Ks as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca 2+ ] i increased maximally activated tail I Ks , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, I Ks recorded during a normal physiological Ca 2+ transient was similar to I Ks measured with [Ca 2+ ] i clamped at 500-600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca 2+ ] i regulates I Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca 2+ ] i , in the submembrane or junctional cleft space, is not required to maximize [Ca 2+ ] i -dependent I Ks activation during normal Ca 2+ transients. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Quantitative analysis of the Ca2+‐dependent regulation of delayed rectifier K+ current I Ks in rabbit ventricular myocytes

PubMed Central

Bartos, Daniel C.; Morotti, Stefano; Ginsburg, Kenneth S.; Grandi, Eleonora

2017-01-01

Key points [Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (I Ks) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol.Rabbit ventricular rapidly activating delayed rectifier K+ current (I Kr) amplitude and voltage dependence were unaffected by high [Ca2+]i.When measuring or simulating I Ks during an action potential, I Ks was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm. Abstract The slowly activating delayed rectifier K+ current (I Ks) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) and β‐adrenergic receptor (β‐AR) stimulation modulate I Ks amplitude and kinetics, but details of these important I Ks regulators and their interaction are limited. We assessed the [Ca2+]i dependence of I Ks in steady‐state conditions and with dynamically changing membrane potential and [Ca2+]i during an AP. I Ks was recorded from freshly isolated rabbit ventricular myocytes using whole‐cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+]i, we found that raising [Ca2+]i from 100 to 600 nm produced similar increases in I Ks as did β‐AR activation, and the effects appeared additive. Both β‐AR activation and high [Ca2+]i increased maximally activated tail I Ks, negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well‐established mathematical model of the rabbit myocyte. In both AP‐clamp experiments and simulations, I Ks recorded during a normal physiological Ca2+ transient was similar to I Ks measured with [Ca2+]i clamped at 500–600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+]i regulates I Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+]i, in the submembrane or junctional cleft space, is not required to maximize [Ca2+]i‐dependent I Ks activation during normal Ca2+ transients. PMID:28008618

Control of Gastric H,K-ATPase Activity by Cations, Voltage and Intracellular pH Analyzed by Voltage Clamp Fluorometry in Xenopus Oocytes

PubMed Central

Dürr, Katharina L.; Tavraz, Neslihan N.; Friedrich, Thomas

2012-01-01

Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E1P and E2P states and measured Rb+ uptake under various ionic and pH conditions. The steady-state E1P/E2P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb+ uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E1P/E2P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V0.5, the voltage, at which the E1P/E2P ratio is 50∶50, by −100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E1P→E2P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb+ uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E1P→E2P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na+ profoundly alters the voltage-dependent E1P/E2P distribution indicating that Na+ ions can act as surrogates for protons regarding the E2P→E1P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ∼0.5 and ∼0.2, respectively. PMID:22448261

Depolarized inactivation overcomes impaired activation to produce DRG neuron hyperexcitability in a Nav1.7 mutation in a patient with distal limb pain.

PubMed

Huang, Jianying; Yang, Yang; Dib-Hajj, Sulayman D; van Es, Michael; Zhao, Peng; Salomon, Jody; Drenth, Joost P H; Waxman, Stephen G

2014-09-10

Sodium channel Nav1.7, encoded by SCN9A, is expressed in DRG neurons and regulates their excitability. Genetic and functional studies have established a critical contribution of Nav1.7 to human pain disorders. We have now characterized a novel Nav1.7 mutation (R1279P) from a female human subject with distal limb pain, in which depolarized fast inactivation overrides impaired activation to produce hyperexcitability and spontaneous firing in DRG neurons. Whole-cell voltage-clamp recordings in human embryonic kidney (HEK) 293 cells demonstrated that R1279P significantly depolarizes steady-state fast-, slow-, and closed-state inactivation. It accelerates deactivation, decelerates inactivation, and facilitates repriming. The mutation increases ramp currents in response to slow depolarizations. Our voltage-clamp analysis showed that R1279P depolarizes channel activation, a change that was supported by our multistate structural modeling. Because this mutation confers both gain-of-function and loss-of-function attributes on the Nav1.7 channel, we tested the impact of R1279P expression on DRG neuron excitability. Current-clamp studies reveal that R1279P depolarizes resting membrane potential, decreases current threshold, and increases firing frequency of evoked action potentials within small DRG neurons. The populations of spontaneously firing and repetitively firing neurons were increased by expressing R1279P. These observations indicate that the dominant proexcitatory gating changes associated with this mutation, including depolarized steady-state fast-, slow-, and closed-state inactivation, faster repriming, and larger ramp currents, override the depolarizing shift of activation, to produce hyperexcitability and spontaneous firing of nociceptive neurons that underlie pain. Copyright © 2014 the authors 0270-6474/14/3412328-13$15.00/0.

Effects of pine needle extract on pacemaker currents in interstitial cells of Cajal from the murine small intestine.

PubMed

Cheong, Hyeonsook; Paudyal, Dilli Parasad; Jun, Jae Yeoul; Yeum, Cheol Ho; Yoon, Pyung Jin; Park, Chan Guk; Kim, Man Yoo; So, Insuk; Kim, Ki Whan; Choi, Seok

2005-10-31

Extracts of pine needles (Pinus densiflora Sieb. et Zucc.) have diverse physiological and pharmacological actions. In this study we show that pine needle extract alters pacemaker currents in interstitial cells of Cajal (ICC) by modulating ATP-sensitive K+ channels and that this effect is mediated by prostaglandins. In whole cell patches at 30 degrees , ICC generated spontaneous pacemaker potentials in the current clamp mode (I = 0), and inward currents (pacemaker currents) in the voltage clamp mode at a holding potential of -70 mV. Pine needle extract hyperpolarized the membrane potential, and in voltage clamp mode decreased both the frequency and amplitude of the pacemaker currents, and increased the resting currents in the outward direction. It also inhibited the pacemaker currents in a dose-dependent manner. Because the effects of pine needle extract on pacemaker currents were the same as those of pinacidil (an ATP-sensitive K+ channel opener) we tested the effect of glibenclamide (an ATP-sensitive K+ channels blocker) on ICC exposed to pine needle extract. The effects of pine needle extract on pacemaker currents were blocked by glibenclamide. To see whether production of prostaglandins (PGs) is involved in the inhibitory effect of pine needle extract on pacemaker currents, we tested the effects of naproxen, a non-selective cyclooxygenase (COX-1 and COX-2) inhibitor, and AH6809, a prostaglandin EP1 and EP2 receptor antagonist. Naproxen and AH6809 blocked the inhibitory effects of pine needle extract on ICC. These results indicate that pine needle extract inhibits the pacemaker currents of ICC by activating ATP-sensitive K+ channels via the production of PGs.

Direct block of native and cloned (Kir2.1) inward rectifier K+ channels by chloroethylclonidine

PubMed Central

Barrett-Jolley, R; Dart, C; Standen, N B

1999-01-01

We have investigated the inhibition of inwardly rectifying potassium channels by the α-adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two-electrode voltage-clamp of rat isolated flexor digitorum brevis muscle and whole-cell patch-clamp of cell lines transfected with Kir2.1 (IRK1).In skeletal muscle and at a membrane potential of −50 mV, chloroethylclonidine (CEC), an agonist at α2-adrenergic receptors and an antagonist at α1x-receptors, was found to inhibit the inward rectifier current with a Ki of 30 μM.The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of α1-(prazosin) or α2-(rauwolscine) antagonists.The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at −50 mV c.f. ∼10% block at −190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be ∼8 s at 0 mV, and the rate of unblock was described by the relationship τ=exp((Vm+149)/22) s.This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine.The data suggest that the clonidine-like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of α-receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site. PMID:10516659

Modulation of voltage-gated conductances of retinal horizontal cells by UV-excited TiO2 nanoparticles.

PubMed

Meshik, Xenia; Choi, Min; Baker, Adam; Malchow, R Paul; Covnot, Leigha; Doan, Samuel; Mukherjee, Souvik; Farid, Sidra; Dutta, Mitra; Stroscio, Michael A

2017-04-01

This study examines the ability of optically-excited titanium dioxide nanoparticles to influence voltage-gated ion channels in retinal horizontal cells. Voltage clamp recordings were obtained in the presence and absence of TiO 2 and ultraviolet laser excitation. Significant current changes were observed in response to UV light, particularly in the -40 mV to +40 mV region where voltage-gated Na + and K + channels have the highest conductance. Cells in proximity to UV-excited TiO 2 exhibited a left-shift in the current-voltage relation of around 10 mV in the activation of Na + currents. These trends were not observed in control experiments where cells were excited with UV light without being exposed to TiO 2 . Electrostatic force microscopy confirmed that electric fields can be induced in TiO 2 with UV light. Simulations using the Hodgkin-Huxley model yielded results which agreed with the experimental data and showed the I-V characteristics of individual ion channels in the presence of UV-excited TiO 2 . Copyright © 2016 Elsevier Inc. All rights reserved.

Travelling wave ultrasonic motor using the B08 flexural mode of a circular membrane.

PubMed

Rayner, P J; Whatmore, R W

2001-05-01

This paper describes the design, construction, and performance of a piezoelectric motor that uses the travelling B08 mode of an 80-mm diameter circular membrane to drive a rotor by frictional contact. The motor is of a thin planar design, giving high torque of up to 0.33 Nm at low speed and has been developed as a design that can be made with lithographic techniques for miniaturization. Investigations of the free stator with a vibration pattern imager and impedance analyzer gave the resonance frequency, mode, and electromechanical coupling of the stator. Motor speed as a function of frequency for a constant voltage and performance charts of speed, output power, and efficiency against torque are presented for a particular input voltage and rotor pre-load. The effects of two different lead zirconate titanate (PZT) ring dimensions have been investigated. Excitation of the B09 mode has been observed, incommensurate with the piezoelectric excitation of the stator. This is discussed with relation to edge-clamping of the stator. Single standing wave motoring was observed, believed to arise from asymmetry of the stator and its perturbation of the B08 resonance mode. Sources of power loss, including frame vibration and friction interface slip, are considered and discussed.

A novel measuring method of clamping force for electrostatic chuck in semiconductor devices

NASA Astrophysics Data System (ADS)

Kesheng, Wang; Jia, Cheng; Yin, Zhong; Linhong, Ji

2016-04-01

Electrostatic chucks are one of the core components of semiconductor devices. As a key index of electrostatic chucks, the clamping force must be controlled within a reasonable range. Therefore, it is essential to accurately measure the clamping force. To reduce the negative factors influencing measurement precision and repeatability, this article presents a novel method to measure the clamping force and we elaborate both the principle and the key procedure. A micro-force probe component is introduced to monitor, adjust, and eliminate the gap between the wafer and the electrostatic chuck. The contact force between the ruby probe and the wafer is selected as an important parameter to characterize de-chucking, and we have found that the moment of de-chucking can be exactly judged. Moreover, this article derives the formula calibrating equivalent action area of backside gas pressure under real working conditions, which can effectively connect the backside gas pressure at the moment of de-chucking and the clamping force. The experiments were then performed on a self-designed measuring platform. The de-chucking mechanism is discussed in light of our analysis of the experimental data. Determination criteria for de-chucking point are summed up. It is found that the relationship between de-chucking pressure and applied voltage conforms well to quadratic equation. Meanwhile, the result reveals that actual de-chucking behavior is much more complicated than the description given in the classical empirical formula. Project supported by No. 02 National Science and Technology Major Project of China (No. 2011ZX02403-004).

Voltage Gating of Shaker K+ Channels

PubMed Central

Rodríguez, Beatriz M.; Sigg, Daniel; Bezanilla, Francisco

1998-01-01

Ionic (Ii) and gating currents (Ig) from noninactivating Shaker H4 K+ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2–22°C. The time course of Ii elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main Ii component is highly temperature dependent (Q10 > 4) and mildly voltage dependent, having a valence times the fraction of electric field (z) of 0.2–0.3 eo. The Ig On response obtained between −60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main Ig component of decay is highly temperature dependent (Q10 > 4) and has a z between 1.6 and 2.8 eo in the voltage range from −60 to −10 mV, and ∼0.45 eo at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at −50 mV reactivates the gating charge with a high temperature dependence (Q10 > 4). In contrast, the reactivation occurring between −90 and −50 mV has a Q10 = 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8°C and the unitary conductance has a low temperature dependence with a Q10 of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change. PMID:9689029

μ-Opioid receptor activation inhibits N- and P-type Ca2+ channel currents in magnocellular neurones of the rat supraoptic nucleus

PubMed Central

Soldo, Brandi L; Moises, Hylan C

1998-01-01

The whole-cell voltage-clamp technique was used to examine opioid regulation of Ba2+ currents (IBa) through voltage-sensitive Ca2+ channels in isolated magnocellular supraoptic neurones (MNCs). The effects of local application of μ-, δ- or κ-opioid receptor selective agonists were examined on specific components of high voltage-activated (HVA) IBa, pharmacologically isolated by use of Ca2+ channel-subtype selective antagonists. The μ-opioid receptor selective agonist, DAMGO, suppressed HVA IBa (in 64/71 neurones) in a naloxone-reversible and concentration-dependent manner (EC50 = 170 nm, Emax = 19.5 %). The DAMGO-induced inhibition was rapid in onset, associated with kinetic slowing and voltage dependent, being reversed by strong depolarizing prepulses. Low-voltage activated (LVA) IBa was not modulated by DAMGO. Administration of κ- (U69 593) or δ-selective (DPDPE) opioid receptor agonists did not affect IBa. However, immunostaining of permeabilized MNCs with an antibody specific for κ1-opioid receptors revealed the presence of this opioid receptor subtype in a large number of isolated somata. μ-Opioid-induced inhibition in IBa was largely abolished after blockade of N-type and P-type channel currents by ω-conotoxin GVIA (1 μm) and ω-agatoxin IVA (100 nm), respectively. Quantitation of antagonist effects on DAMGO-induced reductions in IBa revealed that N- and P-type channels contributed roughly equally to the μ-opioid sensitive portion of total IBa. These results indicate that μ-opioid receptors are negatively coupled to N- and P-type Ca2+ channels in the somatodendritic regions of MNCs, possibly via a membrane-delimited G-protein-dependent pathway. They also support a scheme in which opioids may act in part to modulate cellular activity and regulate neurosecretory function by their direct action on the neuroendocrine neurones of the hypothalamic supraoptic neucleus. PMID:9824718

Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: A natural protocol and its use for patch clamp experiments

PubMed Central

Leng, San-Hua; Lu, Fu-Er

2005-01-01

AIM: To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS: The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine cells with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels (KATP), voltage-dependent potassium channels (KV), and voltage-dependent calcium channels (KCA) in β-cells were identified by patch clamp technique. RESULTS: After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly (MTT value from 0.024±0.003 to 0.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L). Then MTT value and insulin secretion increased quickly from d 5 to d 10 (MTT value from 0.028±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L), then reached high plateau (MTT value >0.052±0.008, insulin secretion >18.3±2.6 mIU/L). In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d (MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L), but afterwards they reduced gradually (MTT value <0.031±0.011, insulin secretion <8.2±1.5 mIU/L), and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, KV, and KCA. CONCLUSION: Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells. PMID:16437601

Push-pull with recovery stage high-voltage DC converter for PV solar generator

NASA Astrophysics Data System (ADS)

Nguyen, The Vinh; Aillerie, Michel; Petit, Pierre; Pham, Hong Thang; Vo, Thành Vinh

2017-02-01

A lot of systems are basically developed on DC-DC or DC-AC converters including electronic switches such as MOS or bipolar transistors. The limits of efficiency are quickly reached when high output voltages and high input currents are needed. This work presents a new high-efficiency-high-step-up based on push-pull DC-DC converter integrating recovery stages dedicated to smart HVDC distributed architecture in PV solar energy production systems. Appropriate duty cycle ratio assumes that the recovery stage work with parallel charge and discharge to achieve high step-up voltage gain. Besides, the voltage stress on the main switch is reduced with a passive clamp circuit and thus, low on-state resistance Rdson of the main switch can be adopted to reduce conduction losses. Thus, the efficiency of a basic DC-HVDC converter dedicated to renewable energy production can be further improved with such topology. A prototype converter is developed, and experimentally tested for validation.

Analysis of a modular generator for high-voltage, high-frequency pulsed applications, using low voltage semiconductors (< 1 kV) and series connected step-up (1:10) transformers.

PubMed

Redondo, L M; Fernando Silva, J; Margato, E

2007-03-01

This article discusses the operation of a modular generator topology, which has been developed for high-frequency (kHz), high-voltage (kV) pulsed applications. The proposed generator uses individual modules, each one consisting of a pulse circuit based on a modified forward converter, which takes advantage of the required low duty cycle to operate with a low voltage clamp reset circuit for the step-up transformer. This reduces the maximum voltage on the semiconductor devices of both primary and secondary transformer sides. The secondary winding of each step-up transformer is series connected, delivering a fraction of the total voltage. Each individual pulsed module is supplied via an isolation transformer. The assembled modular laboratorial prototype, with three 5 kV modules, 800 V semiconductor switches, and 1:10 step-up transformers, has 80% efficiency, and is capable of delivering, into resistive loads, -15 kV1 A pulses with 5 micros width, 10 kHz repetition rate, with less than 1 micros pulse rise time. Experimental results for resistive loads are presented and discussed.

Comparison of clamping technique in robotic partial nephrectomy: does unclamped partial nephrectomy improve perioperative outcomes and renal function?

PubMed

Krane, L Spencer; Mufarrij, Patrick W; Manny, Theodore B; Hemal, Ashok K

2013-02-01

Partial nephrectomy without renal vascular occlusion has been introduced to improve outcomes in patients undergoing robotic partial nephrectomy (RPN). We prospectively evaluated unclamped RPN at our institution and compared this to other clamping techniques in a non-randomized fashion. Ninety-five consecutive patients who successfully completed RPN between June 2010 and October 2011 are included in this analysis. All RPNs were performed by a single surgeon. Clamping technique was artery and vein (AV), artery alone (AO) or unclamped (U) without hypotensive anesthesia. Clamping decision was based on surgeon preference and feasibility of minimizing ischemia. All patients had bilateral functional renal units. Eighteen (19%), 58 (61%) and 19 (20%) patients had AV, AO and U technique respectively. Preoperative characteristics including age (p = 0.43), body mass index (p = 0.40) and RENAL nephromety distribution (p = 0.10) were similar. In AV and AO, mean warm ischemia time were 19 and 17 minutes and similar between the two cohorts (p = 0.39). Mean glomerular filtration rate (GFR) and overall percentage decrease in GFR at time of at last follow up were (64, 69, 81, p = 0.12) and (6%, 6%,and 2%,p = 0.79) for AV, AO and U respectively. Median follow up for last serum creatinine was 113 days and was similar between all cohorts (p = 0.37). Complication rate (p = 0.37), positive margin rate (p = 0.84), and change in hemoglobin concentration postoperatively (p = 0.94) were similar between cohorts. Unclamped partial nephrectomy is possible in patients undergoing RPN. In this study, it does not significantly alter perioperative or postoperative renal function or change rate of complications. Minimal ischemia, irrespective of clamping technique, in patients with bilateral renal units does not appear to adversely effect intermediate term renal function in these patients.

Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres.

PubMed

Rannou, F; Droguet, M; Giroux-Metges, M A; Pennec, Y; Gioux, M; Pennec, J P

2009-11-01

The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocity of muscle fibres. The functional properties of the muscle fibre are also conditioned by its membrane excitability through the electrophysiological properties of sodium voltage-gated channels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus (PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibres are identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slow type I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared (Student's t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms are identified in PL and only type I in Sol single fibres. In PL, maximal sodium current (I(max)), maximal sodium conductance (g(Na,max)) and time constants of activation and inactivation ((m) and (h)) increase according to the scheme I-->IIa-->IIx-->IIb (P < 0.05). (m) values related to sodium channel type and/or function, are similar in Sol I and PL IIb fibres (P = 0.97) despite different contractile properties. The voltage dependence of activation (V(a,1/2)) shows a shift towards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL (P < 0.05). These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle like PL, while slow fibres of postural muscle such as soleus could be recruited in the same ways as IIb fibres in a fast muscle.

Differential effects of tetracaine on two kinetic components of calcium release in frog skeletal muscle fibres.

PubMed Central

Pizarro, G; Csernoch, L; Uribe, I; Ríos, E

1992-01-01

1. Intramembrane charge movements and changes in intracellular calcium concentration were recorded simultaneously in voltage clamped cut skeletal muscle fibres of the frog in the presence and absence of tetracaine. 2. Extracellular application of 20 microM tetracaine reduced the increase in myoplasmic [Ca2+]. The effect on the underlying calcium release flux from the sarcoplasmic reticulum was to suppress the peak of the release while sparing the steady level attained at the end of 100 ms clamp depolarizations. 3. While the peak of the release flux at corresponding voltages was reduced by 62% after the addition of tetracaine, the rate of inactivation was the same when the pulses elicited release fluxes of similar amplitude. 4. Higher concentrations of tetracaine, 0.2 mM, abolished the calcium signal in stretched fibres whereas in slack fibres this concentration left a non-inactivating calcium release flux. 5. Lowering the extracellular pH antagonized the effect of the drug both on charge movements and on calcium signals. The permanently charged analogue tetracaine methobromide lacked effects on excitation-contraction coupling. 6. These results imply that the two kinetic components of calcium release flux have very different tetracaine sensitivities. They are also consistent with an intracellular site of action of the drug at low concentration. Taken together they strongly suggest that the inactivating and non-inactivating components of calcium release correspond to different pathways: one that inactivates, is sensitive to tetracaine and is controlled by calcium, and another that does not inactivate, is much less sensitive to tetracaine and is directly controlled by voltage. PMID:1297844

An In Vivo Study of Low-Dose Intra-Articular Tranexamic Acid Application with Prolonged Clamping Drain Method in Total Knee Replacement: Clinical Efficacy and Safety.

PubMed

Sa-ngasoongsong, Paphon; Chanplakorn, Pongsthorn; Wongsak, Siwadol; Uthadorn, Krisorn; Panpikoon, Tanapong; Jittorntam, Paisan; Aryurachai, Katcharin; Angchaisukisiri, Pantap; Kawinwonggowit, Viroj

2015-01-01

Recently, combined intra-articular tranexamic acid (IA-TXA) injection with clamping drain method showed efficacy for blood loss and transfusion reduction in total knee replacement (TKR). However, until now, none of previous studies revealed the effect of this technique on pharmacokinetics, coagulation, and fibrinolysis. An experimental study was conducted, during 2011-2012, in 30 patients undergoing unilateral TKR. Patients received IA-TXA application and then were allocated into six groups regarding clamping drain duration (2-, 4-, 6-, 8-, 10-, and 12-hours). Blood and drainage fluid were collected to measure tranexamic acid (TXA) level and related coagulation and fibrinolytic markers. Postoperative complication was followed for one year. There was no significant difference of serum TXA level at 2 hour and 24 hour among groups (p < 0.05). Serum TXA level at time of clamp release was significantly different among groups with the highest level at 2 hour (p < 0.0001). There was no significant difference of TXA level in drainage fluid, postoperative blood loss, blood transfusion, and postoperative complications (p < 0.05). Low-dose IA-TXA application in TKR with prolonged clamping drain method is a safe and effective blood conservative technique with only minimal systemic absorption and without significant increase in systemic absorption over time.

A novel method of temperature compensation for piezoresistive microcantilever-based sensors.

PubMed

Han, Jianqiang; Wang, Xiaofei; Yan, Tianhong; Li, Yan; Song, Meixuan

2012-03-01

Microcantilever with integrated piezoresistor has been applied to in situ surface stress measurement in the field of biochemical sensors. It is well known that piezoresistive cantilever-based sensors are sensitive to ambient temperature changing due to highly temperature-dependent piezoresistive effect and mismatch in thermal expansion of composite materials. This paper proposes a novel method of temperature drift compensation for microcantilever-based sensors with a piezoresistive full Wheatstone bridge integrated at the clamped ends by subtracting the amplified output voltage of the reference cantilever from the output voltage of the sensing cantilever through a simple temperature compensating circuit. Experiments show that the temperature drift of microcantilever sensors can be significantly reduced by the method.

n-Alkanols potentiate sodium channel inactivation in squid giant axons.

PubMed Central

Oxford, G S; Swenson, R P

1979-01-01

The effects of n-octanol and n-decanol on nerve membrane sodium channels were examined in internally perfused, voltage-clamped squid giant axons. Both n-octanol and n-decanol almost completely eliminated the residual sodium conductance at the end of 8-ms voltage steps. In contrast, peak sodium conductance was only partially reduced. This block of peak and residual sodium conductance was very reversible and seen with both internal and external alkanol application. The differential sensitivity of peak and residual conductance to alkanol treatment was eliminated after internal pronase treatment, suggesting that n-octanol and n-decanol enhance the normal inactivation mechanism rather than directly blocking channels in a time-dependent manner. PMID:233577

[Protective effect of Uncaria rhynchophylla total alkaloids pretreatment on hippocampal neurons after acute hypoxia].

PubMed

Liu, Wei; Zhang, Zhao-qin; Zhao, Xiao-min; Gao, Yun-sheng

2006-05-01

To investigate the effect of Uncaria rhynchophylla total alkaloids (RTA) pretreatment on the voltage-gated sodium currents of the rat hippocampal neurons after acute hypoxia. Primary cultured hippocampal neurons were divided into RTA pre-treated and non-pretreated groups. Patch clamp whole-cell recording was used to compare the voltage-gated sodium current amplitude and threshold with those before hypoxia. After acute hypoxia, sodium current amplitude was significantly decreased and its threshold was upside. RTA pretreatment could inhibit the reduction of sodium current amplitude. RTA pretreatment alleviates the acute hypoxia-induced change of sodium currents, which may be one of the mechanisms for protective effect of RTA on cells.

Gating of the two-pore cation channel AtTPC1 in the plant vacuole is based on a single voltage-sensing domain.

PubMed

Jaślan, D; Mueller, T D; Becker, D; Schultz, J; Cuin, T A; Marten, I; Dreyer, I; Schönknecht, G; Hedrich, R

2016-09-01

The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembranes. Each subunit consists of two homologous Shaker-like halves, with 12 transmembrane domains in total (S1-S6, S7-S12). In plants, TPC1 channels reside in the vacuolar membrane, and upon voltage stimulation, give rise to the well-known slow-activating SV currents. Here, we combined bioinformatics, structure modelling, site-directed mutagenesis, and in planta patch clamp studies to elucidate the molecular mechanisms of voltage-dependent channel gating in TPC1 in its native plant background. Structure-function analysis of the Arabidopsis TPC1 channel in planta confirmed that helix S10 operates as the major voltage-sensing site, with Glu450 and Glu478 identified as possible ion-pair partners for voltage-sensing Arg537. The contribution of helix S4 to voltage sensing was found to be negligible. Several conserved negative residues on the luminal site contribute to calcium binding, stabilizing the closed channel. During evolution of plant TPC1s from two separate Shaker-like domains, the voltage-sensing function in the N-terminal Shaker-unit (S1-S4) vanished. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

Decrement of GABAA receptor-mediated inhibitory postsynaptic currents in dentate granule cells in epileptic hippocampus.

PubMed

Isokawa, M

1996-05-01

1. Inhibitory postsynaptic currents (IPSCs) were studied in hippocampal dentate granule cells (DGCs) in the pilocarpine model and human temporal lobe epilepsy, with the use of the whole cell patch-clamp recording technique in slice preparations. 2. In the pilocarpine model, hippocampal slices were prepared from rats that were allowed to experience spontaneous seizures for 2 mo. Human hippocampal specimens were obtained from epileptic patients who underwent surgical treatment for medically intractable seizures. 3. IPSCs were generated by single perforant path stimulation and recorded at a membrane potential (Vm) of 0 mV near the reversal potential of glutamate excitatory postsynaptic currents in the voltage-clamp recording. IPSCs were pharmacologically identified as gamma-aminobutyric acid-A (GABAA) IPSCs by 10 microM bicuculline methiodide. 4. During low-frequency stimulation, IPSCs were not different in amplitude among non-seizure-experienced rat hippocampi, human nonsclerotic hippocampi, seizure-experienced rat hippocampi, and human sclerotic hippocampi. In the last two groups of DGCs, current-clamp recordings indicated the presence of prolonged excitatory postsynaptic potentials (EPSPs) mediated by the N-methyl-D-aspartate (NMDA) receptor. 5. High-frequency stimulation, administered at Vm = -30 mV to activate NMDA currents, reduced GABAA IPSC amplitude specifically in seizure-experienced rat hippocampi (t = 2.5, P < 0.03) and human sclerotic hippocampi (t = 7.7, P < 0.01). This reduction was blocked by an NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV) (50 microM). The time for GABAA IPSCs to recover to their original amplitude was also shortened by the application of APV. 6. I conclude that, when intensively activated, NMDA receptor-mediated excitatory transmission may interact with GABAergic synaptic inhibition in DGCs in seizure-experienced hippocampus to transiently reduce GABA(A) receptor-channel function. Such interactions may contribute to give rise to epileptic excitation in chronically seizure-prone hippocampus.

Characterization of Ryanodine Receptor Type 1 Single Channel Activity Using “On-Nucleus” Patch Clamp

PubMed Central

Wagner, Larry E.; Groom, Linda A.; Dirksen, Robert T.; Yule, David I.

2014-01-01

In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca2+ release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca2+] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ~750 pS or 450 pS in symmetrical 250 mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ~40 % of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca2+, and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation. PMID:24972488

Electrophysiological and Electrochemical Methods Development for the Detection of Biologically Active Chemical Agents

DTIC Science & Technology

1988-11-01

Bilayer ........................................... 14 5. Current-Voltage Curve for Gramacidin in a Lecithin -Sphingomyelin Patch Bilayer... lecithin (Avanti). 9 2. MATERIALS 2.1 Patch Microprobe Instrumentation. The basis of the microprobe system is an AxoPatch Patch- Clamping Amplifier System...histogram of 1024 events cut above 2 pA. Events sampled are thought to be from the same single gramacidin channel in a lecithin : sphingomyelin (5:1) patch

Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology

PubMed Central

Lee, Claire Shuk-Kwan; Tong, Benjamin Chun-Kit; Cheng, Cecily Wing-Hei; Hung, Harry Chun-Hin; Cheung, King-Ho

2016-01-01

Lysosomal calcium (Ca2+) release mediated by NAADP triggers signalling cascades that regulate many cellular processes. The identification of two-pore channel 2 (TPC2) as the NAADP receptor advances our understanding of lysosomal Ca2+ signalling, yet the lysosome is not amenable to traditional patch-clamp electrophysiology. Previous attempts to record TPC2 single-channel activity put TPC2 outside its native environment, which not reflect TPC2’s true physiological properties. To test the feasibility of using nuclear membrane electrophysiology for TPC2 channel characterization, we constructed a stable human TPC2-expressing DT40TKO cell line that lacks endogenous InsP3R and RyR (DT40TKO-hTPC2). Immunostaining revealed hTPC2 expression on the ER and nuclear envelope. Intracellular dialysis of NAADP into Fura-2-loaded DT40TKO-hTPC2 cells elicited cytosolic Ca2+ transients, suggesting that hTPC2 was functionally active. Using nuclear membrane electrophysiology, we detected a ~220 pS single-channel current activated by NAADP with K+ as the permeant ion. The detected single-channel recordings displayed a linear current-voltage relationship, were sensitive to Ned-19 inhibition, were biphasically regulated by NAADP concentration, and regulated by PKA phosphorylation. In summary, we developed a cell model for the characterization of the TPC2 channel and the nuclear membrane patch-clamp technique provided an alternative approach to rigorously investigate the electrophysiological properties of TPC2 with minimal manipulation. PMID:26838264

Inhibition of spontaneous activity of rabbit atrioventricular node cells by KB-R7943 and inhibitors of sarcoplasmic reticulum Ca2+ ATPase

PubMed Central

Cheng, Hongwei; Smith, Godfrey L.; Hancox, Jules C.; Orchard, Clive H.

2011-01-01

The atrioventricular node (AVN) can act as a subsidiary cardiac pacemaker if the sinoatrial node fails. In this study, we investigated the effects of the Na–Ca exchange (NCX) inhibitor KB-R7943, and inhibition of the sarcoplasmic reticulum calcium ATPase (SERCA), using thapsigargin or cyclopiazonic acid (CPA), on spontaneous action potentials (APs) and [Ca2+]i transients from cells isolated from the rabbit AVN. Spontaneous [Ca2+]i transients were monitored from undialysed AVN cells at 37 °C using Fluo-4. In separate experiments, spontaneous APs and ionic currents were recorded using the whole-cell patch clamp technique. Rapid application of 5 μM KB-R7943 slowed or stopped spontaneous APs and [Ca2+]i transients. However, in voltage clamp experiments in addition to blocking NCX current (INCX) KB-R7943 partially inhibited L-type calcium current (ICa,L). Rapid reduction of external [Na+] also abolished spontaneous activity. Inhibition of SERCA (using 2.5 μM thapsigargin or 30 μM CPA) also slowed or stopped spontaneous APs and [Ca2+]i transients. Our findings are consistent with the hypothesis that sarcoplasmic reticulum (SR) Ca2+ release influences spontaneous activity in AVN cells, and that this occurs via [Ca2+]i-activated INCX; however, the inhibitory action of KB-R7943 on ICa,L means that care is required in the interpretation of data obtained using this compound. PMID:21163524

Synergistic Effect of Light and Fusicoccin on Stomatal Opening 1

PubMed Central

Assmann, Sarah M.; Schwartz, Amnon

1992-01-01

Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K+ channel blocker, tetraethyl-ammonium+ (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (−60 millivolts) at which K+ channels are inactivated. These results are indicative of FC activation of a H+ pump. FC effects on the magnitude of inward and outward K+ currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H+ ATPase of guard cells is a primary locus for the FC effect on stomatal apertures. PMID:16668799

Biomechanical evaluation of a new fixation device for the thoracic spine.

PubMed

Hongo, Michio; Ilharreborde, Brice; Gay, Ralph E; Zhao, Chunfeng; Zhao, Kristin D; Berglund, Lawrence J; Zobitz, Mark; An, Kai-Nan

2009-08-01

The technology used in surgery for spinal deformity has progressed rapidly in recent years. Commonly used fixation techniques may include monofilament wires, sublaminar wires and cables, and pedicle screws. Unfortunately, neurological complications can occur with all of these, compromising the patients' health and quality of life. Recently, an alternative fixation technique using a metal clamp and polyester belt was developed to replace hooks and sublaminar wiring in scoliosis surgery. The goal of this study was to compare the pull-out strength of this new construct with sublaminar wiring, laminar hooks and pedicle screws. Forty thoracic vertebrae from five fresh frozen human thoracic spines (T5-12) were divided into five groups (8 per group), such that BMD values, pedicle diameter, and vertebral levels were equally distributed. They were then potted in polymethylmethacrylate and anchored with metal screws and polyethylene bands. One of five fixation methods was applied to the right side of the vertebra in each group: Pedicle screw, sublaminar belt with clamp, figure-8 belt with clamp, sublaminar wire, or laminar hook. Pull-out strength was then assessed using a custom jig in a servohydraulic tester. The mean failure load of the pedicle screw group was significantly larger than that of the figure-8 clamp (P = 0.001), sublaminar belt (0.0172), and sublaminar wire groups (P = 0.04) with no significant difference in pull-out strength between the latter three constructs. The most common mode of failure was the fracture of the pedicle. BMD was significantly correlated with failure load only in the figure-8 clamp and pedicle screw constructs. Only the pedicle screw had a statistically significant higher failure load than the sublaminar clamp. The sublaminar method of applying the belt and clamp device was superior to the figure-8 method. The sublaminar belt and clamp construct compared favorably to the traditional methods of sublaminar wires and laminar hooks, and should be considered as an alternative fixation device in the thoracic spine.

Biomechanical evaluation of a new fixation device for the thoracic spine

PubMed Central

Hongo, Michio; Ilharreborde, Brice; Zhao, Chunfeng; Zhao, Kristin D.; Berglund, Lawrence J.; Zobitz, Mark; An, Kai-Nan

2009-01-01

The technology used in surgery for spinal deformity has progressed rapidly in recent years. Commonly used fixation techniques may include monofilament wires, sublaminar wires and cables, and pedicle screws. Unfortunately, neurological complications can occur with all of these, compromising the patients’ health and quality of life. Recently, an alternative fixation technique using a metal clamp and polyester belt was developed to replace hooks and sublaminar wiring in scoliosis surgery. The goal of this study was to compare the pull-out strength of this new construct with sublaminar wiring, laminar hooks and pedicle screws. Forty thoracic vertebrae from five fresh frozen human thoracic spines (T5–12) were divided into five groups (8 per group), such that BMD values, pedicle diameter, and vertebral levels were equally distributed. They were then potted in polymethylmethacrylate and anchored with metal screws and polyethylene bands. One of five fixation methods was applied to the right side of the vertebra in each group: Pedicle screw, sublaminar belt with clamp, figure-8 belt with clamp, sublaminar wire, or laminar hook. Pull-out strength was then assessed using a custom jig in a servohydraulic tester. The mean failure load of the pedicle screw group was significantly larger than that of the figure-8 clamp (P = 0.001), sublaminar belt (0.0172), and sublaminar wire groups (P = 0.04) with no significant difference in pull-out strength between the latter three constructs. The most common mode of failure was the fracture of the pedicle. BMD was significantly correlated with failure load only in the figure-8 clamp and pedicle screw constructs. Only the pedicle screw had a statistically significant higher failure load than the sublaminar clamp. The sublaminar method of applying the belt and clamp device was superior to the figure-8 method. The sublaminar belt and clamp construct compared favorably to the traditional methods of sublaminar wires and laminar hooks, and should be considered as an alternative fixation device in the thoracic spine. PMID:19404687

Arterial waves in humans during peripheral vascular surgery.

PubMed

Khir, A W; Henein, M Y; Koh, T; Das, S K; Parker, K H; Gibson, D G

2001-12-01

The purpose of this study was to investigate the effect of aortic clamping on arterial waves during peripheral vascular surgery. We measured pressure and velocity simultaneously in the ascending aorta, in ten patients (70+/-5 years) with aortic-iliac disease intra-operatively. Pressure was measured using a catheter tip manometer, and velocity was measured using Doppler ultrasound. Data were collected before aortic clamping, during aortic clamping and after unclamping. Hydraulic work in the aortic root was calculated from the measured data, the reflected waves were determined by wave-intensity analysis and wave speed was determined by the PU-loop (pressure-velocity-loop) method; a new technique based on the 'water-hammer' equation. The wave speed is approx. 32% (P<0.05) higher during clamping than before clamping. Although the peak intensity of the reflected wave does not alter with clamping, it arrives 30 ms (P<0.05) earlier and its duration is 25% (P<0.05) longer than before clamping. During clamping, left ventricule (LV) hydraulic systolic work and the energy carried by the reflected wave increased by 27% (P<0.05) and 20% (P<0.05) respectively, compared with before clamping. The higher wave speed during clamping explains the earlier arrival of the reflected waves suggesting an increase in the afterload, since the LV has to overcome earlier reflected compression waves. The longer duration of the reflected wave during clamping is associated with an increase in the total energy carried by the wave, which causes an increase in hydraulic work. Increased hydraulic work during clamping may increase LV oxygen consumption, provoke myocardial ischaemia and hence contribute to the intra-operative impairment of LV function known in patients with peripheral vascular disease.

Altered ion channel conductance and ionic selectivity induced by large imposed membrane potential pulse.

PubMed Central

Chen, W; Lee, R C

1994-01-01

The effects of large magnitude transmembrane potential pulses on voltage-gated Na and K channel behavior in frog skeletal muscle membrane were studied using a modified double vaseline-gap voltage clamp. The effects of electroconformational damage to ionic channels were separated from damage to lipid bilayer (electroporation). A 4 ms transmembrane potential pulse of -600 mV resulted in a reduction of both Na and K channel conductivities. The supraphysiologic pulses also reduced ionic selectivity of the K channels against Na+ ions, resulting in a depolarization of the membrane resting potential. However, TTX and TEA binding effects were unaltered. The kinetics of spontaneous reversal of the electroconformational damage of channel proteins was found to be dependent on the magnitude of imposed membrane potential pulse. These results suggest that muscle and nerve dysfunction after electrical shock may be in part caused by electroconformational damage to voltage-gated ion channels. PMID:7948676

Voltage activation and hysteresis of the non-selective voltage-dependent channel in the intact human red cell.

PubMed

Bennekou, Poul; Barksmann, Trine L; Jensen, Lars R; Kristensen, Berit I; Christophersen, Palle

2004-05-01

Suspension of intact human red cells in media with low chloride and sodium concentrations (isotonic sucrose substitution) results in strongly inside positive membrane potentials, which activate the voltage-dependent non-selective cation (NSVDC) channel. By systematic variation of the initial Nernst potentials for chloride (degree of ion substitution) as well as the chloride conductance (block by NS1652), and by exploiting the interplay between the Ca(2+)-permeable NSVDC channel, the Ca(2+)-activated K+ channel (the Gárdos channel) and the Ca(2+)-pump, a graded activation of the NSVDC channel was achieved. Under these conditions, it was shown that the NSVDC channels exist in two states of activation depending on the initial conditions for the activation. The hysteretic behaviour, which in patch clamp experiments has been found for the individual channel unit, is thus retained at the cellular level and can be demonstrated with red cells in suspension.

Mechanical nonlinearity elimination with a micromechanical clamped-free semicircular beams resonator

NASA Astrophysics Data System (ADS)

Chen, Dongyang; Chen, Xuying; Wang, Yong; Liu, Xinxin; Guan, Yangyang; Xie, Jin

2018-04-01

This paper reports a micro-machined clamped-free semicircular beam resonator aiming to eliminate the nonlinearity that widely exists in traditional mechanical resonators. Cubic coefficients over vibration displacement due to axial extension of the beams are analyzed through theoretical modelling, and the corresponding frequency effect is demonstrated. With the device working in the elastic vibration mode, the cubic coefficients are eliminated by using a free end to release the nonlinear extension of beams and thus the inside axial stress. The amplitude-frequency (A-f) effect is overcome in a large region of source power, and the coefficient of frequency softening is linearized in a large region of polarization voltage. As a result, the resonator can be driven at larger vibration amplitude to achieve a high signal to noise ratio and power handling performance.

AAO2: a general purpose CCD controller for the AAT

NASA Astrophysics Data System (ADS)

Waller, Lew; Barton, John; Mayfield, Don; Griesbach, Jason

2004-09-01

The Anglo-Australian Observatory has developed a 2nd generation optical CCD controller to replace an earlier controller used now for almost twenty years. The new AAO2 controller builds on the considerable experience gained with the first controller, the new technologies now available and the techniques developed and successfully implemented in AAO's IRIS2 detector controller. The AAO2 controller has been designed to operate a wide variety of detectors and to achieve as near to detector limited performance as possible. It is capable of reading out CCDs with one, two or four output amplifiers, each output having its own video processor and high speed 16-bit ADC. The video processor is a correlated double sampler that may be switched between low noise dual slope integration or high speed clamp and sample modes. Programmable features include low noise DAC biases, horizontal clocks with DAC controllable levels and slopes and vertical clocks with DAC controllable arbitrary waveshapes. The controller uses two DSPs; one for overall control and the other for clock signal generation, which is highly programmable, with downloadable sequences of waveform patterns. The controller incorporates a precision detector temperature controller and provides accurate exposure time control. Telemetry is provided of all DAC generated voltages, many derived voltages, power supply voltages, detector temperature and detector identification. A high speed, full duplex fibre optic interface connects the controller to a host computer. The modular design uses six to ten circuit boards, plugged in to common backplanes. Two backplanes separate noisy digital signals from low noise analog signals.

Structural Determinants for Functional Coupling Between the β and α Subunits in the Ca2+-activated K+ (BK) Channel

PubMed Central

Orio, Patricio; Torres, Yolima; Rojas, Patricio; Carvacho, Ingrid; Garcia, Maria L.; Toro, Ligia; Valverde, Miguel A.; Latorre, Ramon

2006-01-01

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. The most remarkable effects of β1 and β2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by α and β1 or β2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of β1 but not β2. Here we reveal the molecular regions in these β subunits that determine their differential functional coupling with the pore-forming α-subunit. We made chimeric constructs between β1 and β2 subunits, and BK channels formed by α and chimeric β subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the β1 and β2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these β subunits. Moreover, the intracellular domains of β1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the α-subunit to be the target of the modulation by the β1-subunit. PMID:16446507

Calcium current in isolated neonatal rat ventricular myocytes.

PubMed Central

Cohen, N M; Lederer, W J

1987-01-01

1. Calcium currents (ICa) from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the 'whole-cell' voltage-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Examination of ICa was limited to one calcium channel type, 'L' type (Nilius, Hess, Lansman & Tsien, 1985), by appropriate voltage protocols. 2. We measured transient and steady-state components of ICa, and could generally describe ICa in terms of the steady-state activation (d infinity) and inactivation (f infinity) parameters. 3. We observed that the reduction of ICa by the calcium channel antagonist D600 can be explained by both a shift of d infinity to more positive potentials as well as a slight reduction of ICa conductance. D600 did not significantly alter either the rate of inactivation of ICa or the voltage dependence of f infinity. 4. The calcium channel modulator BAY K8644 shifted both d infinity and f infinity to more negative potentials. Additionally, BAY K8644 increased the rate of inactivation at potentials between +5 and +55 mV. Furthermore, BAY K8644 also increased ICa conductance, a change consistent with a promotion of 'mode 2' calcium channel activity (Hess, Lansman & Tsien, 1984). 5. We conclude that, as predicted by d infinity and f infinity, there is a significant steady-state component of ICa ('window current') at plateau potentials in neonatal rat heart cells. Modulation of the steady-state and transient components of ICa by various agents can be attributed both to specific alterations in d infinity and f infinity and to more complicated alterations in the mode of calcium channel activity. PMID:2451004

From zebrafish to mammal: functional evolution of prestin, the motor protein of cochlear outer hair cells.

PubMed

Tan, Xiaodong; Pecka, Jason L; Tang, Jie; Okoruwa, Oseremen E; Zhang, Qian; Beisel, Kirk W; He, David Z Z

2011-01-01

Prestin is the motor protein of cochlear outer hair cells. It belongs to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Members of this family serve two fundamentally distinct functions. Although most members transport different anion substrates across a variety of epithelia, prestin (SLC26A5) is unique, functioning as a voltage-dependent motor protein. Recent evidence suggests that prestin orthologs from zebrafish and chicken are electrogenic divalent/chloride anion exchangers/transporters with no motor function. These studies appear to suggest that prestin was evolved from an anion transporter. We examined the motor and transport functions of prestin and its orthologs from four different species in the vertebrate lineage, to gain insights of how these two physiological functions became distinct. Somatic motility, voltage-dependent nonlinear capacitance (NLC), and transporter function were measured in transfected human embryonic kidney (HEK) cells using voltage-clamp and anion uptake techniques. Zebrafish and chicken prestins both exhibited weak NLC, with peaks significantly shifted in the depolarization (right) direction. This was contrasted by robust NLC with peaks left shifted in the platypus and gerbil. The platypus and gerbil prestins retained little transporter function compared with robust anion transport capacities in the zebrafish and chicken orthologs. Somatic motility was detected only in the platypus and gerbil prestins. There appears to be an inverse relationship between NLC and anion transport functions, whereas motor function appears to have emerged only in mammalian prestin. Our results suggest that motor function is an innovation of therian prestin and is concurrent with diminished transporter capabilities.

Pseudomonas fluorescens lipopolysaccharide inhibits both delayed rectifier and transient A-type K+ channels of cultured rat cerebellar granule neurons.

PubMed

Mezghani-Abdelmoula, Sana; Chevalier, Sylvie; Lesouhaitier, Olivier; Orange, Nicole; Feuilloley, Marc G J; Cazin, Lionel

2003-09-05

Pseudomonas fluorescens is a Gram-negative bacillus closely related to the pathogen P. aeruginosa known to provoke infectious disorders in the central nervous system (CNS). The endotoxin lipopolysaccharide (LPS) expressed by the bacteria is the first infectious factor that can interact with the plasma membrane of host cells. In the present study, LPS extracted from P. fluorescens MF37 was examined for its actions on delayed rectifier and A-type K(+) channels, two of the main types of voltage-activated K(+) channels involved in the action potential firing. Current recordings were performed in cultured rat cerebellar granule neurons at days 7 or 8, using the whole-cell patch-clamp technique. A 3-h incubation with LPS (200 ng/ml) markedly depressed both the delayed rectifier (I(KV)) and transient A-type (I(A)) K(+) currents evoked by depolarizations above 0 and -40 mV, respectively. The percent decrease of I(KV) and I(A) ( approximately 30%) did not vary with membrane potential, suggesting that inhibition of both types of K(+) channels by LPS was voltage-insensitive. The endotoxin did neither modify the steady-state voltage-dependent activation properties of I(KV) and I(A) nor the steady-state inactivation of I(A). The present results suggest that, by inhibiting I(KV) and I(A), LPS applied extracellulary increases the action potential firing in cerebellar granule neurons. It is concluded that P. fluorescens MF37 may provoke in the CNS disorders associated with sever alterations of membrane ionic channel functions.

Concentration-jump analysis of voltage-dependent conductances activated by glutamate and kainate in neurons of the avian cochlear nucleus.

PubMed Central

Raman, I M; Trussell, L O

1995-01-01

We have examined the mechanisms underlying the voltage sensitivity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors in voltage-clamped outside-out patches and whole cells taken from the nucleus magnocellularis of the chick. Responses to either glutamate or kainate had outwardly rectifying current-voltage relations. The rate and extent of desensitization during prolonged exposure to agonist, and the rate of deactivation after brief exposure to agonist, decreased at positive potentials, suggesting that a kinetic transition was sensitive to membrane potential. Voltage dependence of the peak conductance and of the deactivation kinetics persisted when desensitization was reduced with aniracetam or blocked with cyclothiazide. Furthermore, the rate of recovery from desensitization to glutamate was not voltage dependent. Upon reduction of extracellular divalent cation concentration, kainate-evoked currents increased but preserved rectifying current-voltage relations. Rectification was strongest at lower kainate concentrations. Surprisingly, nonstationary variance analysis of desensitizing responses to glutamate or of the current deactivation after kainate removal revealed an increase in the mean single-channel conductance with more positive membrane potentials. These data indicate that the rectification of the peak response to a high agonist concentration reflects an increase in channel conductance, whereas rectification of steady-state current is dominated by voltage-sensitive channel kinetics. Images FIGURE 2 FIGURE 3 PMID:8580330

Temporary clamping of external carotid artery in convexity, parasagittal and temporal base meningioma.

PubMed

Yadav, Yad Ram; Parihar, Vijay; Agarwal, Moneet; Bhatele, Pushp Raj

2012-01-01

The management of intraoperative bleeding during removal of a large hyper vascular meningioma is crucial for safe and efficient surgery. Preoperative embolization of meningioma is the best way to reduce vascularity of meningiomas but this technique is not readily available, costly and has its own limitations. The study is aimed to evaluate the use of temporary clamping of external carotid artery to reduce blood loss and operating time during excision of large convexity, parasagittal or temporal base meningiomas. A prospective study of 115 consecutively operated meningiomas of size 5 cms or more were operated from January 2002 to December2010. Temporary clamping of external carotid artery was done in 61 while 51 cases were managed without clamping. There was significant reduction of blood loss, operative time and blood transfusion given in the temporary clipping group compared to non clipping group. There was stitch abscess in two patients each in clamping, and non clamping group. There was no scalp necrosis or mortality in any of the group. Temporary clamping of external carotid artery is a safe, simple and cost-effective alternative to embolization for the surgery of large meningiomas. This can be practiced at all the centers.

Hyperpolarization-activated current (I(h)) in vestibular calyx terminals: characterization and role in shaping postsynaptic events.

PubMed

Meredith, Frances L; Benke, Tim A; Rennie, Katherine J

2012-12-01

Calyx afferent terminals engulf the basolateral region of type I vestibular hair cells, and synaptic transmission across the vestibular type I hair cell/calyx is not well understood. Calyces express several ionic conductances, which may shape postsynaptic potentials. These include previously described tetrodotoxin-sensitive inward Na(+) currents, voltage-dependent outward K(+) currents and a K(Ca) current. Here, we characterize an inwardly rectifying conductance in gerbil semicircular canal calyx terminals (postnatal days 3-45), sensitive to voltage and to cyclic nucleotides. Using whole-cell patch clamp, we recorded from isolated calyx terminals still attached to their type I hair cells. A slowly activating, noninactivating current (I(h)) was seen with hyperpolarizing voltage steps negative to the resting potential. External Cs(+) (1-5 mM) and ZD7288 (100 μM) blocked the inward current by 97 and 83 %, respectively, confirming that I(h) was carried by hyperpolarization-activated, cyclic nucleotide gated channels. Mean half-activation voltage of I(h) was -123 mV, which shifted to -114 mV in the presence of cAMP. Activation of I(h) was well described with a third order exponential fit to the current (mean time constant of activation, τ, was 190 ms at -139 mV). Activation speeded up significantly (τ=136 and 127 ms, respectively) when intracellular cAMP and cGMP were present, suggesting that in vivo I(h) could be subject to efferent modulation via cyclic nucleotide-dependent mechanisms. In current clamp, hyperpolarizing current steps produced a time-dependent depolarizing sag followed by either a rebound afterdepolarization or an action potential. Spontaneous excitatory postsynaptic potentials (EPSPs) became larger and wider when I(h) was blocked with ZD7288. In a three-dimensional mathematical model of the calyx terminal based on Hodgkin-Huxley type ionic conductances, removal of I(h) similarly increased the EPSP, whereas cAMP slightly decreased simulated EPSP size and width.

Mechanics of hydrogenated amorphous carbon deposits from electron-beam-induced deposition of a paraffin precursor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, W.; Dikin, D.A.; Chen, X.

2005-07-01

Many experiments on the mechanics of nanostructures require the creation of rigid clamps at specific locations. In this work, electron-beam-induced deposition (EBID) has been used to deposit carbon films that are similar to those that have recently been used for clamping nanostructures. The film deposition rate was accelerated by placing a paraffin source of hydrocarbon near the area where the EBID deposits were made. High-resolution transmission electron microscopy, electron-energy-loss spectroscopy, Raman spectroscopy, secondary-ion-mass spectrometry, and nanoindentation were used to characterize the chemical composition and the mechanics of the carbonaceous deposits. The typical EBID deposit was found to be hydrogenated amorphousmore » carbon (a-C:H) having more sp{sup 2}- than sp{sup 3}-bonded carbon. Nanoindentation tests revealed a hardness of {approx}4 GPa and an elastic modulus of 30-60 GPa, depending on the accelerating voltage. This reflects a relatively soft film, which is built out of precursor molecular ions impacting the growing surface layer with low energies. The use of such deposits as clamps for tensile tests of poly(acrylonitrile)-based carbon nanofibers loaded between opposing atomic force microscope cantilevers is presented as an example application.« less

Non-linear Membrane Properties in Entorhinal Cortical Stellate Cells Reduce Modulation of Input-Output Responses by Voltage Fluctuations

PubMed Central

Fernandez, Fernando R.; Malerba, Paola; White, John A.

2015-01-01

The presence of voltage fluctuations arising from synaptic activity is a critical component in models of gain control, neuronal output gating, and spike rate coding. The degree to which individual neuronal input-output functions are modulated by voltage fluctuations, however, is not well established across different cortical areas. Additionally, the extent and mechanisms of input-output modulation through fluctuations have been explored largely in simplified models of spike generation, and with limited consideration for the role of non-linear and voltage-dependent membrane properties. To address these issues, we studied fluctuation-based modulation of input-output responses in medial entorhinal cortical (MEC) stellate cells of rats, which express strong sub-threshold non-linear membrane properties. Using in vitro recordings, dynamic clamp and modeling, we show that the modulation of input-output responses by random voltage fluctuations in stellate cells is significantly limited. In stellate cells, a voltage-dependent increase in membrane resistance at sub-threshold voltages mediated by Na+ conductance activation limits the ability of fluctuations to elicit spikes. Similarly, in exponential leaky integrate-and-fire models using a shallow voltage-dependence for the exponential term that matches stellate cell membrane properties, a low degree of fluctuation-based modulation of input-output responses can be attained. These results demonstrate that fluctuation-based modulation of input-output responses is not a universal feature of neurons and can be significantly limited by subthreshold voltage-gated conductances. PMID:25909971

Non-linear Membrane Properties in Entorhinal Cortical Stellate Cells Reduce Modulation of Input-Output Responses by Voltage Fluctuations.

PubMed

Fernandez, Fernando R; Malerba, Paola; White, John A

2015-04-01

The presence of voltage fluctuations arising from synaptic activity is a critical component in models of gain control, neuronal output gating, and spike rate coding. The degree to which individual neuronal input-output functions are modulated by voltage fluctuations, however, is not well established across different cortical areas. Additionally, the extent and mechanisms of input-output modulation through fluctuations have been explored largely in simplified models of spike generation, and with limited consideration for the role of non-linear and voltage-dependent membrane properties. To address these issues, we studied fluctuation-based modulation of input-output responses in medial entorhinal cortical (MEC) stellate cells of rats, which express strong sub-threshold non-linear membrane properties. Using in vitro recordings, dynamic clamp and modeling, we show that the modulation of input-output responses by random voltage fluctuations in stellate cells is significantly limited. In stellate cells, a voltage-dependent increase in membrane resistance at sub-threshold voltages mediated by Na+ conductance activation limits the ability of fluctuations to elicit spikes. Similarly, in exponential leaky integrate-and-fire models using a shallow voltage-dependence for the exponential term that matches stellate cell membrane properties, a low degree of fluctuation-based modulation of input-output responses can be attained. These results demonstrate that fluctuation-based modulation of input-output responses is not a universal feature of neurons and can be significantly limited by subthreshold voltage-gated conductances.

An In Vivo Study of Low-Dose Intra-Articular Tranexamic Acid Application with Prolonged Clamping Drain Method in Total Knee Replacement: Clinical Efficacy and Safety

PubMed Central

Sa-ngasoongsong, Paphon; Chanplakorn, Pongsthorn; Wongsak, Siwadol; Uthadorn, Krisorn; Panpikoon, Tanapong; Jittorntam, Paisan; Aryurachai, Katcharin; Angchaisukisiri, Pantap; Kawinwonggowit, Viroj

2015-01-01

Background. Recently, combined intra-articular tranexamic acid (IA-TXA) injection with clamping drain method showed efficacy for blood loss and transfusion reduction in total knee replacement (TKR). However, until now, none of previous studies revealed the effect of this technique on pharmacokinetics, coagulation, and fibrinolysis. Materials and Methods. An experimental study was conducted, during 2011-2012, in 30 patients undergoing unilateral TKR. Patients received IA-TXA application and then were allocated into six groups regarding clamping drain duration (2-, 4-, 6-, 8-, 10-, and 12-hours). Blood and drainage fluid were collected to measure tranexamic acid (TXA) level and related coagulation and fibrinolytic markers. Postoperative complication was followed for one year. Results. There was no significant difference of serum TXA level at 2 hour and 24 hour among groups (p < 0.05). Serum TXA level at time of clamp release was significantly different among groups with the highest level at 2 hour (p < 0.0001). There was no significant difference of TXA level in drainage fluid, postoperative blood loss, blood transfusion, and postoperative complications (p < 0.05).  Conclusions. Low-dose IA-TXA application in TKR with prolonged clamping drain method is a safe and effective blood conservative technique with only minimal systemic absorption and without significant increase in systemic absorption over time. PMID:26583092

Resurgent current of voltage-gated Na+ channels

PubMed Central

Lewis, Amanda H; Raman, Indira M

2014-01-01

Resurgent Na+ current results from a distinctive form of Na+ channel gating, originally identified in cerebellar Purkinje neurons. In these neurons, the tetrodotoxin-sensitive voltage-gated Na+ channels responsible for action potential firing have specialized mechanisms that reduce the likelihood that they accumulate in fast inactivated states, thereby shortening refractory periods and permitting rapid, repetitive, and/or burst firing. Under voltage clamp, step depolarizations evoke transient Na+ currents that rapidly activate and quickly decay, and step repolarizations elicit slower channel reopening, or a ‘resurgent’ current. The generation of resurgent current depends on a factor in the Na+ channel complex, probably a subunit such as NaVβ4 (Scn4b), which blocks open Na+ channels at positive voltages, competing with the fast inactivation gate, and unblocks at negative voltages, permitting recovery from an open channel block along with a flow of current. Following its initial discovery, resurgent Na+ current has been found in nearly 20 types of neurons. Emerging research suggests that resurgent current is preferentially increased in a variety of clinical conditions associated with altered cellular excitability. Here we review the biophysical, molecular and structural mechanisms of resurgent current and their relation to the normal functions of excitable cells as well as pathophysiology. PMID:25172941

Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains.

PubMed

Hsu, Eric J; Zhu, Wandi; Schubert, Angela R; Voelker, Taylor; Varga, Zoltan; Silva, Jonathan R

2017-03-06

Functional eukaryotic voltage-gated Na + (Na V ) channels comprise four domains (DI-DIV), each containing six membrane-spanning segments (S1-S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1-S4), which together form a voltage-sensing domain (VSD). A critical Na V channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair Na V channel inactivation. These locations include the DIII-DIV linker, the DIII S4-S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII-DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery. © 2017 Hsu et al.

Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain

PubMed Central

Haddad, Georges A.

2011-01-01

The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor. PMID:21518834

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

NASA Astrophysics Data System (ADS)

DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.

2003-04-01

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

Evaluation of insulin secretion and action in New World camelids.

PubMed

Firshman, Anna M; Cebra, Christopher K; Schanbacher, Barbara J; Seaquist, Elizabeth R

2013-01-01

To measure and compare insulin secretion and sensitivity in healthy alpacas and llamas via glucose clamping techniques. 8 llamas and 8 alpacas. Hyperinsulinemic euglycemic clamping (HEC) and hyperglycemic clamping (HGC) were performed on each camelid in a crossover design with a minimum 48-hour washout period between clamping procedures. The HEC technique was performed to measure insulin sensitivity. Insulin was infused IV at 6 mU/min/kg for 4 hours, and an IV infusion of glucose was adjusted to maintain blood glucose concentration at 150 mg/dL. Concentrations of blood glucose and plasma insulin were determined throughout. The HGC technique was performed to assess insulin secretion in response to exogenous glucose infusion. An IV infusion of glucose was administered to maintain blood glucose concentration at 320 mg/dL for 3 hours, and concentrations of blood glucose and plasma insulin were determined throughout. Alpacas and llamas were not significantly different with respect to whole-body insulin sensitivity during HEC or in pancreatic β-cell response during HGC. Alpacas and llamas had markedly lower insulin sensitivity during HEC and markedly lower pancreatic β-cell response during HGC, in comparison with many other species. New World camelids had lower glucose-induced insulin secretion and marked insulin resistance in comparison with other species. This likely contributes to the disorders of fat and glucose metabolism that are common to camelids.

From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology

PubMed Central

Py, Christophe; Martina, Marzia; Diaz-Quijada, Gerardo A.; Luk, Collin C.; Martinez, Dolores; Denhoff, Mike W.; Charrier, Anne; Comas, Tanya; Monette, Robert; Krantis, Anthony; Syed, Naweed I.; Mealing, Geoffrey A. R.

2011-01-01

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells. PMID:22007170

From understanding cellular function to novel drug discovery: the role of planar patch-clamp array chip technology.

PubMed

Py, Christophe; Martina, Marzia; Diaz-Quijada, Gerardo A; Luk, Collin C; Martinez, Dolores; Denhoff, Mike W; Charrier, Anne; Comas, Tanya; Monette, Robert; Krantis, Anthony; Syed, Naweed I; Mealing, Geoffrey A R

2011-01-01

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions - including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.

[Difference in action sites between mecamylamine and hexamethonium on nicotinic receptors of sympathetic neurons].

PubMed

Liu, Wei; Zheng, Jian-Quan; Liu, Zhen-Wei; Li, Li-Jun; Wan, Qin; Liu, Chuan-Gui

2002-12-25

To compare the difference in action sites between mecamylamine (MEC) and hexamethonium (HEX) on nicotinic receptors of sympathetic neurons, we investigated the effects of MEC and HEX on the nicotine-induced currents in cultured superior cervical ganglion neurons by whole-cell patch clamp technique. The IC(50) of MEC and HEX for antagonizing the effect of 0.08 mmol/L nicotine was 0.0012 and 0.0095 mmol/L, respectively. Both MEC and HEX accelerated the desensitization of nicotinic receptors. Furthermore, by comparing their effects at holding potentials 30, 70 and 110 mV, it was indicated that their suppressing effect on the nicotine-induced currents was voltage-dependent. However, different from that of HEX, the inhibitory effect of MEC increased with administering the mixture of MEC and nicotine at intervals of 3 min, indicating a use-dependent effect of MEC. It is concluded that the action site of MEC on nicotinic receptors of sympathetic neurons is different from that of HEX.

Conductivity noise in transmembrane ion channels due to ion concentration fluctuations via diffusion.

PubMed

Mak, D O; Webb, W W

1997-03-01

A Green's function approach is developed from first principles to evaluate the power spectral density of conductance fluctuations caused by ion concentration fluctuations via diffusion in an electrolyte system. This is applied to simple geometric models of transmembrane ion channels to obtain an estimate of the magnitude of ion concentration fluctuation noise in the channel current. Pure polypeptide alamethicin forms stable ion channels with multiple conductance states in artificial phospholipid bilayers isolated onto tips of micropipettes with gigaohm seals. In the single-channel current recorded by voltage-clamp techniques, excess noise was found after the background instrumental noise and the intrinsic Johnson and shot noises were removed. The noise que to ion concentration fluctuations via diffusion was isolated by the dependence of the excess current noise on buffer ion concentration. The magnitude of the concentration fluctuation noise derived from experimental data lies within limits estimated using our simple geometric channel models. Variation of the noise magnitude for alamethicin channels in various conductance states agrees with theoretical prediction.

Nelumbo nucifera leaves extracts inhibit mouse airway smooth muscle contraction.

PubMed

Yang, Xiao; Xue, Lu; Zhao, Qingyang; Cai, Congli; Liu, Qing-Hua; Shen, Jinhua

2017-03-20

Alkaloids extracted from lotus leaves (AELL) can relax vascular smooth muscle. However, whether AELL has a similar relaxant role on airway smooth muscle (ASM) remains unknown. This study aimed to explore the relaxant property of AELL on ASM and the underlying mechanism. Alkaloids were extracted from dried lotus leaves using the high temperature rotary evaporation extraction method. The effects of AELL on mouse ASM tension were studied using force measuring and patch-clamp techniques. It was found that AELL inhibited the high K + or acetylcholine chloride (ACh)-induced precontraction of mouse tracheal rings by 64.8 ± 2.9%, or 48.8 ± 4.7%, respectively. The inhibition was statistically significant and performed in a dose-dependent manner. Furthermore, AELL-induced smooth muscle relaxation was partially mediated by blocking voltage-dependent Ca 2+ channels (VDCC) and non-selective cation channels (NSCC). AELL, which plays a relaxant role in ASM, might be a new complementary treatment to treat abnormal contractions of the trachea and asthma.

Functional analysis of tight junction organization.

PubMed

DiBona, D R

1985-01-01

The functional basis of tight junction design has been examined from the point of view that this rate-limiting barrier to paracellular transport is a multicompartment system. Review of the osmotic sensitivity of these structures points to the need for this sort of analysis for meaningful correlation of structure and function under a range of conditions. A similar conclusion is drawn with respect to results from voltage-clamping protocols where reversal of spontaneous transmural potential difference elicits parallel changes in both structure and function in much the same way as does reversal of naturally occurring osmotic gradients. In each case, it becomes necessary to regard the junction as a functionally polarized structure to account for observations of its rectifying properties. Lastly, the details of experimentally-induced junction deformation are examined in light of current theories of its organization; arguments are presented in favor of the view that the primary components of intramembranous organization (as viewed with freeze-fracture techniques) are lipidic rather than proteinaceous.

Dissection of the Mechanical Impedance Components of the Outer Hair Cell Using a Chloride-Channel Blocker

NASA Astrophysics Data System (ADS)

Harasztosi, Csaba; Gummer, Anthony W.

2011-11-01

The voltage-dependent chloride-channel blocker anthracene-9-carboxylic acid (9AC) has been found to reduce the imaginary but not the real part of the mechanical impedance of the organ of Corti, suggesting that the effective stiffness of outer hair cells (OHCs) is reduced by 9AC. To examine whether 9AC interacts directly with the motor protein prestin to reduce the membrane component of the impedance, the patch-clamp technique in whole-cell configuration was used to measure the nonlinear capacitance (NLC) of isolated OHCs and, as control, prestin-transfected human embryonic kidney 293 (HEK293) cells. Extracellular application of 9AC significantly reduced the NLC of both OHCs and HEK293 cells. Intracellular 9AC did not influence the blocking effect of the extracellular applied drug. These results suggest that 9AC interacts directly with prestin, reducing the effective stiffness of the motor, and that the interaction is extracellular.

High voltage load resistor array

DOEpatents

Lehmann, Monty Ray [Smithfield, VA

2005-01-18

A high voltage resistor comprising an array of a plurality of parallel electrically connected resistor elements each containing a resistive solution, attached at each end thereof to an end plate, and about the circumference of each of the end plates, a corona reduction ring. Each of the resistor elements comprises an insulating tube having an electrode inserted into each end thereof and held in position by one or more hose clamps about the outer periphery of the insulating tube. According to a preferred embodiment, the electrode is fabricated from stainless steel and has a mushroom shape at one end, that inserted into the tube, and a flat end for engagement with the end plates that provides connection of the resistor array and with a load.

Röntgen’s electrode-free elastomer actuators without electromechanical pull-in instability

PubMed Central

Keplinger, Christoph; Kaltenbrunner, Martin; Arnold, Nikita; Bauer, Siegfried

2010-01-01

Electrical actuators made from films of dielectric elastomers coated on both sides with stretchable electrodes may potentially be applied in microrobotics, tactile and haptic interfaces, as well as in adaptive optical elements. Such actuators with compliant electrodes are sensitive to the pull-in electromechanical instability, limiting operational voltages and attainable deformations. Electrode-free actuators driven by sprayed-on electrical charges were first studied by Röntgen in 1880. They withstand much higher voltages and deformations and allow for electrically clamped (charge-controlled) thermodynamic states preventing electromechanical instabilities. The absence of electrodes allows for direct optical monitoring of the actuated elastomer, as well as for designing new 3D actuator configurations and adaptive optical elements. PMID:20173097

Two-Stage Rumen Cannulation Technique in Dairy Cows.

PubMed

Martineau, Roger; Proulx, Julien G; Côrtes, Cristiano; Brito, Andre F; Duffield, Todd F

2015-07-01

To describe a 2-stage rumen cannulation technique for dairy cows. Case series. 172 dairy cows from 2 research institutions. The 2-stage rumen cannulation technique first exteriorized a rumen segment within a wooden clamp, fixing the clamp to the skin with 6 mattress sutures. After 1 week, the necrotic rumen segment was removed, leaving a rumen fistula in which a 7.5 cm cannula was inserted. This was replaced by a 10 cm cannula a further 1 week later. The surgery took an average of 30 minutes. At least 1 assistant is required for the technique. The overall complication frequency was 7/172 (4%). One cow and 1 heifer aborted less than 10 days after surgery. Two late-pregnant heifers died from peritonitis after insertion of the 7.5 cm cannula because of incomplete adhesion of the rumen to the abdominal wall. The exteriorized rumen segment slipped back in the abdomen in 3 cows but was successfully re-clamped prior to insertion of the 7.5 cm cannula. A high success rate was achieved with this 2-stage cannulation technique. Postoperative complications were attributed to delayed adhesion of the rumen, perhaps because of stress-related factors (e.g., transport, mixing with other animals, transition period). © 2015 by The American College of Veterinary Surgeons.

Kinetics of Inhibitory Feedback from Horizontal Cells to Photoreceptors: Implications for an Ephaptic Mechanism

PubMed Central

Warren, Ted J.; Van Hook, Matthew J.; Tranchina, Daniel

2016-01-01

Inhibitory feedback from horizontal cells (HCs) to cones generates center-surround receptive fields and color opponency in the retina. Mechanisms of HC feedback remain unsettled, but one hypothesis proposes that an ephaptic mechanism may alter the extracellular electrical field surrounding photoreceptor synaptic terminals, thereby altering Ca2+ channel activity and photoreceptor output. An ephaptic voltage change produced by current flowing through open channels in the HC membrane should occur with no delay. To test for this mechanism, we measured kinetics of inhibitory feedback currents in Ambystoma tigrinum cones and rods evoked by hyperpolarizing steps applied to synaptically coupled HCs. Hyperpolarizing HCs stimulated inward feedback currents in cones that averaged 8–9 pA and exhibited a biexponential time course with time constants averaging 14–17 ms and 120–220 ms. Measurement of feedback-current kinetics was limited by three factors: (1) HC voltage-clamp speed, (2) cone voltage-clamp speed, and (3) kinetics of Ca2+ channel activation or deactivation in the photoreceptor terminal. These factors totaled ∼4–5 ms in cones meaning that the true fast time constants for HC-to-cone feedback currents were 9–13 ms, slower than expected for ephaptic voltage changes. We also compared speed of feedback to feedforward glutamate release measured at the same cone/HC synapses and found a latency for feedback of 11–14 ms. Inhibitory feedback from HCs to rods was also significantly slower than either measurement kinetics or feedforward release. The finding that inhibitory feedback from HCs to photoreceptors involves a significant delay indicates that it is not due to previously proposed ephaptic mechanisms. SIGNIFICANCE STATEMENT Lateral inhibitory feedback from horizontal cells (HCs) to photoreceptors creates center-surround receptive fields and color-opponent interactions. Although underlying mechanisms remain unsettled, a longstanding hypothesis proposes that feedback is due to ephaptic voltage changes that regulate photoreceptor synaptic output by altering Ca2+ channel activity. Ephaptic processes should occur with no delay. We measured kinetics of inhibitory feedback currents evoked in photoreceptors with voltage steps applied to synaptically coupled HCs and found that feedback is too slow to be explained by ephaptic voltage changes generated by current flowing through continuously open channels in HC membranes. By eliminating the proposed ephaptic mechanism for HC feedback regulation of photoreceptor Ca2+ channels, our data support earlier proposals that synaptic cleft pH changes are more likely responsible. PMID:27683904

Enhanced optical gain clamping for upstream packet based traffic on hybrid WDM/TDM-PON using fiber Bragg grating

NASA Astrophysics Data System (ADS)

Neto, B.; Klingler, A.; Reis, C.; Dionísio, R. P.; Nogueira, R. N.; Teixeira, A. L. J.; André, P. S.

2011-03-01

In this paper, we propose a method to mitigate the temporal power transients arising from Erbium doped fiber amplifiers (EDFAs) on packeted/bursty scenario. The technique, applicable on hybrid WDM/TDM-PON for extended reach, is based on a low power clamping provided by a distributed feedback (DFB) laser and a fiber Bragg grating (FBG). An improvement in the data signal Q factor was achieved keeping the clamping control signal with a low power, accompanied by a maximum reduction in the gain excursion of 1.12 dB.

Endogenous channels in HEK cells and potential roles in HCN ionic current measurements.

PubMed

Varghese, Anthony; Tenbroek, Erica M; Coles, James; Sigg, Daniel C

2006-01-01

A transformed line of human embryonic kidney epithelial cells (HEK 293) is commonly used as an expression system for exogenous ion channel genes. Previously, it has been shown that these cells contain mRNAs for a variety of ion channels. Expression of some of these genes has been confirmed at the protein level. Patch-clamp electrophysiology experiments confirm the presence of multiple ion channels and molecular data agree with pharmacological profiles of identified channels. In this work, we show that endogenous voltage-gated potassium channels in HEK cells are a significant source of outward current at positive potentials. We show that both non-transfected HEK cells and HEK cells transfected with hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have a significant amount of voltage-gated potassium (K(V)) current when certain tail current voltage-clamp protocols are used to assay HCN current activation. Specifically, tail current protocols that use a depolarized holding potential of -40 mV followed by hyperpolarizing pulses (-80 to -140 mV) and then a tail pulse potential of +20 mV indicate K(V) channels undergo closed-state inactivation at the more depolarized holding potential of -40 mV, followed by recovery from inactivation (but no activation) at hyperpolarizing potentials and high amount of activation at the positive tail potential. Our results indicate that pulse protocols with positive tail pulses are inaccurate assays for HCN current in certain HEK cells. Surprisingly, HEK-293 cells were found to contain mRNA for HCN2 and HCN3 although we have not detected a significant and consistent endogenous I(f)-like current in these cells.

Enhanced excitability and down-regulated voltage-gated potassium channels in colonic drg neurons from neonatal maternal separation rats.

PubMed

Luo, Jia-Lie; Qin, Hong-Yan; Wong, Chun-Kit; Tsang, Suk-Ying; Huang, Yu; Bian, Zhao-Xiang

2011-05-01

Irritable bowel syndrome (IBS), characterized mainly by abdominal pain, is a functional bowel disorder. The present study aimed to examine changes in the excitability and the activity of the voltage-gated K(+) channel in dorsal root ganglia (DRG) neurons innervating the colon of rats subjected to neonatal maternal separation (NMS). Colonic DRG neurons from NMS rats as identified by FAST DiI™ labeling showed an increased cell size compared with those from nonhandled (NH) rats. Whole cell current-clamp recordings showed that colonic DRG neurons from NMS rats displayed: 1) depolarized resting membrane potential; 2) increased input resistance; 3) a dramatic reduction in rheobase; and 4) a significant increase in the number of action potentials evoked at twice rheobase. Whole cell voltage-clamp recordings revealed that neurons from both groups exhibited transient A-type (I(A)) and delayed rectifier (I(K)) K(+) currents. Compared with NH rat neurons, the averaged density of I(K) was significantly reduced in NMS rat neurons. Furthermore, the Kv1.2 expression was significantly decreased in NMS rat colonic DRG neurons. These results suggest that NMS increases the excitability of colonic DRG neurons mainly by suppressing the I(K) current, which is likely accounted for by the downregulation of the Kv1.2 expression and somal hypertrophy. This study demonstrates the alteration of delayed rectifier K current and Kv1.2 expression in DRG neurons from IBS model rats, representing a molecular mechanism underlying visceral pain and sensitization in IBS, suggesting the potential of Kv1.2 as a therapeutic target for the treatment of IBS. Copyright © 2011 American Pain Society. Published by Elsevier Inc. All rights reserved.

Defective Fast Inactivation Recovery of Nav1.4 in Congenital Myasthenic Syndrome

PubMed Central

Arnold, W. David; Feldman, Daniel H.; Ramirez, Sandra; He, Liuyuan; Kassar, Darine; Quick, Adam; Klassen, Tara L.; Lara, Marian; Nguyen, Joanna; Kissel, John T.; Lossin, Christoph; Maselli, Ricardo A.

2015-01-01

Objective To describe the unique phenotype and genetic findings in a 57-year-old female with a rare form of congenital myasthenic syndrome (CMS) associated with longstanding muscle fatigability, and to investigate the underlying pathophysiology. Methods We used whole-cell voltage clamping to compare the biophysical parameters of wild-type and Arg1457His-mutant Nav1.4. Results Clinical and neurophysiological evaluation revealed features consistent with CMS. Sequencing of candidate genes indicated no abnormalities. However, analysis of SCN4A, the gene encoding the skeletal muscle sodium channel Nav1.4, revealed a homozygous mutation predicting an arginine-to-histidine substitution at position 1457 (Arg1457His), which maps to the channel’s voltage sensor, specifically D4/S4. Whole-cell patch clamp studies revealed that the mutant required longer hyperpolarization to recover from fast inactivation, which produced a profound use-dependent current attenuation not seen in the wild type. The mutant channel also had a marked hyperpolarizing shift in its voltage dependence of inactivation as well as slowed inactivation kinetics. Interpretation We conclude that Arg1457His compromises muscle fiber excitability. The mutant fast-inactivates with significantly less depolarization, and it recovers only after extended hyperpolarization. The resulting enhancement in its use dependence reduces channel availability, which explains the patient’s muscle fatigability. Arg1457His offers molecular insight into a rare form of CMS precipitated by sodium channel inactivation defects. Given this channel’s involvement in other muscle disorders such as paramyotonia congenita and hyperkalemic periodic paralysis, our study exemplifies how variations within the same gene can give rise to multiple distinct dysfunctions and phenotypes, revealing residues important in basic channel function. PMID:25707578

A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K2P) from a marine sponge

PubMed Central

Wells, Gregory D.; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J.; Pritchard, Erica N.; Leys, Sally P.; Logothetis, Diomedes E.; Boland, Linda M.

2012-01-01

SUMMARY A cDNA encoding a potassium channel of the two-pore domain family (K2P, KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK2P cannot be placed into any of the established functional groups of mammalian K2P channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK2P. In whole cells, non-inactivating, voltage-independent, outwardly rectifying K+ currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC50 ∼30 μmol l–1), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK2P but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K2P channels, the sponge K2P channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pKa 8.18) activated the AquK2P channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K2P channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K2P channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA. PMID:22723483

A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K₂P) from a marine sponge.

PubMed

Wells, Gregory D; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J; Pritchard, Erica N; Leys, Sally P; Logothetis, Diomedes E; Boland, Linda M

2012-07-15

A cDNA encoding a potassium channel of the two-pore domain family (K(2P), KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK(2P) cannot be placed into any of the established functional groups of mammalian K(2P) channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK(2P). In whole cells, non-inactivating, voltage-independent, outwardly rectifying K(+) currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC(50) ∼30 μmol l(-1)), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK(2P) but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K(2P) channels, the sponge K(2P) channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pK(a) 8.18) activated the AquK(2P) channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K(2P) channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K(2P) channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA.

Regulation of Kv2.1 K+ conductance by cell surface channel density

PubMed Central

Fox, Philip D.; Loftus, Rob J.; Tamkun, Michael M.

2013-01-01

The Kv2.1 voltage-gated K+ channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are non-conducting. Using TIRF microscopy the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared to K+ channel conductance measured by whole-cell voltage-clamp of the same cell. This approach indicated that as channel density increases non-clustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the non-conducting state with 17% conducting K+ at higher surface densities. The non-conducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immuno-fluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared to the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 days, respectively. Together these data indicate that the non-conducting state depends primarily on surface density as opposed to cluster location and that this non-conducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K+ conductance further supports a non-conducting role for Kv2.1 in excitable tissues. PMID:23325261

Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sujeong, E-mail: crystalee@gmail.com; Lee, Hyang-Ae, E-mail: hyangaelee@gmail.com; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799

2016-04-01

The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effectsmore » of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD{sub 90}) and 50% (APD{sub 50}) repolarization, reduced the maximum upstroke velocity (dV/dt{sub max}) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including I{sub Kr}, I{sub Ks}, I{sub Na}, and I{sub Ca}. Among them, I{sub Kr} and I{sub Na} showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K{sup +} channels. - Highlights: • Nefazodone prolonged APD and decreased upstroke velocity of APs in hiPSC-CMs. • Nefazodone inhibited cardiac ion channels, especially I{sub Kr} and I{sub Na}, in hiPSC-CMs. • Nefazodone-induced AP changes are mainly the result of I{sub Kr} and I{sub Na} inhibition. • hiPSC-CMs are sensitive model to validate nefazodone-induced cardiotoxicity. • hiPSC-CMs provide an integrated approach for evaluating mechanism of drug actions.« less

Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

PubMed

Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

2012-06-01

The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

Welding and joining: A compilation

NASA Technical Reports Server (NTRS)

1975-01-01

A compilation is presented of NASA-developed technology in welding and joining. Topics discussed include welding equipment, techniques in welding, general bonding, joining techniques, and clamps and holding fixtures.

Control of Grid Connected Photovoltaic System Using Three-Level T-Type Inverter

NASA Astrophysics Data System (ADS)

Zorig, Abdelmalik; Belkeiri, Mohammed; Barkat, Said; Rabhi, Abdelhamid

2016-08-01

Three-level T-Type inverter (3LT2I) topology has numerous advantageous compared to three-level neutral-point-clamped (NPC) inverter. The main benefits of 3LT2I inverter are the efficiency, inverter cost, switching losses, and the quality of output voltage waveforms. In this paper, a photovoltaic distributed generation system based on dual-stage topology of DC-DC boost converter and 3LT2I is introduced. To that end, a decoupling control strategy of 3LT2I is proposed to control the current injected into the grid, reactive power compensation, and DC-link voltage. The resulting system is able to extract the maximum power from photovoltaic generator, to achieve sinusoidal grid currents, and to ensure reactive power compensation. The voltage-balancing control of two split DC capacitors of the 3LT2I is achieved using three-level space vector modulation with balancing strategy based on the effective use of the redundant switching states of the inverter voltage vectors. The proposed system performance is investigated at different operating conditions.

Ionic Dependence of Reversal Voltage of the Light Response in Limulus Ventral Photoreceptors

PubMed Central

Brown, J. E.; Mote, M. I.

1974-01-01

The light-induced current as measured using a voltage clamp (holding voltage at resting potential) is attenuated when sodium ions in the bathing solution, Nao, are replaced by Tris, choline, or Li or when NaCl is replaced by sucrose. After replacement of NaCl by sucrose, the reversal voltage, V rev, for the light response becomes more negative. In this case, the slope of the V rev vs. log Nao near Nao = 425 mM is approximately 55 mV/decade increase of Nao (mean for 13 cells). The slope decreases at lower values of Nao. Choline is not impermeant and partially substitutes for Na; the slope of V rev vs. log Nao is 20 mV/decade (mean for three cells). V rev does not change when Na is replaced by Li. Decreases in the bath concentrations of Ca, Mg, Cl, or K do not affect V rev. When Nao = 212 mM, V rev becomes more positive when Ko is increased. Thus, light induces a change in membrane permeability to Na and probably also to K. PMID:4817353

Inhibitory effects of magnolol on voltage-gated Na+ and K+ channels of NG108-15 cells.

PubMed

Gong, Chi-Li; Wong, Kar-Lok; Cheng, Ka-Shun; Kuo, Chang-Shin; Chao, Chia-Chia; Tsai, Min-Fan; Leung, Yuk-Man

2012-05-05

Magnolol, a polyphenolic compound isolated from Houpu, a Chinese herb from the bark of Magnolia officinalis, has been reported to have in vitro and in vivo neuroprotective effects. In spite of these reported beneficial effects, studies on the direct impact of magnolol on neuronal ion channels have been scarce. Whether magnolol affects voltage-gated Na(+) channels (VGSC) and voltage-gated K(+) (Kv) channels is unknown. Using the whole-cell voltage-clamp method, we studied the effects of magnolol on voltage-gated ion channels in neuronal NG108-15 cells. Magnolol inhibited VGSC channels with mild state-dependence (IC(50) of 15 and 30 μM, at holding potentials of -70 and -100 mV, respectively). No frequency-dependence was observed in magnolol block. Magnolol caused a left-shift of 18 mV in the steady-state inactivation curve but did not affect the voltage-dependence of activation. Magnolol inhibited Kv channels with an IC(50) of 21 μM, and it caused a 20-mV left-shift in the steady-state inactivation curve without affecting the voltage-dependence of activation. In conclusion, magnolol is an inhibitor of both VGSC and Kv channels and these inhibitory effects may in part contribute to some of the reported neuroprotective effects of magnolol. Copyright © 2012 Elsevier B.V. All rights reserved.

Local anesthetics disrupt energetic coupling between the voltage-sensing segments of a sodium channel.

PubMed

Muroi, Yukiko; Chanda, Baron

2009-01-01

Local anesthetics block sodium channels in a state-dependent fashion, binding with higher affinity to open and/or inactivated states. Gating current measurements show that local anesthetics immobilize a fraction of the gating charge, suggesting that the movement of voltage sensors is modified when a local anesthetic binds to the pore of the sodium channel. Here, using voltage clamp fluorescence measurements, we provide a quantitative description of the effect of local anesthetics on the steady-state behavior of the voltage-sensing segments of a sodium channel. Lidocaine and QX-314 shifted the midpoints of the fluorescence-voltage (F-V) curves of S4 domain III in the hyperpolarizing direction by 57 and 65 mV, respectively. A single mutation in the S6 of domain IV (F1579A), a site critical for local anesthetic block, abolished the effect of QX-314 on the voltage sensor of domain III. Both local anesthetics modestly shifted the F-V relationships of S4 domain IV toward hyperpolarized potentials. In contrast, the F-V curve of the S4 domain I was shifted by 11 mV in the depolarizing direction upon QX-314 binding. These antagonistic effects of the local anesthetic indicate that the drug modifies the coupling between the voltage-sensing domains of the sodium channel. Our findings suggest a novel role of local anesthetics in modulating the gating apparatus of the sodium channel.

Effect of Complete Syndesmotic Disruption and Deltoid Injuries and Different Reduction Methods on Ankle Joint Contact Mechanics.

PubMed

LaMothe, Jeremy; Baxter, Josh R; Gilbert, Susannah; Murphy, Conor I; Karnovsky, Sydney C; Drakos, Mark C

2017-06-01

Syndesmotic injuries can be associated with poor patient outcomes and posttraumatic ankle arthritis, particularly in the case of malreduction. However, ankle joint contact mechanics following a syndesmotic injury and reduction remains poorly understood. The purpose of this study was to characterize the effects of a syndesmotic injury and reduction techniques on ankle joint contact mechanics in a biomechanical model. Ten cadaveric whole lower leg specimens with undisturbed proximal tibiofibular joints were prepared and tested in this study. Contact area, contact force, and peak contact pressure were measured in the ankle joint during simulated standing in the intact, injured, and 3 reduction conditions: screw fixation with a clamp, screw fixation without a clamp (thumb technique), and a suture-button construct. Differences in these ankle contact parameters were detected between conditions using repeated-measures analysis of variance. Syndesmotic disruption decreased tibial plafond contact area and force. Syndesmotic reduction did not restore ankle loading mechanics to values measured in the intact condition. Reduction with the thumb technique was able to restore significantly more joint contact area and force than the reduction clamp or suture-button construct. Syndesmotic disruption decreased joint contact area and force. Although the thumb technique performed significantly better than the reduction clamp and suture-button construct, syndesmotic reduction did not restore contact mechanics to intact levels. Decreased contact area and force with disruption imply that other structures are likely receiving more loads (eg, medial and lateral gutters), which may have clinical implications such as the development of posttraumatic arthritis.

A novel monolithic piezoelectric actuated flexure-mechanism based wire clamp for microelectronic device packaging.

PubMed

Liang, Cunman; Wang, Fujun; Tian, Yanling; Zhao, Xingyu; Zhang, Hongjie; Cui, Liangyu; Zhang, Dawei; Ferreira, Placid

2015-04-01

A novel monolithic piezoelectric actuated wire clamp is presented in this paper to achieve fast, accurate, and robust microelectronic device packaging. The wire clamp has compact, flexure-based mechanical structure and light weight. To obtain large and robust jaw displacements and ensure parallel jaw grasping, a two-stage amplification composed of a homothetic bridge type mechanism and a parallelogram leverage mechanism was designed. Pseudo-rigid-body model and Lagrange approaches were employed to conduct the kinematic, static, and dynamic modeling of the wire clamp and optimization design was carried out. The displacement amplification ratio, maximum allowable stress, and natural frequency were calculated. Finite element analysis (FEA) was conducted to evaluate the characteristics of the wire clamp and wire electro discharge machining technique was utilized to fabricate the monolithic structure. Experimental tests were carried out to investigate the performance and the experimental results match well with the theoretical calculation and FEA. The amplification ratio of the clamp is 20.96 and the working mode frequency is 895 Hz. Step response test shows that the wire clamp has fast response and high accuracy and the motion resolution is 0.2 μm. High speed precision grasping operations of gold and copper wires were realized using the wire clamper.

The use of virtual ground to control transmembrane voltages and measure bilayer currents in serial arrays of droplet interface bilayers

NASA Astrophysics Data System (ADS)

Sarles, Stephen A.

2013-09-01

The droplet interface bilayer (DIB) is a simple technique for constructing a stable lipid bilayer at the interface of two lipid-encased water droplets submerged in oil. Networks of DIBs formed by connecting more than two droplets constitute a new form of modular biomolecular smart material, where the transduction properties of a single lipid bilayer can affect the actions performed at other interface bilayers in the network via diffusion through the aqueous environments of shared droplet connections. The passive electrical properties of a lipid bilayer and the arrangement of droplets that determine the paths for transport in the network require specific electrical control to stimulate and interrogate each bilayer. Here, we explore the use of virtual ground for electrodes inserted into specific droplets in the network and employ a multichannel patch clamp amplifier to characterize bilayer formation and ion-channel activity in a serial DIB array. Analysis of serial connections of DIBs is discussed to understand how assigning electrode connections to the measurement device can be used to measure activity across all lipid membranes within a network. Serial arrays of DIBs are assembled using the regulated attachment method within a multi-compartment flexible substrate, and wire-type electrodes inserted into each droplet compartment of the substrate enable the application of voltage and measurement of current in each droplet in the array.

Rapid communication between neurons and astrocytes in primary cortical cultures.

PubMed

Murphy, T H; Blatter, L A; Wier, W G; Baraban, J M

1993-06-01

The identification of neurotransmitter receptors and voltage-sensitive ion channels on astrocytes (reviewed by Barres, 1991) has renewed interest in how these cells respond to neuronal activity. To investigate the physiology of neuron astrocyte signaling, we have employed primary cortical cultures that contain both neuronal and glial cells. As the neurons in these cultures exhibit synchronous spontaneous synaptic activity, we have used both calcium imaging and whole-cell recording techniques to identify physiological activity in astrocytes related to neuronal activity. Whole-cell voltage-clamp records from astrocytes revealed rapid inward currents that coincide with bursts of electrical activity in neighboring neurons. Calcium imaging studies demonstrate that these currents in astrocytes are not always associated with slowly propagating calcium waves. Inclusion of the dye Lucifer yellow within patch pipettes confirmed that astrocytes are extensively coupled to each other but not to adjacent neurons, indicating that the currents observed are not due to gap junction connections between these cell types. These currents do not reflect widespread diffusion of glutamate or potassium released during neuronal activity since a population of small, round, multipolar presumed glial cells that are not dye coupled to adjacent cells did not display electrical currents coincident with neuronal firing, even though they respond to locally applied glutamate and potassium. These findings indicate that, in addition to the relatively slow signaling conveyed by calcium waves, astrocytes also display rapid electrical responses to neuronal activity.

VEGF attenuated increase of outward delayed-rectifier potassium currents in hippocampal neurons induced by focal ischemia via PI3-K pathway.

PubMed

Wu, K W; Yang, P; Li, S S; Liu, C W; Sun, F Y

2015-07-09

We recently indicated that the vascular endothelial growth factor (VEGF) protects neurons against hypoxic death via enhancement of tyrosine phosphorylation of Kv1.2, an isoform of the delayed-rectifier potassium channels through activation of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway. The present study investigated whether VEGF could attenuate ischemia-induced increase of the potassium currents in the hippocampal pyramidal neurons of rats after ischemic injury. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) to induce brain ischemia. The whole-cell patch-clamp technique was used to record the potassium currents of hippocampal neurons in brain slices from the ischemically injured brains of the rats 24h after MCAO. We detected that transient MCAO caused a significant increase of voltage-gated potassium currents (Kv) and outward delayed-rectifier potassium currents (IK), but not outward transient potassium currents (IA), in the ipsilateral hippocampus compared with the sham. Moreover, we found that VEGF could acutely, reversibly and voltage-dependently inhibit the ischemia-induced IK increase. This inhibitory effect of VEGF could be completely abolished by wortmannin, an inhibitor of PI3-K. Our data indicate that VEGF attenuates the ischemia-induced increase of IK via activation of the PI3-K signaling pathway. Published by Elsevier Ltd.

Molecular cloning and functional expression of the K+ channel KV7.1 and the regulatory subunit KCNE1 from equine myocardium.

PubMed

Pedersen, Philip J; Thomsen, Kirsten B; Flak, Jon B; Tejada, Maria A; Hauser, Frank; Trachsel, Dagmar; Buhl, Rikke; Kalbfleisch, Theodore; DePriest, Michael Scott; MacLeod, James N; Calloe, Kirstine; Klaerke, Dan A

2017-08-01

The voltage-gated K + -channel K V 7.1 and the subunit KCNE1, encoded by the KCNQ1 and KCNE1 genes, respectively, are responsible for termination of the cardiac action potential. In humans, mutations in these genes can predispose patients to arrhythmias and sudden cardiac death (SCD). To characterize equine K V 7.1/KCNE1 currents and compare them to human K V 7.1/KCNE1 currents to determine whether K V 7.1/KCNE1 plays a similar role in equine and human hearts. mRNA encoding K V 7.1 and KCNE1 was isolated from equine hearts, sequenced, and cloned into expression vectors. The channel subunits were heterologously expressed in Xenopus laevis oocytes or CHO-K1 cells and characterized using voltage-clamp techniques. Equine K V 7.1/KCNE1 expressed in CHO-K1 cells exhibited electrophysiological properties that are overall similar to the human orthologs; however, a slower deactivation was found which could result in more open channels at fast rates. The results suggest that the equine K V 7.1/KCNE1 channel may be important for cardiac repolarization and this could indicate that horses are susceptible to SCD caused by mutations in KCNQ1 and KCNE1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Activation by intracellular GDP, metabolic inhibition and pinacidil of a glibenclamide-sensitive K-channel in smooth muscle cells of rat mesenteric artery.

PubMed Central

Zhang, H; Bolton, T B

1995-01-01

1. Single-channel recordings were made from cell-attached and isolated patches, and whole-cell currents were recorded under voltage clamp from single smooth muscle cells obtained by enzymic digestion of a small branch of the rat mesenteric artery. 2. In single voltage-clamped cells 1 mM uridine diphosphate (UDP) or guanidine diphosphate (GDP) added to the pipette solution, or pinacidil (100 microM) a K-channel opener (KCO) applied in the bathing solution, evoked an outward current of up to 100pA which was blocked by glibenclamide (10 microM). In single cells from which recordings were made by the 'perforated patch' (nystatin pipette) technique, metabolic inhibition by 1 mM NaCN and 10 mM 2-deoxy-glucose also evoked a similar glibenclamide-sensitive current. 3. Single K-channel activity was observed in cell-attached patches only infrequently unless the metabolism of the cell was inhibited, whereupon channel activity blocked by glibenclamide was seen; pinacidil applied to the cell evoked similar glibenclamide-sensitive channel activity. If the patch was pulled off the cell to form an isolated inside-out patch, similar glibenclamide-sensitive single-channel currents were observed in the presence of UDP and/or pinacidil to those seen in cell-attached mode; channel conductance was 20 pS (60:130 K-gradient) and openings showed no voltage-dependence and noisy inward currents, typical of the nucleoside diphosphate (NDP) activated K-channel (KNDP) seen previously in rabbit portal vein. 4. Formation of an isolated inside-out patch into an ATP-free solution did not increase the probability of channel opening which declined with time even when some single-channel activity had occurred in the cell-attached mode before detachment. However, application of 1 mM UDP or GDP, but not ATP, to inside-out patches evoked single-channel activity. Application of ATP-free solution to isolated patches, previously exposed to ATP and in which channel activity had been seen, did not evoke channel activity. 5. It is concluded that small conductance K-channels (KNDP) open in smooth muscle cells from this small artery in response to UDP or GDP acting from the inside, or pinacidil acting from the outside; the same channels open during inhibition of metabolism presumably mainly due to the rise in nucleoside diphosphates, but a fall in the ATP concentration on the inside of the channel did not by itself evoke channel activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7735693

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

PubMed

Annecchino, Luca A; Morris, Alexander R; Copeland, Caroline S; Agabi, Oshiorenoya E; Chadderton, Paul; Schultz, Simon R

2017-08-30

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Industrializing electrophysiology: HT automated patch clamp on SyncroPatch® 96 using instant frozen cells.

PubMed

Polonchuk, Liudmila

2014-01-01

Patch-clamping is a powerful technique for investigating the ion channel function and regulation. However, its low throughput hampered profiling of large compound series in early drug development. Fortunately, automation has revolutionized the area of experimental electrophysiology over the past decade. Whereas the first automated patch-clamp instruments using the planar patch-clamp technology demonstrated rather a moderate throughput, few second-generation automated platforms recently launched by various companies have significantly increased ability to form a high number of high-resistance seals. Among them is SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich, Germany), a fully automated giga-seal patch-clamp system with the highest throughput on the market. By recording from up to 96 cells simultaneously, the SyncroPatch(®) 96 allows to substantially increase throughput without compromising data quality. This chapter describes features of the innovative automated electrophysiology system and protocols used for a successful transfer of the established hERG assay to this high-throughput automated platform.

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation

PubMed Central

Yu, Alec; Zhu, Wandi; Silva, Jonathan R.; Ruben, Peter C.

2017-01-01

E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation. PMID:28898267

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation.

PubMed

Peters, Colin H; Yu, Alec; Zhu, Wandi; Silva, Jonathan R; Ruben, Peter C

2017-01-01

E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation.

A retrospective analysis of laparoscopic partial nephrectomy with segmental renal artery clamping and factors that predict postoperative renal function.

PubMed

Li, Pu; Qin, Chao; Cao, Qiang; Li, Jie; Lv, Qiang; Meng, Xiaoxin; Ju, Xiaobing; Tang, Lijun; Shao, Pengfei

2016-10-01

To evaluate the feasibility and efficiency of laparoscopic partial nephrectomy (LPN) with segmental renal artery clamping, and to analyse the factors affecting postoperative renal function. We conducted a retrospective analysis of 466 consecutive patients undergoing LPN using main renal artery clamping (group A, n = 152) or segmental artery clamping (group B, n = 314) between September 2007 and July 2015 in our department. Blood loss, operating time, warm ischaemia time (WIT) and renal function were compared between groups. Univariable and multivariable linear regression analyses were applied to assess the correlations of selected variables with postoperative glomerular filtration rate (GFR) reduction. Volumetric data and estimated GFR of a subset of 60 patients in group B were compared with GFR to evaluate the correlation between these functional variables and preserved renal function after LPN. The novel technique slightly increased operating time, WIT and intra-operative blood loss (P < 0.001), while it provided better postoperative renal function (P < 0.001) compared with the conventional technique. The blocking method and tumour characteristics were independent factors affecting GFR reduction, while WIT was not an independent factor. Correlation analysis showed that estimated GFR presented better correlation with GFR compared with kidney volume (R(2) = 0.794 cf. R(2) = 0.199) in predicting renal function after LPN. LPN with segmental artery clamping minimizes warm ischaemia injury and provides better early postoperative renal function compared with clamping the main renal artery. Kidney volume has a significantly inferior role compared with eGFR in predicting preserved renal function. © 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.

Effects of Imperfect Dynamic Clamp: Computational and Experimental Results

PubMed Central

Bettencourt, Jonathan C.; Lillis, Kyle P.; White, John A.

2008-01-01

In the dynamic clamp technique, a typically nonlinear feedback system delivers electrical current to an excitable cell that represents the actions of “virtual” ion channels (e.g., channels that are gated by local membrane potential or by electrical activity in neighboring biological or virtual neurons). Since the conception of this technique, there have been a number of different implementations of dynamic clamp systems, each with differing levels of flexibility and performance. Embedded hardware-based systems typically offer feedback that is very fast and precisely timed, but these systems are often expensive and sometimes inflexible. PC-based systems, on the other hand, allow the user to write software that defines an arbitrarily complex feedback system, but real-time performance in PC-based systems can be deteriorated by imperfect real-time performance. Here we systematically evaluate the performance requirements for artificial dynamic clamp knock-in of transient sodium and delayed rectifier potassium conductances. Specifically we examine the effects of controller time step duration, differential equation integration method, jitter (variability in time step), and latency (the time lag from reading inputs to updating outputs). Each of these control system flaws is artificially introduced in both simulated and real dynamic clamp experiments. We demonstrate that each of these errors affect dynamic clamp accuracy in a way that depends on the time constants and stiffness of the differential equations being solved. In simulations, time steps above 0.2 ms lead to catastrophic alteration of spike shape, but the frequency-vs.-current relationship is much more robust. Latency (the part of the time step that occurs between measuring membrane potential and injecting re-calculated membrane current) is a crucial factor as well. Experimental data are substantially more sensitive to inaccuracies than simulated data. PMID:18076999

[L-glutamate-activated conductivity in the membrane of isolated pyramidal neurons of the rat hippocampus].

PubMed

Kiksin, N I; Tsyndrenko, A Ia

1985-01-01

Isolated pyramidal neurons from rat hippocampus were investigated under conditions of intracellular perfusion and voltage clamp using the method of rapid application of extracellular solution. It was found that the L-glutamate-activated current was carried by sodium and potassium ions with PK+/PNa+ ratio about 0.6. The dose-response relationship demonstrated one to one interaction between L-glutamate and membrane receptor with Kd value about 1.1 mmol/l.

Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology.

PubMed

Abbott, Jeffrey; Ye, Tianyang; Ham, Donhee; Park, Hongkun

2018-03-20

Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane, clarifying how the nanoelectrode attains intracellular access. This understanding will be translated into a circuit model for the nanobio interface, which we will then use to lay out the strategies for improving the interface. The intracellular interface of the nanoelectrode is currently inferior to that of the patch clamp electrode; reaching this benchmark will be an exciting challenge that involves optimization of electrode geometries, materials, chemical modifications, electroporation protocols, and recording/stimulation electronics, as we describe in the Account. Another important theme of this Account, beyond the optimization of the individual nanoelectrode-cell interface, is the scalability of the nanoscale electrodes. We will discuss this theme using a recent development from our groups as an example, where an array of ca. 1000 nanoelectrode pixels fabricated on a CMOS integrated circuit chip performs parallel intracellular recording from a few hundreds of cardiomyocytes, which marks a new milestone in electrophysiology.

Fault-tolerant drive electronics for a Xinetics deformable mirror at GeMS DM0

NASA Astrophysics Data System (ADS)

Barberio, Michael J.

2016-07-01

Gemini South is replacing one of the (3) CILAS DMs with a 349-actuator Xinetics DM in its GeMS MCAO system. Xinetics mirrors operate over a 40-100V dynamic range and require that inter-actuator stroke differences are limited to half-scale; each actuator must be within 30V of its neighbor to prevent mechanical stress and possible face sheet separation. A robust way to implement this protection is to use high power transient voltage suppressors (TVSs) as a 2D-mesh between the amplifiers and mirror, but this has system implications. A sustained clamp condition dissipates significant power in the devices, and if an actuator fails as short (which occurred once with the DM in a thermal chamber), the system is subject to a cascade failure event as multiple outputs drive the shorted actuator through the TVS network. This latter risk is readily resolved by using series fuses to the DM. In this third-generation driver, current sensing and logic inhibit amplifier outputs after a sustained TVS clamp condition or shorted output, and LED indicators show the location. Redundant thermal sensing is used on modular TVS row and column boards. A second 2D-mesh of high impedance resistors after the fuses will hold an unpowered channel to the average voltage of its neighbors, with a negligible influence function. A Failure Modes and Effects Analysis shows significant fault tolerance.

Stable operation of a high-power piezoelectric transformer comprising two identical bolt-clamped Langevin-type transducers and a stepped horn

NASA Astrophysics Data System (ADS)

Adachi, Kazunari; Suzuki, Kohei; Shibamata, Yuki

2018-06-01

We previously developed a 100 W piezoelectric transformer comprising two identical bolt-clamped Langevin-type transducers (BLTs) and a stepped horn whose cross-sectional area ratio determines the specified step-up voltage transformation ratio. Unlike conventional piezoelectric transformers, this transformer is driven at a frequency quite near its mechanical resonance, and thus can be mechanically held firmly at its clearly identified vibratory node without mechanical energy loss. However, it has been revealed that the high-power operation of the transformer often becomes very unstable owing to the “jumping and dropping” phenomena first found by Takahashi and Hirose [Jpn. J. Appl. Phys. 31, 3055 (1992)]. To avoid this instability, we have investigated the peculiar phenomena, and found that they can be attributed to a heavily distorted electric field inside the piezoelectric ceramic disks of the BLT on the primary side of the transformer being driven by a low-impedance voltage source near the mechanical resonance. The resultant concentration of the electric field leads to the local reversal of piezoelectric polarization in every half period of the vibration, viz., the instability. Consequently, we have developed a scheme for the steady high-power operation of this type of piezoelectric transformer and examined its validity experimentally. The method has eventually improved the linearity and power transfer efficiency of the transformer significantly.

‘Sleepy’ inward rectifier channels in guinea-pig cardiomyocytes are activated only during strong hyperpolarization

PubMed Central

Liu, Gong Xin; Daut, Jürgen

2002-01-01

K+ channels of isolated guinea-pig cardiomyocytes were studied using the patch-clamp technique. At transmembrane potentials between −120 and −220 mV we observed inward currents through an apparently novel channel. The novel channel was strongly rectifying, no outward currents could be recorded. Between −200 and −160 mV it had a slope conductance of 42.8 ± 3.0 pS (s.d.; n = 96). The open probability (Po) showed a sigmoid voltage dependence and reached a maximum of 0.93 at −200 mV, half-maximal activation was approximately −150 mV. The voltage dependence of Po was not affected by application of 50 μm isoproterenol. The open-time distribution could be described by a single exponential function, the mean open time ranged between 73.5 ms at −220 mV and 1.4 ms at −160 mV. At least two exponential components were required to fit the closed time distribution. Experiments with different external Na+, K+ and Cl− concentrations suggested that the novel channel is K+ selective. Extracellular Ba2+ ions gave rise to a voltage-dependent reduction in Po by inducing long closed states; Cs+ markedly reduced mean open time at −200 mV. In cell-attached recordings the novel channel frequently converted to a classical inward rectifier channel, and vice versa. This conversion was not voltage dependent. After excision of the patch, the novel channel always converted to a classical inward rectifier channel within 0–3 min. This conversion was not affected by intracellular Mg2+, phosphatidylinositol (4,5)-bisphosphate or spermine. Taken together, our findings suggest that the novel K+ channel represents a different ‘mode’ of the classical inward rectifier channel in which opening occurs only at very negative potentials. PMID:11897847

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

PubMed Central

Emirandetti, Amanda; Lewicka, Michalina; Hermanson, Ola; Fisahn, André

2010-01-01

Background Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. Methodology/Principal Findings In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. Conclusions/Significance Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture. PMID:21079795

Difference of acute dissociation and 1-day culture on the electrophysiological properties of rat dorsal root ganglion neurons.

PubMed

Song, Yuanlong; Zhang, Miaomiao; Tao, Xiaoqing; Xu, Zifen; Zheng, Yunjie; Zhu, Minjie; Zhang, Liangpin; Qiao, Jinhan; Gao, Linlin

2018-01-19

The dissociated dorsal root ganglion (DRG) neurons with or without culture were widely used for investigation of their electrophysiological properties. The culture procedures, however, may alter the properties of these neurons and the effects are not clear. In the present study, we recorded the action potentials (AP) and the voltage-gated Na + , K + , and Ca 2+ currents with patch clamp technique and measured the mRNA of Nav1.6-1.9 and Cav2.1-2.2 with real-time PCR technique from acutely dissociated and 1-day (1-d) cultured DRG neurons. The effects of the nerve growth factor (NGF) on the expression of Nav1.6-1.9 and Cav2.1-2.2 were evaluated. The neurons were classified as small (DRG-S), medium (DRG-M), and large (DRG-L), according to their size frequency distribution pattern. We found 1-d culture increased the AP size but reduced the excitability, and reduced the voltage-gated Na + and Ca 2+ currents and their corresponding mRNA expression in all types of neurons. The lack of NGF in the culture medium may contribute to the reduced Na + and Ca 2+ current, as the application of NGF recovered some of the reduced transcripts (Nav1.9, Cav2.1, and Cav2.2). 1-d culture showed neuron-type specific effects on some of the AP properties: it increased the maximum AP depolarizing rate (MDR) and hyperpolarized the resting membrane potential (RP) in DRG-M and DRG-L neurons, but slowed the maximum AP repolarizing rate (MRR) in DRG-S neurons. In conclusion, the 1-d cultured neurons had different properties with those of the acutely dissociated neurons, and lack of NGF may contribute to some of these differences.

BKCa currents are enriched in a subpopulation of adult rat cutaneous nociceptive dorsal root ganglion neurons

PubMed Central

Zhang, Xiu-Lin; Mok, Lee-Peng; Katz, Elizabeth J; Gold, Michael S.

2010-01-01

The biophysical properties and distribution of voltage-dependent, Ca2+-modulated K+ (BKCa) currents among subpopulations of acutely dissociated DiI labeled cutaneous sensory neurons from the adult rat were characterized with whole cell patch clamp techniques. BKCa currents were isolated from total K+ current with iberiotoxin, charybdotoxin, or paxilline. There was considerable variability in biophysical properties of BKCa currents. There was also variability in the distribution of BKCa current among subpopulations of cutaneous DRG neurons. While present in each of the subpopulations defined by cell body size, IB4 binding or capsaicin sensitivity, BKCa current was present in vast majority (>90%) of small diameter IB4+ neurons but was present in only a minority of neurons in subpopulations defined by other criteria (i.e., small diameter IB4−). Current clamp analysis indicated that in IB4+ neurons, BKCa currents contribute to the repolarization of the action potential and adaptation in response to sustained membrane depolarization, while playing little role in the determination of action potential threshold. RT-PCR analysis of mRNA collected from whole DRG revealed the presence of multiple splice variants of the BKCa channel α-subunit, rslo and all 4 of the accessory β subunits, suggesting that heterogeneity in the biophysical and pharmacological properties of BKCa current in cutaneous neurons, reflects, at least in part, the differential distribution of splice variants and/or β subunits. Because even a small decrease in BKCa current appears to have a dramatic influence on excitability, modulation of this current may contribute to sensitization of nociceptive afferents observed following tissue injury. PMID:20105244

Low K+-induced hyperpolarizations trigger transient depolarizations and action potentials in rabbit ventricular myocytes

PubMed Central

Akuzawa-Tateyama, M; Tateyama, M; Ochi, R

1998-01-01

The effects of large reductions of [K+]o on membrane potential were studied in isolated rabbit ventricular myocytes using the whole-cell patch clamp technique.Decreasing [K+]o from the normal level of 5.4 mm to 0.1 mm increased resting membrane potential (Vrest) from −75.6 ± 0.3 to −140.3 ± 1.9 mV (means ± s.e.m; n = 127), induced irregular, transient depolarizations with mean maximal amplitudes of 19.5 ± 1.5 mV and elicited action potentials in 56.7 % of trials. The action potentials exhibited overshoots of 37.9 ± 1.5 mV (n = 72) and sustained plateaux.Addition of 0.1 mm La3+ in the presence of 0.1 mm[K+]o significantly increased Vrest but decreased the amplitude of transient depolarizations and suppressed the firing of action potentials.Replacement of external Na+ or Cl− with N-methyl-D-glucamine or aspartate, respectively, or internal dialysis with 10 mm EGTA or BAPTA had little effect on low [K+]o-induced membrane potential changes.Hyperpolarizing voltage clamp pulses to potentials between −110 and −200 mV activated irregular inward currents that increased in amplitude and frequency with increasing hyperpolarization and were depressed by 0.1 mm La3+.The generation of transient depolarizations by low [K+]o can be explained as being a consequence of decreasing the inward rectifier K+ current (IK1) and the appearance of inward currents reflecting electroporation resulting from strong electric fields across the membrane. PMID:9824717

A simple method for characterizing passive and active neuronal properties: application to striatal neurons.

PubMed

Lepora, Nathan F; Blomeley, Craig P; Hoyland, Darren; Bracci, Enrico; Overton, Paul G; Gurney, Kevin

2011-11-01

The study of active and passive neuronal dynamics usually relies on a sophisticated array of electrophysiological, staining and pharmacological techniques. We describe here a simple complementary method that recovers many findings of these more complex methods but relies only on a basic patch-clamp recording approach. Somatic short and long current pulses were applied in vitro to striatal medium spiny (MS) and fast spiking (FS) neurons from juvenile rats. The passive dynamics were quantified by fitting two-compartment models to the short current pulse data. Lumped conductances for the active dynamics were then found by compensating this fitted passive dynamics within the current-voltage relationship from the long current pulse data. These estimated passive and active properties were consistent with previous more complex estimations of the neuron properties, supporting the approach. Relationships within the MS and FS neuron types were also evident, including a graduation of MS neuron properties consistent with recent findings about D1 and D2 dopamine receptor expression. Application of the method to simulated neuron data supported the hypothesis that it gives reasonable estimates of membrane properties and gross morphology. Therefore detailed information about the biophysics can be gained from this simple approach, which is useful for both classification of neuron type and biophysical modelling. Furthermore, because these methods rely upon no manipulations to the cell other than patch clamping, they are ideally suited to in vivo electrophysiology. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Analysis of whole-cell currents by patch clamp of guinea-pig myenteric neurones in intact ganglia

PubMed Central

Rugiero, François; Gola, Maurice; Kunze, Wolf A A; Reynaud, Jean-Claude; Furness, John B; Clerc, Nadine

2002-01-01

Whole-cell patch-clamp recordings taken from guinea-pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of −47 ± 6 mV and input resistances (Rin) of 713 ± 49 MΩ at voltages ranging from −90 to −40 mV. At more negative levels, activation of a time-independent, caesium-sensitive, inward-rectifier current (IKir) decreased Rin to 103 ± 10 MΩ. AH neurones had resting potentials of −57 ± 4 mV and Rin was 502 ± 27 MΩ. Rin fell to 194 ± 16 MΩ upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, Ih, and to IKir. Resting potential and Rin exhibited a low sensitivity to changes in [K+]o in both AH and S neurones. This indicates that both cells have a low background K+ permeability. The cationic current, Ih, contributed about 20 % to the resting conductance of AH neurones. It had a half-activation voltage of −72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e-fold change. Ih has relatively fast, voltage-dependent kinetics, with on and off time constants in the range of 50–350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium-dependent current that was poorly sensitive to TTX. In AH neurones, the post-action-potential slow hyperpolarizing current, IAHP, displayed large variation from cell to cell. IAHP appeared to be highly Ca2+ sensitive, since its activation with either membrane depolarization or caffeine (1 mm) was not prevented by perfusing the cell with 10 mm BAPTA. We determined the identity of the Ca2+ channels linked to IAHP. Action potentials of AH neurones that were elongated by TEA (10 mm) were similarly shortened and IAHP was suppressed with each of the three Ω-conotoxins GVIA, MVIIA and MVIIC (0.3–0.5 μm), but not with Ω-agatoxin IVA (0.2 μm). There was no additivity between the effects of the three conotoxins, which indicates the presence of N- but not of P/Q-type Ca2+ channels. A residual Ca2+ current, resistant to all toxins, but blocked by 0.5 mm Cd2+, could not generate IAHP. This patch-clamp study, performed on intact ganglia, demonstrates that the AH neurones of the guinea-pig duodenum are under the control of four major currents, IAHP, Ih, an N-type Ca2+ current and a slowly inactivating Na+ current. PMID:11790812

Dynamic Clamp in Cardiac and Neuronal Systems Using RTXI

PubMed Central

Ortega, Francis A.; Butera, Robert J.; Christini, David J.; White, John A.; Dorval, Alan D.

2016-01-01

The injection of computer-simulated conductances through the dynamic clamp technique has allowed researchers to probe the intercellular and intracellular dynamics of cardiac and neuronal systems with great precision. By coupling computational models to biological systems, dynamic clamp has become a proven tool in electrophysiology with many applications, such as generating hybrid networks in neurons or simulating channelopathies in cardiomyocytes. While its applications are broad, the approach is straightforward: synthesizing traditional patch clamp, computational modeling, and closed-loop feedback control to simulate a cellular conductance. Here, we present two example applications: artificial blocking of the inward rectifier potassium current in a cardiomyocyte and coupling of a biological neuron to a virtual neuron through a virtual synapse. The design and implementation of the necessary software to administer these dynamic clamp experiments can be difficult. In this chapter, we provide an overview of designing and implementing a dynamic clamp experiment using the Real-Time eXperiment Interface (RTXI), an open- source software system tailored for real-time biological experiments. We present two ways to achieve this using RTXI’s modular format, through the creation of a custom user-made module and through existing modules found in RTXI’s online library. PMID:25023319

Water transport by Na+-coupled cotransporters of glucose (SGLT1) and of iodide (NIS). The dependence of substrate size studied at high resolution

PubMed Central

Zeuthen*, Thomas; Belhage, Bo; Zeuthen, Emil

2006-01-01

The relation between substrate and water transport was studied in Na+-coupled cotransporters of glucose (SGLT1) and of iodide (NIS) expressed in Xenopus oocytes. The water transport was monitored from changes in oocyte volume at a resolution of 20 pl, more than one order of magnitude better than previous investigations. The rate of cotransport was monitored as the clamp current obtained from two-electrode voltage clamp. The high resolution data demonstrated a fixed ratio between the turn-over of the cotransporter and the rate of water transport. This applied to experiments in which the rate of cotransport was changed by isosmotic application of substrate, by rapid changes in clamp voltage, or by poisoning. Transport of larger substrates gave rise to less water transport. For the rabbit SGLT1, 378 ± 20 (n = 18 oocytes) water molecules were cotransported along with the 2 Na+ ions and the glucose-analogue α-MDG (MW 194); using the larger sugar arbutin (MW 272) this number was reduced by a factor of at least 0.86 ± 0.03 (15). For the human SGLT1 the respective numbers were 234 ± 12 (18) and 0.85 ± 0.8 (7). For NIS, 253 ± 16 (12) water molecules were cotransported for each 2 Na+ and 1 thiocyanate (SCN−, MW 58), with I− as anion (MW 127) only 162 ± 11 (19) water molecules were cotransported. The effect of substrate size suggests a molecular mechanism for water cotransport and is opposite to what would be expected from unstirred layer effects. Data were analysed by a model which combined cotransport and osmosis at the membrane with diffusion in the cytoplasm. The combination of high resolution measurements and precise modelling showed that water transport across the membrane can be explained by cotransport of water in the membrane proteins and that intracellular unstirred layers effects are minute. PMID:16322051

Voltage-Gated Na+ Channels are Modulated by Glucose and Involved in Regulating Cellular Insulin Content of INS-1 Cells.

PubMed

Chen, Chong; Wang, Songhua; Hu, Qingjuan; Zeng, Lvming; Peng, Hailong; Liu, Chao; Huang, Li-Ping; Song, Hao; Li, Yuping; Yao, Li-Hua; Meng, Wei

2018-01-01

Islet beta cells (β-cells) are unique cells that play a critical role in glucose homeostasis by secreting insulin in response to increased glucose levels. Voltage-gated ion channels in β-cells, such as K+ and Ca2+ channels, contribute to insulin secretion. The response of voltage-gated Na+ channels (VGSCs) in β-cells to the changes in glucose levels remains unknown. This work aims to determine the role of extracellular glucose on the regulation of VGSC. The effect of glucose on VGSC currents (INa) was investigated in insulin-secreting β-cell line (INS-1) cells of rats using whole-cell patch clamp techniques, and the effects of glucose on insulin content and cell viability were determined using Enzyme-Linked Immunosorbent Assay (ELISA) and Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) assay methods respectively. Our results show that extracellular glucose application can inhibit the peak of INa in a concentration-dependent manner. Glucose concentration of 18 mM reduced the amplitude of INa, suppressed the INa of steady-state activation, shifted the steady-state inactivation curves of INa to negative potentials, and prolonged the time course of INa recovery from inactivation. Glucose also enhanced the activity-dependent attenuation of INa and reduced the fraction of activated channels. Furthermore, 18 mM glucose or low concentration of tetrodotoxin (TTX, a VGSC-specific blocker) partially inhibited the activity of VGSC and also improved insulin synthesis. These results revealed that extracellular glucose application enhances the insulin synthesis in INS-1 cells and the mechanism through the partial inhibition on INa channel is involved. Our results innovatively suggest that VGSC plays a vital role in modulating glucose homeostasis. © 2018 The Author(s). Published by S. Karger AG, Basel.

Characterisation of a cell swelling-activated K+-selective conductance of Ehrlich mouse ascites tumour cells

PubMed Central

Niemeyer, María Isabel; Hougaard, Charlotte; Hoffmann, Else K; Jørgensen, Finn; Stutzin, Andrés; Sepúlveda, Francisco V

2000-01-01

The K+ and Cl− currents activated by hypotonic cell swelling were studied in Ehrlich ascites tumour cells using the whole-cell recording mode of the patch-clamp technique. Currents were measured in the absence of added intracellular Ca2+ and with strong buffering of Ca2+. K+ current activated by cell swelling was measured as outward current at the Cl− equilibrium potential (ECl) under quasi-physiological gradients. It could be abolished by replacing extracellular Na+ with K+, thereby cancelling the driving force. Replacement with other cations suggested a selectivity sequence of K+ > Rb+ > NH4≈ Na+≈ Li+; Cs+ appeared to be inhibitory. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and +20 mV with a permeability coefficient of around 10−6 cm s−1 with both physiological and high-K+ extracellular solutions. The class III antiarrhythmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner with an IC50 of 32 μM. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease response of Ehrlich cells. Cell swelling-activated K+ currents of Ehrlich cells are voltage and calcium insensitive and are resistant to a range of K+ channel inhibitors. These characteristics are similar to those of the so-called background K+ channels. Noise analysis of whole-cell current was consistent with a unitary conductance of 5.5 pS for the single channels underlying the K+ current evoked by cell swelling, measured at 0 mV under a quasi-physiological K+ gradient. PMID:10790156

Sweet taste transduction in hamster: sweeteners and cyclic nucleotides depolarize taste cells by reducing a K+ current.

PubMed

Cummings, T A; Daniels, C; Kinnamon, S C

1996-03-01

1. The gigaseal voltage-clamp technique was used to record responses of hamster taste receptor cells to synthetic sweeteners and cyclic nucleotides. Voltage-dependent currents and steady-state currents were monitored during bath exchanges of saccharin, two high-potency sweeteners, 8-chlorophenylthio-adenosine 3',5'-cyclic monophosphate (8cpt-cAMP), and dibutyryl-guanosine 3',5'-cyclic monophosphate (db-cGMP). 2. Of the 237 fungiform taste cells studied, only one in eight was sweet responsive. Outward currents, both voltage-dependent and resting, were reduced by all of the sweeteners tested in sweet-responsive taste cells, whereas these currents were unaffected by sweeteners in sweet-unresponsive taste cells. 3. In every sweet-responsive cell tested, 8cpt-cAMP and db-cGMP mimicked the response to the sweeteners, but neither nucleotide elicited responses in sweet-unresponsive cells. Thus there was a one-to-one correlation between sweet responsivity and cyclic nucleotide responsivity. 4. Sweet responses showed cross adaptation with cyclic nucleotide responses. This indicates that the same ion channel is modulated by sweeteners and cyclic nucleotides. 5. The sweetener- and cyclic nucleotide-blocked current had an apparent reversal potential of -50 mV, which was close to the potassium reversal potential in these experiments. In addition, there was no effect of sweeteners and cyclic nucleotides in the presence of the K+ channel blocker tetraethylammonium bromide (TEA). These data suggest that block of a resting, TEA-sensitive K+ current is the final common step leading to taste cell depolarization during sweet transduction. 6. These data, together with data from a previous study (Cummings et al. 1993), suggest that both synthetic sweeteners and sucrose utilize second-messenger pathways that block a resting K+ conductance to depolarize the taste cell membrane.

A large Ca2+-dependent channel formed by recombinant ADP/ATP carrier from Neurospora crassa resembles the mitochondrial permeability transition pore.

PubMed

Brustovetsky, Nickolay; Tropschug, Maximilian; Heimpel, Simone; Heidkämper, Doerthe; Klingenberg, Martin

2002-10-01

Strong support for the central role of the ADP/ATP carrier (AAC) in the mitochondrial permeability transition (mPT) is provided by the single-channel current measurements in patch-clamp experiments with the isolated reconstituted AAC. In previous work [Brustovetsky, N., and Klingenberg, M. (1996) Biochemistry 35, 8483-8488], this technique was applied to the AAC isolated from bovine heart mitochondria. Here we used recombinant AAC (rAAC) from Neurospora crassa expressed in E. coli, since AAC from mammalian sources cannot be expresssed in E. coli. The rAAC is free from residual mitochondrial components which might associate with the AAC in preparation from bovine heart. Ca(2+)-dependent channels with up to 600 pS are obtained, which are gated at >150 mV. The channel corresponds to a preferential matrix-outside orientation of rAAC in the patch membrane as shown with carboxyatractylate and a polar gating asymmetry. The channel is inhibited by ADP and bongkrekate, not by carboxyatractylate. Cyclophilin, isolated from Neurospora crassa, suppresses the gating, thus increasing conductivity at high positive voltage. Cyclosporin A abolishes the cyclophilin effect. ADP does not eliminate the cyclophilin effect but produces fast large-amplitude flickering of the channel without a stable decrease of the channel conductance. Also the pro-oxidant tert-butyl hydroperoxide reversibly suppresses voltage gating of the channel. The results show that the AAC can be a conducting component of the mPT pore, exhibiting similar characteristics as the mPT pore (response to Ca(2+), BKA, ADP), with a cyclophilin and pro-oxidant-sensitive gating at high voltage.

The ionic bases of the action potential in isolated mouse cardiac Purkinje cell.

PubMed

Vaidyanathan, Ravi; O'Connell, Ryan P; Deo, Makarand; Milstein, Michelle L; Furspan, Philip; Herron, Todd J; Pandit, Sandeep V; Musa, Hassan; Berenfeld, Omer; Jalife, José; Anumonwo, Justus M B

2013-01-01

Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse. To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs. Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data. APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (≈-40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca(2+) currents (I(Ca,T)) were present in PCs but not VMs. Computer simulations suggest that I(Ca,T) and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs. Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of I(Ca,T) are important determinants of the longer and relatively negative plateau phase of the APs. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Activation of muscarinic M3 receptors inhibits large-conductance voltage- and Ca2+-activated K+ channels in rat urinary bladder smooth muscle cells

PubMed Central

Parajuli, Shankar P.

2013-01-01

Large conductance voltage- and Ca2+-activated K+ (BK) channels are key regulators of detrusor smooth muscle (DSM) contraction and relaxation during urine voiding and storage. Here, we explored whether BK channels are regulated by muscarinic receptors (M-Rs) in native freshly isolated rat DSM cells under physiological conditions using the perforated whole cell patch-clamp technique and pharmacological inhibitors. M-R activation with carbachol (1 μM) initially evoked large transient outward BK currents, followed by inhibition of the spontaneous transient outward BK currents (STBKCs) in DSM cells. Carbachol (1 μM) also inhibited the amplitude and frequency of spontaneous transient hyperpolarizations (STHs) and depolarized the DSM cell membrane potential. Selective inhibition of the muscarinic M3 receptors (M3-Rs) with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 0.1 μM), but not muscarinic M2 receptors with methoctramine (1 μM), blocked the carbachol inhibitory effects on STBKCs. Furthermore, blocking the inositol 1,4,5-triphosphate (IP3) receptors with xestospongin-C (1 μM) inhibited the carbachol-induced large transient outward BK currents without affecting carbachol inhibitory effects on STBKCs. Upon pharmacological inhibition of all known cellular sources of Ca2+ for BK channel activation, carbachol (1 μM) did not affect the voltage-step-induced steady-state BK currents, suggesting that the muscarinic effects in DSM cells are mediated by mobilization of intracellular Ca2+. In conclusion, our findings provide strong evidence that activation of M3-Rs leads to inhibition of the STBKCs, STHs, and depolarization of DSM cells. Collectively, the data suggest the existence of functional interactions between BK channels and M3-Rs at a cellular level in DSM. PMID:23703523

Constant-frequency, clamped-mode resonant converters

NASA Technical Reports Server (NTRS)

Tsai, Fu-Sheng; Materu, Peter; Lee, Fred C.

1987-01-01

Two novel clamped-mode resonant converters are proposed which operate at a constant frequency while retaining many desired features of conventional series- and parallel-resonant converters. State-plane analysis techniques are used to identify all possible operating modes and define their mode boundaries. Control-to-output characteristics are derived that specify the regions for natural and forced commutation. The predicted operating modes are verified using a prototype circuit.

75 FR 35619 - Airworthiness Directives; Piper Aircraft, Inc. Models PA-32R-301T and PA-46-350P Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-23

... tighten to an July 28, 2010 (the initial torque of effective date of 40 in. lbs. Tap the this AD... installation than by a change in clamp design. Their experience shows proper installation, torque techniques, and pre-torque alignments of components go a long way in preventing clamp failures down the road. We...

Retinovascular physiology and pathophysiology: new experimental approach/new insights

PubMed Central

Puro, Donald G.

2012-01-01

An important challenge in visual neuroscience is understand the physiology and pathophysiology of the intra-retinal vasculature, whose function is required for ophthalmoception by humans and most other mammals. In the quest to learn more about this highly specialized portion of the circulatory system, a newly developed method for isolating vast microvascular complexes from the rodent retina has opened the way for using techniques such as patch-clamping, fluorescence imaging and time-lapse photography to elucidate the functional organization of a capillary network and its pre-capillary arteriole. For example, the ability to obtain dual perforated-patch recordings from well-defined sites within an isolated microvascular complex permitted the first characterization of the electrotonic architecture of a capillary/arteriole unit. This analysis revealed that this operational unit is not simply a homogenous synctium, but has a complex functional organization that is dynamically modulated by extracellular signals such as angiotensin II. Another recent discovery is that a capillary and its pre-capillary arteriole have distinct physiological differences; capillaries have an abundance of ATP-sensitive potassium (KATP) channels and a dearth of voltage-dependent calcium channels (VDCCs) while the converse is true for arterioles. In addition, voltage transmission between abluminal cells and the endothelium is more efficient in the capillaries. Thus, the capillary network is well-equipped to generate and transmit voltages, and the pre-capillary arteriole is well-adapted to transduce a capillary-generated voltage into a change in abluminal cell calcium and thereby, a vasomotor response. Use of microvessels isolated from the diabetic retina has led to new insights concerning retinal vascular pathophysiology. For example, soon after the onset of diabetes, the efficacy of voltage transmission through the endothelium is diminished; arteriolar VDCCs is inhibited, and there is increased vulnerability to purinergic vasotoxicity, which is a newly identified pathobiological mechanism. Other recent studies reveal that KATP channels not only have an essential physiological role in generating vasomotor responses, but their activation substantially boosts the lethality of hypoxia. Thus, the pathophysiology of the retinal microvasculature is closely linked with its physiology. PMID:22333041

Octanol reduces end-plate channel lifetime

PubMed Central

Gage, Peter W.; McBurney, Robert N.; Van Helden, Dirk

1978-01-01

1. Post-synaptic effects of n-octanol at concentrations of 0·1-1 mM were examined in toad sartorius muscles by use of extracellular and voltage-clamp techniques. 2. Octanol depressed the amplitude and duration of miniature end-plate currents and hence depressed neuromuscular transmission. 3. The decay of miniature end-plate currents remained exponential in octanol solutions even when the time constant of decay (τD) was decreased by 80-90%. 4. The lifetime of end-plate channels, obtained by analysis of acetylcholine noise, was also decreased by octanol. The average lifetime measured from noise spectra agreed reasonably well with the time constant of decay of miniature end-plate currents, both in control solution and in octanol solutions. 5. Octanol caused a reduction in the conductance of end-plate channels. Single channel conductance was on average about 25 pS in control solution and 20 pS in octanol. 6. In most cells the normal voltage sensitivity of the decay of miniature end-plate currents was retained in octanol solutions. The lifetime of end-plate channels measured from acetylcholine noise also remained voltage-sensitive in octanol solutions. In some experiments in which channel lifetime was exceptionally reduced the voltage sensitivity was less than normal. 7. In octanol solutions, τD was still very sensitive to temperature changes in most cells although in some the temperature sensitivity of τD was clearly reduced. Changes in τD with temperature could generally be fitted by the Arrhenius equation suggesting that a single step reaction controlled the decay of currents both in control and in octanol solutions. In some cells in which τD became less than 0·3 ms, the relationship between τD and temperature became inconsistent with the Arrhenius equation. 8. As the decay of end-plate currents in octanol solutions remains exponential, and the voltage and temperature sensitivity can be unchanged even when τD is significantly reduced, it seems likely that octanol decreases τD by increasing the rate of the reaction which normally controls the lifetime of end-plate channels. PMID:203674

Self-organised criticality and 1/f noise in single-channel current of voltage-dependent anion channel

NASA Astrophysics Data System (ADS)

Banerjee, J.; Verma, M. K.; Manna, S.; Ghosh, S.

2006-02-01

Noise profile of Voltage Dependent Anion Channel (VDAC) is investigated in open channel state. Single-channel currents through VDAC from mitochondria of rat brain reconstituted into a planar lipid bilayer are recorded under different voltage clamped conditions across the membrane. Power spectrum analysis of current indicates power law noise of 1/f nature. Moreover, this 1/f nature of the open channel noise is seen throughout the range of applied membrane potential from -30 to +30 mV. It is being proposed that 1/f noise in open ion channel arises out of obstruction in the passage of ions across the membrane. The process is recognised as a phenomenon of self-organized criticality (SOC) like sandpile avalanche and other physical systems. Based on SOC it has been theoretically established that the system of ion channel follows power law noise as observed in our experiments. We also show that the first-time return probability of current fluctuations obeys a power law distribution.

Membrane potential dynamics of axons in cultured hippocampal neurons probed by second-harmonic-generation imaging

NASA Astrophysics Data System (ADS)

Nuriya, Mutsuo; Yasui, Masato

2010-03-01

The electrical properties of axons critically influence the nature of communication between neurons. However, due to their small size, direct measurement of membrane potential dynamics in intact and complex mammalian axons has been a challenge. Furthermore, quantitative optical measurements of axonal membrane potential dynamics have not been available. To characterize the basic principles of somatic voltage signal propagation in intact axonal arbors, second-harmonic-generation (SHG) imaging is applied to cultured mouse hippocampal neurons. When FM4-64 is applied extracellularly to dissociated neurons, whole axonal arbors are visualized by SHG imaging. Upon action potential generation by somatic current injection, nonattenuating action potentials are recorded in intact axonal arbors. Interestingly, however, both current- and voltage-clamp recordings suggest that nonregenerative subthreshold somatic voltage changes at the soma are poorly conveyed to these axonal sites. These results reveal the nature of membrane potential dynamics of cultured hippocampal neurons, and further show the possibility of SHG imaging in physiological investigations of axons.

Calibration of PVDF Film Transducers for the Cavitation Impact Measurement

NASA Astrophysics Data System (ADS)

Hujer, Jan; Müller, Miloš

2018-06-01

This paper describes investigation of the influence of the protective layer thickness on the calibration sensitivity of PVDF films sensors for the cavitation impacts measurements. The PVDF film sensor is casted into an aluminium block. The drop ball method is used for the measurement of the relation between impact force and the voltage detected on the PVDF film sensor. The calibration constants are measured for three different protective layers thicknesses. Five different ball weights for 400 mm drop height are used to reach the required impact force range. The ball positions for the evaluation of the impact force are measured with a high speed camera. The voltage signal detected on the PVDF film clamps was measured with a high speed digitizer. The measured signals are analysed in LabVIEW Signal Express.

Single-crystalline graphene radio-frequency nanoswitches

NASA Astrophysics Data System (ADS)

Li, Peng; Cui, Tianhong

2015-07-01

Growth of monolayer single-crystalline graphene (SCG) using the low-pressure chemical vapor deposition method is reported. Graphene’s superb quality and single-crystalline nature were characterized and verified by Raman microscopy, atomic force microscopy, and carrier mobility measurement. Radio-frequency (RF) nanoelectromechanical switches based on coplanar waveguide double-clamped SCG membrane were achieved, and the superb properties of SCG enable the switches to operate at a pull-in voltage as low as 1 V, with switch time in the nanosecond regime. Owing to their single-crystalline nature, the switches’ lifetime (>5000 times) is much longer than that of polycrystalline graphene ones reported. The RF devices exhibit good isolation (-30 dB at 40 GHz (Ka band)), which can be further improved by SCG’s conductivity variation due to actuation voltage.

Induction of Electrode-Cellular Interfaces with ˜ 0.05 μm^2 Contact Areas

NASA Astrophysics Data System (ADS)

Flanders, Bret; Thapa, Prem

2009-10-01

Individual cells of the slime mold Dictyostelium discoideum attach themselves to negatively biased nanoelectrodes that are separated by 30 μm from grounded electrodes. There is a -43 mV voltage-threshold for cell-to-electrode attachment, with negligible probability across the 0 to -38 mV range but probability that approaches 0.7 across the -46 to -100 mV range. A cell initiates contact by extending a pseudopod to the electrode and maintains contact until the voltage is turned off. Scanning electron micrographs of these interfaces show the contact areas to be of the order of 0.05 μm^2. Insight into this straight-forward, reproducible process may lead to new electrode-cellular attachment strategies that complement established approaches, such as blind sampling and patch clamp.

Epidermal growth factor upregulates motility of Mat-LyLu rat prostate cancer cells partially via voltage-gated Na+ channel activity

PubMed Central

Ding, Yanning; Brackenbury, William J.; Onganer, Pinar U.; Montano, Ximena; Porter, Louise M.; Bates, Lucy F.; Djamgoz, Mustafa B. A.

2014-01-01

The main aim of this investigation was to determine whether a functional relationship existed between epidermal growth factor (EGF) and voltage-gated sodium channel (VGSC) upregulation, both associated with strongly metastatic prostate cancer cells. Incubation with EGF for 24 h more than doubled VGSC current density. Similar treatment with EGF significantly and dose-dependently enhanced the cells’ migration through Transwell filters. Both the patch clamp recordings and the migration assay suggested that endogenous EGF played a similar role. Importantly, co-application of EGF and tetrodotoxin, a highly selective VGSC blocker, abolished 65% of the potentiating effect of EGF. It is suggested that a significant portion of the EGF-induced enhancement of migration occurred via VGSC activity. PMID:17960590

Automated and manual patch clamp data of human induced pluripotent stem cell-derived dopaminergic neurons.

PubMed

Franz, Denise; Olsen, Hervør Lykke; Klink, Oliver; Gimsa, Jan

2017-04-25

Human induced pluripotent stem cells can be differentiated into dopaminergic neurons (Dopa.4U). Dopa.4U neurons expressed voltage-gated Na V and K V channels and showed neuron-like spontaneous electrical activity. In automated patch clamp measurements with suspended Dopa.4U neurons, delayed rectifier K + current (delayed K V ) and rapidly inactivating A-type K + current (fast K V ) were identified. Examination of the fast K V current with inhibitors yielded IC 50 values of 0.4 mM (4-aminopyridine) and 0.1 mM (tetraethylammonium). In manual patch clamp measurements with adherent Dopa.4U neurons, fast K V current could not be detected, while the delayed K V current showed an IC 50 of 2 mM for 4-aminopyridine. The Na V channels in adherent and suspended Dopa.4U neurons showed IC 50 values for tetrodotoxin of 27 and 2.9 nM, respectively. GABA-induced currents that could be observed in adherent Dopa.4U neurons could not be detected in suspended cells. Application of current pulses induced action potentials in approx. 70 % of the cells. Our results proved the feasibility of automated electrophysiological characterization of neuronal cells.

The sliding-helix voltage sensor

PubMed Central

Peyser, Alexander; Nonner, Wolfgang

2012-01-01

The voltage sensor (VS) domain of voltage-gated ion channels underlies electrical excitability of living cells. We simulate a mesoscale model of the VS domain to determine the functional consequences of some of its physical elements. Our mesoscale model is based on VS charges, linear dielectrics and whole-body motion, applied to an S4 ‘sliding helix’. The electrostatics under voltage-clamped boundary conditions are solved consistently using a boundary element method. Based on electrostatic configurational energy, statistical-mechanical expectations of the experimentally observable relation between displaced charge and membrane voltage are predicted. Consequences of the model are investigated for variations of: S4 configuration (α- and 310-helical), countercharge alignment with S4 charges, protein polarizability, geometry of the gating canal, screening of S4 charges by the baths, and fixed charges located at the bath interfaces. The sliding helix VS domain has an inherent electrostatic stability in the explored parameter space: countercharges present in the region of weak dielectric always retain an equivalent S4 charge in that region but allow sliding movements displacing 3 to 4 e0. That movement is sensitive to small energy variations (< 2kT) along the path dependent on a number of electrostatic parameters tested in our simulations. These simulations show how the slope of the relation between displaced charge and voltage could be tuned in a channel. PMID:22907204

Xanthine derivatives without PDE effect stimulate voltage-activated chloride conductance of toad skin.

PubMed

Nagel, Wolfram; Katz, Uri

2003-02-01

The effect of xanthine derivatives on the voltage-activated Cl(-) conductance (G(Cl)) of amphibian skin was analyzed. 3-Isobutyl-1-methylxanthine (IBMX) and the recently synthesized xanthine derivatives 3,7-dimethyl-1-propyl xanthine (X-32) and 3,7-dimethyl-1-isobutyl xanthine (X-33), which lack inhibitory effects on phosphodiesterases in CHO and Calu-3 cells, increased voltage-activated G(Cl) without effect on baseline conductance at inactivating voltage. Half-maximal stimulation of G(Cl) occurred at 108 +/- 9 microM for X-32 and X-33 after apical or basolateral application. The stimulation of G(Cl), which occurs only in the presence of Cl(-) in the mucosal solution, is caused by a shift of the voltage sensitivity to lower clamp potentials and an increase of the maximally activated level. Furosemide reversed both the shift of sensitivity and the increase in magnitude. These patterns are fundamentally different from those seen after application of membrane-permeant, nonmetabolized analogs of cAMP, and they indicate that the xanthines stimulate G(Cl) directly. This notion is strengthened by the lack of influence on intracellular cAMP content, which is consistent with the observations in CHO and Calu-3 cells. We propose that the xanthine derivatives increase the voltage sensitivity of a regulative component in the conductive Cl(-) pathway across amphibian skin.

Measuring Feedforward Inhibition and Its Impact on Local Circuit Function.

PubMed

Hull, Court

2017-05-01

This protocol describes a series of approaches to measure feedforward inhibition in acute brain slices from the cerebellar cortex. Using whole-cell voltage and current clamp recordings from Purkinje cells in conjunction with electrical stimulation of the parallel fibers, these methods demonstrate how to measure the relationship between excitation and inhibition in a feedforward circuit. This protocol also describes how to measure the impact of feedforward inhibition on Purkinje cell excitability, with an emphasis on spike timing. © 2017 Cold Spring Harbor Laboratory Press.

Design, Control, and Modeling of a New Voltage Source Converter for HVDC System

NASA Astrophysics Data System (ADS)

Mohan, Madhan; Singh, Bhim; Ketan Panigrahi, Bijaya

2013-05-01

Abstract: A New Voltage Source Converter (VSC) based on neutral clamped three-level circuit is proposed for High Voltage DC (HVDC) system. The proposed VSC is designed in a multipulse configuration. The converter is operated by Fundamental Frequency Switching (FFS). A new control method is developed for achieving all the necessary control aspects of HVDC system such as independent real and reactive power control, bidirectional real and reactive power control. The basic of the control method is varying the pulse width and by keeping the dc link voltage constant. The steady state and dynamic performances of HVDC system interconnecting two different frequencies network are demonstrated for active and reactive power control. Total number of transformers used in this system are reduced to half in comparison with the two-level VSCs for both active and reactive power control. The performance of the HVDC system is improved in terms of reduced harmonics level even at fundamental frequency switching. The harmonic performance of the designed converter is also studied for different value of the dead angle (β), and the optimized range of the dead angle is achieved for varying reactive power requirement. Simulation results are presented for the designed three level multipulse voltage source converters with the proposed control algorithm.

The NH2 terminus regulates voltage-dependent gating of CALHM ion channels.

PubMed

Tanis, Jessica E; Ma, Zhongming; Foskett, J Kevin

2017-08-01

Calcium homeostasis modulator protein-1 (CALHM1) and its Caenorhabditis elegans (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca 2+ (Ca 2+ o ) but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion, and point mutants to assess the role of the NH 2 terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 NH 2 termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca 2+ o , deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the NH 2 -terminal helix also decreased voltage dependence but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence, respectively, in 0 Ca 2+ In 2 mM Ca 2+ o , ceCLHM-1 NH 2 -terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca 2+ o indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels. Copyright © 2017 the American Physiological Society.

Membrane Potential Controls the Efficacy of Catecholamine-induced β1-Adrenoceptor Activity*

PubMed Central

Birk, Alexandra; Rinne, Andreas; Bünemann, Moritz

2015-01-01

G protein-coupled receptors (GPCRs) are membrane-located proteins and, therefore, are exposed to changes in membrane potential (VM) in excitable tissues. These changes have been shown to alter receptor activation of certain Gi-and Gq-coupled GPCRs. By means of a combination of whole-cell patch-clamp and Förster resonance energy transfer (FRET) in single cells, we demonstrate that the activation of the Gs-coupled β1-adrenoreceptor (β1-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by VM. This voltage-dependence is also transmitted to G protein and arrestin 3 signaling. Voltage-dependence of β2-AR activation, however, was weak compared with β1-AR voltage-dependence. Drug efficacy is a major target of β1-AR voltage-dependence as depolarization attenuated receptor activation, even under saturating concentrations of agonists, with significantly faster kinetics than the deactivation upon agonist withdrawal. Also the efficacy of the endogenous full agonist adrenaline was reduced by depolarization. This is a unique finding since reports of natural full agonists at other voltage-dependent GPCRs only show alterations in affinity during depolarization. Based on a Boltzmann function fit to the relationship of VM and receptor-arrestin 3 interaction we determined the voltage-dependence with highest sensitivity in the physiological range of membrane potential. Our data suggest that under physiological conditions voltage regulates the activity of agonist-occupied β1-adrenoceptors on a very fast time scale. PMID:26408198

Urinary urea nitrogen excretion during the hyperinsulinemic euglycemic clamp in type 1 diabetic patients and healthy subjects.

PubMed

Wohl, P; Krusinová, E; Klementová, M; Wohl, P; Kratochvílová, S; Pelikánová, T

2008-01-01

The hyperinsulinemic euglycemic clamp (HEC) combined with indirect calorimetry (IC) is used for estimation of insulin-stimulated substrate utilization. Calculations are based on urinary urea nitrogen excretion (UE), which is influenced by correct urine collection. The aims of our study were to improve the timing of urine collection during the clamp and to test the effect of insulin on UE in patients with type 1 diabetes (DM1; n=11) and healthy subjects (C; n=11). Urine samples were collected (a) over 24 h divided into 3-h periods and (b) before and during two-step clamp (1 and 10 mIU.kg(-1).min(-1); period 1 and period 2) combined with IC. The UE during the clamp was corrected for changes in urea pool size (UEc). There were no significant differences in 24-h UE between C and DM1 and no circadian variation in UE in either group. During the clamp, serum urea decreased significantly in both groups (p<0.01). Therefore, UEc was significantly lower as compared to UE not adjusted for changes in urea pool size both in C (p<0.001) and DM1 (p<0.001). While UE did not change during the clamp, UEc decreased significantly in both groups (p<0.01). UEc during the clamp was significantly higher in DM1 compared to C both in period 1 (p<0.05) and period 2 (p<0.01). The UE over 24 h and UEc during the clamp were statistically different in both C and DM1. We conclude that urine collection performed during the clamp with UE adjusted for changes in urea pool size is the most suitable technique for measuring substrate utilization during the clamp both in DM1 and C. Urine collections during the clamp cannot be replaced either by 24-h sampling (periods I-VII) or by a single 24-h urine collection. Attenuated insulin-induced decrease in UEc in DM1 implicates the impaired insulin effect on proteolysis.

Noninvasive Intracranial Pressure Monitoring Using Advanced Machine Learning Techniques

DTIC Science & Technology

2013-11-01

drainage requiring removal to prevent infection or to allow computed tomography scan. 3. If clinicians had the ability to predict near-future ICP... drainage of cerebrospinal fluid from the ventricles; however, ICP readings are only accurate when the external drainage system is clamped. ICP clamping... craniotomy for hemorrhage evacuation or a craniectomy for treatment of cerebral edema. Overall in-hospital mortality was 19.4%. Eight hundred and ninety

Laser microsurgery of higher plant cell walls permits patch-clamp access

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Taylor, A. R.; Brownlee, C.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1996-01-01

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Small Molecules for Early Endosome-Specific Patch Clamping.

PubMed

Chen, Cheng-Chang; Butz, Elisabeth S; Chao, Yu-Kai; Grishchuk, Yulia; Becker, Lars; Heller, Stefan; Slaugenhaupt, Susan A; Biel, Martin; Wahl-Schott, Christian; Grimm, Christian

2017-07-20

To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages. Copyright © 2017 Elsevier Ltd. All rights reserved.

Robotic partial nephrectomy with selective parenchymal compression (Simon clamp).

PubMed

Castillo, O A; Rodriguez-Carlin, A; Lopez-Fontana, G; Aleman, E

2013-01-01

To present our initial experience using selective renal parenchymal ischemia, without hilar clamping, in robotic-assisted partial nephrectomy. In four patients with T1a renal tumor we performed robotic-assisted partial nephrectomy, using the Simon's clamp (Aesculap). It provides selective parenchymal compression without the need of vascular clamping. All patients had exofitic renal tumors in polar location. Renal parenchymal reconstruction was done as the standard technique. The median age was 49.6 years (42-59), 3 male and 1 female patient. Median operative time was 71,6 minutes (40-120). Mean stimated bleeding was 250 ml (50-400). Average tumor size was 3,25 cm (1,5-5,3). There were no complications and the average hospital stay was 3,5 days (1-7). The pathology was informed as renal cell carcinoma in three patients and one hemorrhagic cyst. The surgical margins were negative. Our preliminary results shows that selective renal parenchymal compression, with the Simon's clamp, provides an alternative to vascular control in selected patients with polar renal tumors. Copyright © 2012 AEU. Published by Elsevier Espana. All rights reserved.

Temporary bypass for superior vena cava reconstruction with Anthron bypass tubeTM

PubMed Central

Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Kamohara, Ryotaro; Hatachi, Go; Nagayasu, Takeshi

2017-01-01

Total superior vena cava (SVC) clamping for SVC replacement or repair can be used in thoracic surgery. A bypass technique is an option to avoid hemodynamic instability and cerebral venous hypertension and hypoperfusion. The present report describes a venous bypass technique using Anthron bypass tubeTM for total SVC clamping. Indications for this procedure include the need for a temporary bypass between the brachiocephalic vein and atrium for complete tumor resection. This procedure allows the surgeons sufficient time to complete replacement of SVC or partial resection of SVC without adverse effects. Further, it is a relatively simple procedure requiring minimal time. PMID:28840027

Attenuated Ca(2+) release in a mouse model of limb girdle muscular dystrophy 2A.

PubMed

DiFranco, Marino; Kramerova, Irina; Vergara, Julio L; Spencer, Melissa Jan

2016-01-01

Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca(2+) release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca(2+) handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n',n'-tetraacetic acid (EGTA) to measure Ca(2+) fluxes. Muscles were subjected to both current and voltage clamp conditions. Standard and confocal fluorescence microscopy was used to study Ca(2+) release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student's test with significance set at p < 0.05. We found that the peak value of Ca(2+) fluxes elicited by single action potentials was significantly reduced by 15-20 % in C3KO fibers, but the kinetics was unaltered. Ca(2+) release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca(2+) release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca(2+) release in C3KO mice but reduced peak Ca(2+) fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca(2+) release at all triads consistent with a homogenous reduction of functional voltage activated Ca(2+) release sites. Overall, these results suggest that decreased Ca(2+) release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

Cerebral hemodynamic changes and electroencephalography during carotid endarterectomy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Algotsson, L.; Messeter, K.; Rehncrona, S.

Some patients undergoing endarterectomy for occlusive carotid artery disease run a risk of brain ischemia during cross-clamping of the artery. The present study of 15 patients was undertaken to evaluate changes in cerebral blood flow (CBF), as measured with an intravenous (IV) tracer (133Xenon) technique, and to relate CBF changes to changes in the electroencephalogram (EEG). CBF was measured before and after induction of anesthesia, during cross-clamping of the carotid artery, after release of the clamps, and at 24 hours after the operation. All the patients were anesthetized with methohexitone, fentanyl, and nitrous oxide and oxygen. EEG was continuously recordedmore » during the operation. Carotid artery shunts were not used. In 8 patients, cross-clamping of the carotid artery did not influence the EEG. In this group of patients, induction of anesthesia caused a 38% decrease in CBF, which presumably reflects the normal reaction to the anesthetic agent given. There were no further changes in CBF during cross-clamping. In 7 patients, the EEG showed signs of deterioration during the intraoperative vascular occlusion. In these patients, anesthesia did not cause any CBF change, whereas cross-clamping the artery induced a 33% decrease in CBF. In individual patients, the severity of EEG changes correlated with the decrease in CBF. The absence of a change in CBF by anesthesia and a decrease due to cross-clamping of the carotid artery may be explained by the presence of a more advanced cerebrovascular disease and an insufficiency to maintain CBF during cross-clamping.« less

Design of A Grid Integrated PV System with MPPT Control and Voltage Oriented Controller using MATLAB/PLECES

NASA Astrophysics Data System (ADS)

Soreng, Bineeta; Behera, Pradyumna; Pradhan, Raseswari

2017-08-01

This paper presents model of a grid-integrated photovoltaic array with Maximum Power Point Tracker (MPPT) and voltage oriented controller. The MPPT of the PV array is usually an essential part of PV system as MPPT helps the operating point of the solar array to align its maximum power point. In this model, the MPPT along with a DC-DC converter lets a PV generator to produce continuous power, despite of the measurement conditions. The neutral-point-clamped converter (NPC) with a boost converter raises the voltage from the panels to the DC-link. An LCL-filter smoothens the current ripple caused by the PWM modulation of the grid-side inverter. In addition to the MPPT, the system has two more two controllers, such as voltage controller and a current controller. The voltage control has a PI controller to regulate the PV voltage to optimal level by controlling the amount of current injected into the boost stage. Here, the grid-side converter transfers the power from the DC-link into the grid and maintains the DC-link voltage. Three-phase PV inverters are used for off-grid or designed to create utility frequency AC. The PV system can be connected in series or parallel to get the desired output power. To justify the working of this model, the grid-integrated PV system has been designed in MATLAB/PLECS. The simulation shows the P-V curve of implemented PV Array consisting 4 X 20 modules, reactive, real power, grid voltage and current.

Integrative Approach with Electrophysiological and Theoretical Methods Reveals a New Role of S4 Positively Charged Residues in PKD2L1 Channel Voltage-Sensing.

PubMed

Numata, Tomohiro; Tsumoto, Kunichika; Yamada, Kazunori; Kurokawa, Tatsuki; Hirose, Shinichi; Nomura, Hideki; Kawano, Mitsuhiro; Kurachi, Yoshihisa; Inoue, Ryuji; Mori, Yasuo

2017-08-29

Numerical model-based simulations provide important insights into ion channel gating when experimental limitations exist. Here, a novel strategy combining numerical simulations with patch clamp experiments was used to investigate the net positive charges in the putative transmembrane segment 4 (S4) of the atypical, positively-shifted voltage-dependence of polycystic kidney disease 2-like 1 (PKD2L1) channel. Charge-neutralising mutations (K452Q, K455Q and K461Q) in S4 reduced gating charges, positively shifted the Boltzmann-type activation curve [i.e., open probability (P open )-V curve] and altered the time-courses of activation/deactivation of PKD2L1, indicating that this region constitutes part of a voltage sensor. Numerical reconstruction of wild-type (WT) and mutant PKD2L1-mediated currents necessitated, besides their voltage-dependent gating parameters, a scaling factor that describes the voltage-dependence of maximal conductance, G max . Subsequent single-channel conductance (γ) measurements revealed that voltage-dependence of G max in WT can be explained by the inward-rectifying property of γ, which is greatly changed in PKD2L1 mutants. Homology modelling based on PKD2 and Na V Ab structures suggest that such voltage dependence of P open and γ in PKD2L1 could both reflect the charged state of the S4 domain. The present conjunctive experimental and theoretical approaches provide a framework to explore the undetermined mechanism(s) regulating TRP channels that possess non-classical voltage-dependent properties.

Endothelial effects of hemostatic devices for continuous cardioplegia or minimally invasive operations.

PubMed

Perrault, L P; Menasché, P; Wassef, M; Bidouard, J P; Janiak, P; Villeneuve, N; Jacquemin, C; Bloch, G; Vilaine, J P; Vanhoutte, P M

1996-10-01

Improvements in myocardial protection may include the continuous delivery of normothermic blood cardioplegia. Technical aids are required for optimal visualization of the operative field during the performance of coronary anastomoses if cardioplegia is to be given continuously or during minimally invasive operations. However, the effects of the different hemostatic devices on coronary endothelial function are unknown. We compared the effects on endothelial function of two commonly used hemostatic techniques, coronary clamping and gas jet insufflation, with those of a technique using extravascular balloon occlusion to mimic systolic luminal closure by the surrounding myocardium. The three techniques were applied for 15 minutes on porcine epicardial coronary arteries from explanted hearts. For coronary clamping, standard bulldog clamps were used. Gas jet insufflation was applied by blowing oxygen (12 L/min) tangentially at a 45-degree angle 1 cm away from a 3-mm arteriotomy. Extravascular balloon occlusion was achieved with a needle-tipped silicone loop, the midportion of which, once positioned beneath the coronary artery, was inflated to push a myocardial "cushion" against the back of the vessel until its occlusion. Control rings were taken from the same coronary artery. The endothelial function of control and instrumented arterial rings was then studied in organ chambers filled with modified Krebs-Ringer bicarbonate solution. Contractions to potassium chloride and prostaglandin F2 alpha and endothelium-independent relaxation to sin-1, a nitric oxide donor, were unaffected in all groups. Endothelium-dependent relaxation to serotonin was impaired after clamping and preserved after gas jet insufflation and extravascular balloon occlusion. Maximal endothelium-dependent relaxation to serotonin was as follows: for coronary clamping, 63% +/- 6% versus 87% +/- 3% in controls; for gas jet insufflation, 67% +/- 12% versus 88% +/- 7%; and for extraluminal balloon occlusion, 79% +/- 6% versus 85% +/- 5%. Whereas commonly used hemostatic devices may impair endothelial function, extravascular balloon occlusion appears to achieve effective hemostasis while preserving endothelial integrity.

Using a Harmonic Scalpel "Drilling and Clamping" Method to Implement Zero Ischemic Robotic-assisted Partial Nephrectomy: An Observation Case Report Study.

PubMed

Hou, Chen-Pang; Lin, Yu-Hsiang; Hsu, Yu-Chao; Chen, Chien-Lun; Chang, Phei-Lang; Tsui, Ke-Hung

2016-01-01

Robot-assisted partial nephrectomy (RAPN) has gradually become a popular minimally invasive nephron-sparing surgical option for small renal tumors. Ischemic injury should be minimized because it impacts renal function outcomes following partial nephrectomy. Herein, the authors detail the technique and present initial perioperative outcomes of our novel harmonic scalpel "drilling and clamping" method to implement zero-ischemic RAPN. The authors prospectively collected baseline and perioperative data of patients who underwent zero ischemic RAPN performed by our harmonic scalpel "drilling and clamping" method. From April 2012 to December 2014, a total of 19 consecutive zero ischemic RAPN procedures were performed by a single surgeon. For 18 of the 19 patients, RAPN using our harmonic scalpel "Drilling and Clamping" method was successfully completed without the need for hilar clamping. The median tumor size was 3.4 cm (range: 1.8-6.2); operative time was 3.2 hours (range: 1.9-4.5); blood loss was 100 mL (range: 30-950); and postoperative hospital stay was 4 days (3-26). One patient required intraoperative blood transfusion. Two patients had intra or postoperative complications: 1 was converted to traditional laparotomy because of massive bleeding, whereas another had postoperative stress ulcer. Pathology confirmed renal cell carcinoma in 13 patients (63.2%), angiomyolipoma in 6 patients: (31.5%), and oncocytoma in 1 patient (5.3%). Mean pre- and postoperative serum creatinine (0.82 mg/dL and 0.85 mg/dL, respectively), estimated glomerular filtration rate (84.12 and 82.18, respectively), and hemoglobin (13.27 g/dL and 12.71 g/dL, respectively) were comparable. The authors present a novel zero-ischemic technique for RAPN. They believe that this technique is feasible and reproducible.

Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

PubMed

Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

2013-09-01

We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped with various types of high-voltage activated Ca(v) channels and with IP3 receptors for intracellular Ca2+ signaling.

Central action of dendrotoxin: selective reduction of a transient K conductance in hippocampus and binding to localized acceptors.

PubMed

Halliwell, J V; Othman, I B; Pelchen-Matthews, A; Dolly, J O

1986-01-01

Dendrotoxin, a small single-chain protein from the venom of Dendroaspis angusticeps, is highly toxic following intracerebroventricular injection into rats. Voltage-clamp analysis of CA1 neurons in hippocampal slices, treated with tetrodotoxin, revealed that nanomolar concentrations of dendrotoxin reduce selectively a transient, voltage-dependent K conductance. Epileptiform activity known to be induced by dendrotoxin can be attributed to such an action. Membrane currents not affected directly by the toxin include (i) Ca-activated K conductance; (ii) noninactivating voltage-dependent K conductance; (iii) inactivating and noninactivating Ca conductances; (iv) persistent inward (anomalous) rectifier current. Persistence of the effects of the toxin when Cd was included to suppress spontaneous transmitter release indicates a direct action on the neuronal membrane. Using biologically active, 125I-labeled dendrotoxin, protein acceptor sites of high affinity were detected on cerebrocortical synaptosomal membranes and sections of rat brain. In hippocampus, toxin binding was shown autoradiographically to reside in synapse-rich and white matter regions, with lower levels in cell body layers. This acceptor is implicated in the action of toxin because its affinities for dendrotoxin congeners are proportional to their central neurotoxicities and potencies in reducing the transient, voltage-dependent K conductance.

Pitfalls during biomechanical testing - Evaluation of different fixation methods for measuring tendons endurance properties.

PubMed

Hangody, Gy; Pánics, G; Szebényi, G; Kiss, R; Hangody, L; Pap, K

2016-03-01

The goal of the study was to find a proper technique to fix tendon grafts into an INSTRON loading machine. From 8 human cadavers, 40 grafts were collected. We removed the bone-patella tendon-bone grafts, the semitendinosus and gracilis tendons, the quadriceps tendon-bone grafts, the Achilles tendons, and the peroneus longus tendons from each lower extremity. We tested the tendon grafts with five different types of fixation devices: surgical thread (Premicron 3), general mounting clamp, wire mesh, cement fixation, and a modified clamp for an INSTRON loading machine. The mean failure load in case of surgical thread fixation was (381N ± 26N). The results with the general clamp were (527N ± 45N). The wire meshes were more promising (750N ± 21N), but did not reach the outcomes we desired. Easy slippages of the ends of the tendons from the cement encasements were observed (253N ± 18N). We then began to use Shi's clamp that could produce 977N ± 416N peak force. We combined Shi's clamp with freezing of the graft and the rupture of the tendon itself demonstrated an average force of 2198 N ± 773N. We determined that our modified frozen clamp fixed the specimens against high tensile forces.

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

PubMed

Brueggemann, A; George, M; Klau, M; Beckler, M; Steindl, J; Behrends, J C; Fertig, N

2004-01-01

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

Thermoelectric Device Fabrication Using Thermal Spray and Laser Micromachining

NASA Astrophysics Data System (ADS)

Tewolde, Mahder; Fu, Gaosheng; Hwang, David J.; Zuo, Lei; Sampath, Sanjay; Longtin, Jon P.

2016-02-01

Thermoelectric generators (TEGs) are solid-state devices that convert heat directly into electricity. They are used in many engineering applications such as vehicle and industrial waste-heat recovery systems to provide electrical power, improve operating efficiency and reduce costs. State-of-art TEG manufacturing is based on prefabricated materials and a labor-intensive process involving soldering, epoxy bonding, and mechanical clamping for assembly. This reduces their durability and raises costs. Additive manufacturing technologies, such as thermal spray, present opportunities to overcome these challenges. In this work, TEGs have been fabricated for the first time using thermal spray technology and laser micromachining. The TEGs are fabricated directly onto engineering component surfaces. First, current fabrication techniques of TEGs are presented. Next, the steps required to fabricate a thermal spray-based TEG module, including the formation of the metallic interconnect layers and the thermoelectric legs are presented. A technique for bridging the air gap between two adjacent thermoelectric elements for the top layer using a sacrificial filler material is also demonstrated. A flat 50.8 mm × 50.8 mm TEG module is fabricated using this method and its performance is experimentally characterized and found to be in agreement with expected values of open-circuit voltage based on the materials used.

Diverse Short-Term Dynamics of Inhibitory Synapses Converging on Striatal Projection Neurons: Differential Changes in a Rodent Model of Parkinson's Disease

PubMed Central

Herrera-Valdez, Marco A.; Lopez-Huerta, Violeta Gisselle; Galarraga, Elvira

2015-01-01

Most neurons in the striatum are projection neurons (SPNs) which make synapses with each other within distances of approximately 100 µm. About 5% of striatal neurons are GABAergic interneurons whose axons expand hundreds of microns. Short-term synaptic plasticity (STSP) between fast-spiking (FS) interneurons and SPNs and between SPNs has been described with electrophysiological and optogenetic techniques. It is difficult to obtain pair recordings from some classes of interneurons and due to limitations of actual techniques, no other types of STSP have been described on SPNs. Diverse STSPs may reflect differences in presynaptic release machineries. Therefore, we focused the present work on answering two questions: Are there different identifiable classes of STSP between GABAergic synapses on SPNs? And, if so, are synapses exhibiting different classes of STSP differentially affected by dopamine depletion? Whole-cell voltage-clamp recordings on SPNs revealed three classes of STSPs: depressing, facilitating, and biphasic (facilitating-depressing), in response to stimulation trains at 20 Hz, in a constant ionic environment. We then used the 6-hydroxydopamine (6-OHDA) rodent model of Parkinson's disease to show that synapses with different STSPs are differentially affected by dopamine depletion. We propose a general model of STSP that fits all the dynamics found in our recordings. PMID:26167304

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

PubMed

Jonas, E A; Knox, R J; Kaczmarek, L K

1997-07-01

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

The Mechanosensitive Ca2+ Channel as a Central Regular of Prostate Tumor Cell Migration and Invasiveness

DTIC Science & Technology

2011-04-01

activation still needs to be determined (Strotmann et al. 2000). 7.2.4 The Use of MS Enzyme Inhibitors A further strategy for implicating potential MS...invasiveness and metastatic potential . 1.1 Use patch-clamp/pressure clamp techniques, confocal immunofluorescence, Westerns and surface biotinylation...9. Maroto, R. Kurosky, A. Hamill, O.P. Expression and function of canonical transient recptor potential channels in human prostate tumor cells

Ranolazine inhibits shear sensitivity of endogenous Na+ current and spontaneous action potentials in HL-1 cells

PubMed Central

Strege, Peter; Beyder, Arthur; Bernard, Cheryl; Crespo-Diaz, Ruben; Behfar, Atta; Terzic, Andre; Ackerman, Michael; Farrugia, Gianrico

2012-01-01

NaV1.5 is a mechanosensitive voltage-gated Na+ channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na+ current and delayed rectifier (IKr) currents. Recently, ranolazine was also shown to be an inhibitor of NaV1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na+ current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na+ current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine. PMID:23018927

The effects of some inhalation anaesthetics on the sodium current of the squid giant axon.

PubMed Central

Haydon, D A; Urban, B W

1983-01-01

The effects of diethyl ether, methoxyflurane, halothane, dichloromethane and chloroform on the ionic currents and electrical capacity of the squid giant axon have been examined. The peak inward current in voltage-clamped axons was reduced reversibly by each substance. Sodium currents under voltage clamp were recorded in intracellularly perfused axons before, during, and sometimes after exposure to the test substances, and the records were fitted with equations similar to those proposed by Hodgkin & Huxley (1952). Shifts in the dependence of the steady-state activation and inactivation parameters (m infinity and h infinity) on membrane potential, reductions in the peak heights of the activation and inactivation time constants (tau m and tau h) and decreases in the maximum Na conductance (gNa) have been tabulated. For each of the anaesthetics the steady-state inactivation curve was shifted in the hyperpolarizing direction though less markedly than for the hydrocarbons. The steady-state activation curve was in each instance shifted in the depolarizing direction, as for the alcohols and other surface active substances. In common with both the hydrocarbons and the surface active substances the peak time constants were invariably reduced. The membrane capacity at 100 kHz was affected significantly only by methoxyflurane, where decreases of ca. 9% were observed for 3 mM solutions. The extent to which the results can be accounted for in terms of the perturbation of membrane lipid has been discussed. PMID:6312031

Cocaine acute "binge" administration results in altered thalamocortical interactions in mice.

PubMed

Urbano, Francisco J; Bisagno, Verónica; Wikinski, Silvia I; Uchitel, Osvaldo D; Llinás, Rodolfo R

2009-10-15

Abnormalities in both thalamic and cortical areas have been reported in human cocaine addicts with noninvasive functional magnetic resonance imaging. Given the substantial involvement of the thalamocortical system in sensory processing and perception, we defined electrophysiology-based protocols to attempt a characterization of cocaine effects on thalamocortical circuits. Thalamocortical function was studied in vivo and in vitro in mice after cocaine "binge" administration. In vivo awake electroencephalography (EEG) was implemented in mice injected with saline, 1 hour or 24 hours after the last cocaine "binge" injection. In vitro current- and voltage-clamp whole-cell patch-clamp recordings were performed from slices including thalamic relay ventrobasal (VB) neurons. In vivo EEG recordings after cocaine "binge" administration showed a significant increment, compared with saline, in low frequencies while observing no changes in high-frequency gamma activity. In vitro patch recordings from VB neurons after cocaine "binge" administration showed low threshold spikes activation at more negative membrane potentials and increments in both I(h) and low voltage activated T-type calcium currents. Also, a 10-mV negative shift on threshold activation level of T-type current and a remarkable increment in both frequency and amplitudes of gamma-aminobutyric acid-A-mediated minis were observed. Our data indicate that thalamocortical dysfunctions observed in cocaine abusers might be due to two distinct but additive events: 1) increased low frequency oscillatory thalamocortical activity, and 2) overinhibition of VB neurons that can abnormally "lock" the whole thalamocortical system at low frequencies.

Excellent performances of energy harvester using cantilever driving double-clamped 0.7Pb(Mg{sub 1/3}Nb{sub 2/3})O{sub 3}-0.3PbTiO{sub 3} plates and symmetric middle-stops

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Zhou; Xu, Qing; University of Chinese Academy of Sciences, Beijing 100049

We present a high performance nonlinear piezoelectric energy harvester constituted by a cantilever with symmetrically middle-stops and double-clamped piezoelectric plates based on piezoelectric single crystal 0.7Pb(Mg{sub 1/3}Nb{sub 2/3})O{sub 3}-0.3PbTiO{sub 3}. Electrical properties of the device under different excitation frequencies, accelerations, and load resistances are studied systematically. Under a low acceleration of 3 m/s{sup 2} (0.3 g), a peak voltage of 26.2 V and a maximum normalized power of 25.6 mW/g{sup 2} were obtained across a matching impedance of 600 kΩ with favorable bandwidths. The low excitation acceleration and excellent performances indicate that the device can be a promising candidate for energy harvesting in low-power electronicsmore » and wireless sensors.« less

Tetrodotoxin Blockade on Canine Cardiac L-Type Ca2+ Channels Depends on pH and Redox Potential

PubMed Central

Hegyi, Bence; Komáromi, István; Kistamás, Kornél; Ruzsnavszky, Ferenc; Váczi, Krisztina; Horváth, Balázs; Magyar, János; Bányász, Tamás; Nánási, Péter P.; Szentandrássy, Norbert

2013-01-01

Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na+ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca2+ current (ICa) in canine cardiac cells. In the present study, the TTX-sensitivity of ICa was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC50 value obtained under physiological conditions) caused 60% ± 2% inhibition of ICa in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of ICa in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 μM H2O2). Phosphorylation of the channel protein (induced by 3 μM forskolin) failed to modify the inhibiting potency of TTX; an IC50 value of 50 ± 4 μM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels. PMID:23771047

Inhibitory effects of cortisone and hydrocortisone on human Kv1.5 channel currents.

PubMed

Yu, Jing; Park, Mi-Hyeong; Jo, Su-Hyun

2015-01-05

Glucocorticoids are the primary hormones that respond to stress and protect organisms from dangerous situations. The glucocorticoids hydrocortisone and its dormant form, cortisone, affect the cardiovascular system with changes such as increased blood pressure and cardioprotection. Kv1.5 channels play a critical role in the maintenance of cellular membrane potential and are widely expressed in pancreatic β-cells, neurons, myocytes, and smooth muscle cells of the pulmonary vasculature. We examined the electrophysiological effects of both cortisone and hydrocortisone on human Kv1.5 channels expressed in Xenopus oocytes using a two-microelectrode voltage clamp technique. Both cortisone and hydrocortisone rapidly and irreversibly suppressed the amplitude of Kv1.5 channel current with IC50 values of 50.2±4.2μM and 33.4±3.2μM, respectively, while sustained the current trace shape of Kv1.5 current. The inhibitory effect of cortisone on Kv1.5 decreased progressively from -10mV to +30mV, while hydrocortisone׳s inhibition of the channel did not change across the same voltage range. Both cortisone and hydrocortisone blocked Kv1.5 channel currents in a non-use-dependent manner and neither altered the channel׳s steady-state activation or inactivation curves. These results show that cortisone and hydrocortisone inhibited Kv1.5 channel currents differently, and that Kv1.5 channels were more sensitive to hydrocortisone than to cortisone. Copyright © 2014 Elsevier B.V. All rights reserved.

Block of the delayed rectifier current (IK) by the 5-HT3 antagonists ondansetron and granisetron in feline ventricular myocytes.

PubMed Central

de Lorenzi, F G; Bridal, T R; Spinelli, W

1994-01-01

1. We investigated the effects of two 5-HT3 antagonists, ondansetron and granisetron, on the action potential duration (APD) and the delayed rectifier current (IK) of feline isolated ventricular myocytes. Whole-cell current and action potential recordings were performed at 37 degrees C with the patch clamp technique. 2. Ondansetron and granisetron blocked IK with a KD of 1.7 +/- 1.0 and 4.3 +/- 1.7 microM, respectively. At a higher concentration (30 microM), both drugs blocked the inward rectifier (IKl). 3. The block of IK was dependent on channel activation. Both drugs slowed the decay of IK tail currents and produced a crossover with the pre-drug current trace. These results are consistent with block and unblock from the open state of the channel. 4. Granisetron showed an intrinsic voltage-dependence as the block increased with depolarization. The equivalent voltage-dependency of block (delta) was 0.10 +/- 0.04, suggesting that granisetron blocks from the intracellular side at a binding site located 10% across the transmembrane electrical field. 5. Ondansetron (1 microM) and granisetron (3 microM) prolonged APD by about 30% at 0.5 Hz. The prolongation of APD by ondansetron was abolished at faster frequencies (3 Hz) showing reverse rate dependence. 6. In conclusion, the 5-HT3 antagonists, ondansetron and granisetron, are open state blockers of the ventricular delayed rectifier and show a clear class III action. PMID:7834204

Ion channel recordings on an injection-molded polymer chip.

PubMed

Tanzi, Simone; Matteucci, Marco; Christiansen, Thomas Lehrmann; Friis, Søren; Christensen, Mette Thylstrup; Garnaes, Joergen; Wilson, Sandra; Kutchinsky, Jonatan; Taboryski, Rafael

2013-12-21

In this paper, we demonstrate recordings of the ion channel activity across the cell membrane in a biological cell by employing the so-called patch clamping technique on an injection-molded polymer microfluidic device. The findings will allow direct recordings of ion channel activity to be made using the cheapest materials and production platform to date and with the potential for very high throughput. The employment of cornered apertures for cell capture allowed the fabrication of devices without through holes and via a scheme comprising master origination by dry etching in a silicon substrate, electroplating in nickel and injection molding of the final part. The most critical device parameters were identified as the length of the patching capillary and the very low surface roughness on the inside of the capillary. The cross-sectional shape of the orifice was found to be less critical, as both rectangular and semicircular profiles seemed to have almost the same ability to form tight seals with cells with negligible leak currents. The devices were functionally tested using human embryonic kidney cells expressing voltage-gated sodium channels (Nav1.7) and benchmarked against a commercial state-of-the-art system for automated ion channel recordings. These experiments considered current-voltage (IV) relationships for activation and inactivation of the Nav1.7 channels and their sensitivity to a local anesthetic, lidocaine. Both IVs and lidocaine dose-response curves obtained from the injection-molded polymer device were in good agreement with data obtained from the commercial system.

18β-Glycyrrhetinic acid preferentially blocks late Na current generated by ΔKPQ Nav1.5 channels

PubMed Central

Du, Yi-mei; Xia, Cheng-kun; Zhao, Ning; Dong, Qian; Lei, Ming; Xia, Jia-hong

2012-01-01

Aim: To compare the effects of two stereoisomeric forms of glycyrrhetinic acid on different components of Na+ current, HERG and Kv1.5 channel currents. Methods: Wild-type (WT) and long QT syndrome type 3 (LQT-3) mutant ΔKPQ Nav1.5 channels, as well as HERG and Kv1.5 channels were expressed in Xenopus oocytes. In addition, isolated human atrial myocytes were used. Two-microelectrode voltage-clamp technique was used to record the voltage-activated currents. Results: Superfusion of 18β-glycyrrhetinic acid (18β-GA, 1–100 μmol/L) blocked both the peak current (INa,P) and late current (INa,L) generated by WT and ΔKPQ Nav1.5 channels in a concentration-dependent manner, while 18α-glycyrrhetinic acid (18α-GA) at the same concentrations had no effects. 18β-GA preferentially blocked INa,L (IC50=37.2±14.4 μmol/L) to INa,P (IC50=100.4±11.2 μmol/L) generated by ΔKPQ Nav1.5 channels. In human atrial myocytes, 18β-GA (30 μmol/L) inhibited 47% of INa,P and 87% of INa,L induced by Anemonia sulcata toxin (ATX-II, 30 nmol/L). Superfusion of 18β-GA (100 μmol/L) had no effects on HERG and Kv1.5 channel currents. Conclusion: 18β-GA preferentially blocked the late Na current without affecting HERG and Kv1.5 channels. PMID:22609834