Science.gov

Sample records for active voltage clamp

  1. Application of active electrode compensation to perform continuous voltage-clamp recordings with sharp microelectrodes

    NASA Astrophysics Data System (ADS)

    Gómez-González, J. F.; Destexhe, A.; Bal, T.

    2014-10-01

    Objective. Electrophysiological recordings of single neurons in brain tissues are very common in neuroscience. Glass microelectrodes filled with an electrolyte are used to impale the cell membrane in order to record the membrane potential or to inject current. Their high resistance induces a high voltage drop when passing current and it is essential to correct the voltage measurements. In particular, for voltage clamping, the traditional alternatives are two-electrode voltage-clamp technique or discontinuous single electrode voltage-clamp (dSEVC). Nevertheless, it is generally difficult to impale two electrodes in a same neuron and the switching frequency is limited to low frequencies in the case of dSEVC. We present a novel fully computer-implemented alternative to perform continuous voltage-clamp recordings with a single sharp-electrode. Approach. To reach such voltage-clamp recordings, we combine an active electrode compensation algorithm (AEC) with a digital controller (AECVC). Main results. We applied two types of control-systems: a linear controller (proportional plus integrative controller) and a model-based controller (optimal control). We compared the performance of the two methods to dSEVC using a dynamic model cell and experiments in brain slices. Significance. The AECVC method provides an entirely digital method to perform continuous recording and smooth switching between voltage-clamp, current clamp or dynamic-clamp configurations without introducing artifacts.

  2. Novel Low Loss Active Voltage Clamp Circuit for Series Connection of RCGCT thyristors

    NASA Astrophysics Data System (ADS)

    Ito, Hiroshi; Suzuki, Akihiro; Iwata, Akihiko

    This paper describes novel low loss active voltage clamp circuits for the series connection of RCGCT thyristors. For high voltage converters the series connection of power semiconductor devices is an essential technique for direct switching of high voltages. Several protection circuits have been applied to the series connection of RCGCT thyristors such as CRD snubber circuits which suppress over-voltages across RCGCT thyristors, and voltage balancing resistors to equalize voltage sharing in steady states. However, significant losses in these protection circuits lower the converter’s efficiency. We propose novel low-loss protection circuits, which have active voltage clamp snubber circuits and static voltage balancing circuits. The clamp capacitor voltage of the active voltage clamp snubber circuits are designed to be higher than the equally divided DC-Link voltage. This method can reduce the loss of the clamp circuit to no more than 1/10 of that of the conventional CRD snubber. Also the static voltage balancing circuits compensate for the voltage imbalance generated by the difference in the leakage current between the series connection RCGCT thyristors.

  3. Transistorized circuit clamps voltage with 0.1 percent error

    NASA Technical Reports Server (NTRS)

    1965-01-01

    Transistorized clamping circuit clamps either of two voltage levels to input of digital-to-analog resistive matrix with 0.1 percent error. Clamping circuit technique has analog, digital, and hybrid circuit applications.

  4. Voltage-gated calcium and Ca2+-activated chloride channels and Ca2+ transients: voltage-clamp studies of perfused and intact cells of Chara.

    PubMed

    Berestovsky, Genrikh N; Kataev, Anatoly A

    2005-11-01

    The voltage-clamp technique was used to study Ca(2+) and Cl(-) transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about -140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10(3) ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca(2+) tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca(2+) injection into tonoplast-free and intact cells, to activate the chloride channels by Ca(2+) . When cells are perfused with EDTA-containing medium in the presence of excess Mg(2+), this method of injection allows the free submembrane Ca(2+) concentration, [Ca(2+)](c), to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to [see text]. When Ca(2+) is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation-inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m(-2) and tau(inact)~0.5 s at -200 mV). Analysis of our data and theoretical considerations indicate that the [Ca(2+)](c) rise during cell excitation is caused mainly by Ca(2+) entry through plasmalemma Ca channels rather than by Ca(2+) release from intracellular stores.

  5. 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

  6. Voltage clamp experiments on ventricular myocardial fibres

    PubMed Central

    Beeler, G. W.; Reuter, H.

    1970-01-01

    1. A voltage clamp method utilizing a sucrose gap and glass microelectrodes was developed and used to study dog ventricular myocardial fibre bundles. The limitations and the reliability of this method are demonstrated by a series of tests. 2. A dynamic sodium current, excited at membrane potentials more positive than -65 mV, was measured. The equilibrium potential for this large, rapid inward current depends directly on [Na]o, shifting 29·0 ± 2·3 mV (± S.E. of mean), as opposed to a theoretically expected value of 30·6 mV, when [Na]o is reduced to 31% of normal. 3. Sodium current is inactivated by conditioning depolarizations. Complete inactivation occurs with conditioning potentials more positive than -45 mV, and 50% inactivation occurs at about -55 mV. The location of the inactivation curve shifts along the voltage axis, when [Ca]o is varied between 0·2 and 7·2 mM. 4. A second, much smaller and slower net inward current, with a threshold around -30 mV, and an equilibrium potential above +40 mV was also observed. 5. The `steady-state' current—voltage relationship (after 300-600 msec) exhibits inward-going (anomalous) rectification with negative slope between -50 and -25 mV. 6. A small, very slowly developing component of outward current was observed at inside positive potentials. The equilibrium potential for this current, although slightly dependent on [K]o, is neither identical with the potassium equilibrium potential nor with the resting potential in normal Tyrode solution. 7. Anatomical limitations, primarily resistance in the extracellular space within the bundle, prevent complete characterization of the rapid, large sodium current, but do not limit the application of the clamp method to the study of other, smaller and slower currents. The evidence for this is discussed extensively in the Appendix. PMID:5503866

  7. Patch voltage clamp of squid axon membrane.

    PubMed

    Fishman, H M

    1975-12-01

    A small area (patch) of the external surface of a squid axon can be "isolated" electrically from the surrounding bath by means of a pair of concentric glass pipettes. The seawater-filled inner pipette makes contact with the axon and constitutes the external access to the patch. The outer pipette is used to direct flowing sucrose solution over the area surrounding the patch of membrane underlying the inner pipette. Typically, sucrose isolated patches remain in good condition (spike amplitude greater than 90 mV) for periods of approximately one half hour. Patches of axon membrane which had previously been exposed to sucrose solution were often excitable. Membrane survival of sucrose treatment apparently arises from an outflow of ions from the axon and perhaps satellite cells into the interstitial cell space surrounding the exolemma. Estimate of the total access resistance (electrode plus series resistance) to the patch is about 100 komega (7 omega cm2). Patch capacitance ranges from 10-100 pF, which suggests areas of 10(-4) to 10(-5) cm2 and resting patch resistances of 10-100 Momega. Shunt resistance through the interstitial space exposed to sucrose solution, which isolates the patch, is typically 1-2 Momega. These parameters indicate that good potential control and response times can be achieved on a patch. Furthermore, spatial uniformity is demonstrated by measurement of an exoplasmic isopotential during voltage clamp of an axon patch. The method may be useful for other preparations in which limited membrane area is available or in special instances such as in the measurement of membrane conduction noise. PMID:1214276

  8. Voltage clamp effects on bacterial chemotaxis.

    PubMed Central

    Margolin, Y; Eisenbach, M

    1984-01-01

    To examine whether or not sensory signaling in bacteria is by way of fluctuations in membrane potential, we studied the effect of clamping the potential on bacterial chemotaxis. The potential was clamped by valinomycin, a K+ -specific ionophore, in the presence of K+. Despite the clamped potential, sensory signaling did occur: both Escherichia coli and Bacillus subtilis cells were still excitable and adaptable under these conditions. It is concluded that signaling in the excitation and adaptation steps of chemotaxis is not by way of fluctuations in the membrane potential. PMID:6430873

  9. Axon voltage-clamp simulations. I. Methods and tests.

    PubMed Central

    Moore, J W; Ramón, F; Joyner, R W

    1975-01-01

    This is the first in a series of four papers in which we present the numerical simulation of the application of the voltage clamp technique to excitable cells. In this paper we describe the application of the Crank-Nicolson (1947) method for the solution of the parabolic partial differential equations that describe a cylindrical cell in which the ionic conductances are functions of voltage and time (Hodgkin and Huxley, 1952). This method is compared with other methods in terms of accuracy and speed of solution for a propagated action potential. In addition, differential equations representing a simple voltage-clamp electronic circuit are presented. Using the voltage clamp circuit equations, we simulate the voltage clamp of a single isopotential membrane patch and show how the parameters of the circuit affect the transient response of the patch to a step change in the control potential.The stimulation methods presented in this series of papers allow the evaluation of voltage clamp control of an excitable cell or a syncytium of excitable cells. To the extent that membrane parameters and geometrical factors can be determined, the methods presented here provide solutions for the voltage profile as a function of time. PMID:1174640

  10. Voltage-Clamp Studies on Uterine Smooth Muscle

    PubMed Central

    Anderson, Nels C.

    1969-01-01

    These studies have developed and tested an experimental approach to the study of membrane ionic conductance mechanisms in strips of uterine smooth muscle. The experimental and theoretical basis for applying the double sucrose-gap technique is described along with the limitations of this system. Nonpropagating membrane action potentials were produced in response to depolarizing current pulses under current-clamp conditions. The stepwise change of membrane potential under voltage-clamp conditions resulted in a family of ionic currents with voltage- and time-dependent characteristics. In sodium-free solution the peak transient current decreased and its equilibrium potential shifted along the voltage axis toward a more negative internal potential. These studies indicate a sodium-dependent, regenerative excitation mechanism. PMID:5796366

  11. VOLTAGE CLAMP BEHAVIOR OF IRON-NITRIC ACID SYSTEM AS COMPARED WITH THAT OF NERVE MEMBRANE

    PubMed Central

    Tasaki, I.; Bak, A. F.

    1959-01-01

    The current-voltage relation for the surface layer of an iron wire immersed in nitric acid was investigated by the voltage clamp technique. Comparing the phase of nitric acid to the axoplasm and the metallic phase to the external fluid medium for the nerve fiber, a striking analogy was found between the voltage clamp behavior of the iron-nitric acid system and that of the nerve membrane. The current voltage curve was found to consist of three parts: (a) a straight line representing the behavior of the resting (passive) membrane, (b) a straight line representing the fully excited (active) state, and (c) an intermediate zone connecting (a) and (b). It was shown that in the intermediate zone, the surface of iron consisted of a fully active patch (or patches) surrounded by a remaining resting area. The phenomenon corresponding to "repetitive firing of responses under voltage clamp" in the nerve membrane was demonstrated in the intermediate zone. The behavior of the cobalt electrode system was also investigated by the same technique. An attempt was made to interpret the phenomenon of initiation and abolition of an active potential on the basis of the thermodynamics of irreversible processes. PMID:13654740

  12. Pirmenol, a new antiarrhythmic drug with potassium- and sodium-channel blocking activity; a voltage-clamp study in rabbit Purkinje fibres.

    PubMed

    Reichardt, B; Konzen, G; Hauswirth, O

    1990-05-01

    The target of this study was to characterize the effect of pirmenol hydrochloride on the electrophysiological properties of cardiac cells. Action potential studies were carried out using the standard microelectrode technique in isolated rabbit Purkinje fibres. Information about the effect of pirmenol on the fast sodium current was obtained by Vmax-measurement. Furthermore the delayed rectifying current ix was studied by the two microelectrode voltage clamp technique. In concentrations of 0.5-5 mumol/l pirmenol caused a marked prolongation of the action potential duration in isolated rabbit Purkinje fibres. Measurements of the delayed rectifying current ix displayed a strong depression with a KD-value of 1 mumol/l pirmenol. The steady-state current voltage relation showed that pirmenol also caused a reduction of the steady-state sodium window current and/or of the slowly decaying components of the sodium current. In concentrations of greater than or equal to 10 mumol/l pirmenol the action potential duration was diminished again and Vmax was depressed in a use-dependent manner. Furthermore pirmenol caused a depression and a negative shift of the Vmax/Em-relation. Pirmenol blocked sodium channels which recovered from block with a time constant of 6.7 s at a holding potential of -105 mV. Similar to quinidine and sotalol the prolongation of the action potential duration under pirmenol is essentially caused by a diminution of the delayed rectifying current ix. The depression of Vmax is mainly independent from the action potential duration indicating the dominance of an open channel block. Pirmenol is a new drug with class Ia antiarrhythmic action.

  13. Voltage clamping single cells in intact malpighian tubules of mosquitoes.

    PubMed

    Masia, R; Aneshansley, D; Nagel, W; Nachman, R J; Beyenbach, K W

    2000-10-01

    Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage (V(pc)) was -86.7 mV, and input resistance (R(pc)) was 388.5 kOmega (n = 49 cells). In six cells, Ba(2+) (15 mM) had negligible effects on V(pc), but it increased R(pc) from 325.3 to 684.5 kOmega (P < 0.001). In the presence of Ba(2+), leucokinin-VIII (1 microM) increased V(pc) to -101.8 mV (P < 0.001) and reduced R(pc) to 340.2 kOmega (P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652. 0 kOmega; apical membrane resistance, 340.2 kOmega; shunt resistance (R(sh)), 344.3 kOmega; transcellular resistance, 992.2 kOmega. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and R(sh) (3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V(pc) from -94.3 to -10.7 mV (P < 0.001) and reversibly increased R(pc) from 412 to 2,879 kOmega (P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways. PMID:10997925

  14. Stoichiometry and voltage dependence of the sodium pump in voltage- clamped, internally dialyzed squid giant axon

    PubMed Central

    1989-01-01

    The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG- sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K- free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3

  15. Membrane tether formation from voltage-clamped outer hair cells using optical tweezers

    NASA Astrophysics Data System (ADS)

    Qian, Feng; Ermilov, Sergey A.; Murdock, David R.; Brownell, William E.; Anvari, Bahman

    2004-06-01

    Outer hair cells contribute an active mechanical feedback to the vibrations of the cochlear structures resulting in the high sensitivity and frequency selectivity of normal hearing. We have designed and implemented a novel experimental setup that combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement during tether elongation is monitored by a quadrant photodetector to obtain time-resolved measurements of the tethering force. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from outer hair cells and human embryonic kidney cells are presented.

  16. Solutions for transients in arbitrarily branching cables: III. Voltage clamp problems.

    PubMed Central

    Major, G

    1993-01-01

    Branched cable voltage recording and voltage clamp analytical solutions derived in two previous papers are used to explore practical issues concerning voltage clamp. Single exponentials can be fitted reasonably well to the decay phase of clamped synaptic currents, although they contain many underlying components. The effective time constant depends on the fit interval. The smoothing effects on synaptic clamp currents of dendritic cables and series resistance are explored with a single cylinder + soma model, for inputs with different time courses. "Soma" and "cable" charging currents cannot be separated easily when the soma is much smaller than the dendrites. Subtractive soma capacitance compensation and series resistance compensation are discussed. In a hippocampal CA1 pyramidal neurone model, voltage control at most dendritic sites is extremely poor. Parameter dependencies are illustrated. The effects of series resistance compound those of dendritic cables and depend on the "effective capacitance" of the cell. Plausible combinations of parameters can cause order-of-magnitude distortions to clamp current waveform measures of simulated Schaeffer collateral inputs. These voltage clamp problems are unlikely to be solved by the use of switch clamp methods. PMID:8369450

  17. High throughput ion-channel pharmacology: planar-array-based voltage clamp.

    PubMed

    Kiss, Laszlo; Bennett, Paul B; Uebele, Victor N; Koblan, Kenneth S; Kane, Stefanie A; Neagle, Brad; Schroeder, Kirk

    2003-02-01

    Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs. PMID:15090139

  18. Bactridine's effects on DUM cricket neurons under voltage clamp conditions.

    PubMed

    Forsyth, P; Sevcik, C; Martínez, R; Castillo, C; D'Suze, G

    2012-12-01

    We describe the effects of six bactridines (150 nM) on cricket dorsal unpaired median (DUM) neurons. The addition of bactridine 2 to DUM neurons induced a large current component with a reversal potential more negative than -30 mV, most evident at the end of the pulses. This current was completely suppressed when 1 μM amiloride was applied before adding the bactridines. Since the amiloride sensitive current is able to distort the aim of our study, i.e. the effect of bactridines on sodium channels, all experiments were done in the presence of 1 μM amiloride. Most bactridines induced voltage shifts of V(1/2) of the Boltzmann inactivation voltage dependency curves in the hyperpolarizing direction. Bactridines 1, 4 and 6 reduced Na current peak by 65, 80 and 24% of the control, respectively. The sodium conductance blockage by bactridines was voltage independent at potentials >20 mV. Bactridines effect on cricket DUM neurons does not correspond to neither α- nor β-toxins. Most bactridines shifted the inactivation curves in the hyperpolarizing direction without any effects on the activation m(∞)-like curves. Also bactridines differ from other NaScpTx in that they increased an amiloride-sensitive conductance in DUM neurons. Our result suggest that the α/β classification of sodium scorpion toxins is not all encompassing. The present work shows that bactridines target more than one site: insect voltage dependent Na channels and an amiloride-sensitive ionic pathway which is under study. PMID:23085555

  19. Introduction of a modular automated voltage-clamp platform and its correlation with manual human Ether-à-go-go related gene voltage-clamp data.

    PubMed

    Scheel, Olaf; Himmel, Herbert; Rascher-Eggstein, Gesa; Knott, Thomas

    2011-12-01

    In investigating ion channel pharmacology, the manual patch clamp is still considered the gold standard for data quality, notwithstanding the major drawbacks of low throughput and the need for skilled operators. The automated patch clamp platform CytoPatch™ Instrument overcomes these restrictions. Its modular fully automated design makes it possible to obtain scalable throughput without the need for well-trained operators. Its chip design and perfusion system reproduces the manual patch technique, thus ensuring optimal data quality. Further, the use of stably transfected frozen cells, usable immediately after thawing, eliminates the cell quality impairment and low success rates associated with a running cell culture and renders screening costs accurately calculable. To demonstrate the applicability of this platform, 18 blinded compounds were assessed for their impact on the cardiac human Ether-à-go-go related gene K(+) channel. The IC(50) values obtained by the CytoPatch Instrument using the frozen human embryonic kidney 293 cells showed a high correlation (R(2)=0.928) with those obtained from manual patch clamp recordings with human embryonic kidney 293 cells from a running cell culture. Moreover, this correlation extended to sticky compounds such as terfenadine or astemizole. In conclusion, the CytoPatch Instrument operated with frozen cells ready to use directly after thawing provides the same high data quality known from the manual voltage clamp and has the added benefit of enhanced throughput for use in ion channel screening and safety assessment. PMID:21675869

  20. A voltage-clamp study of the light response in solitary rods of the tiger salamander.

    PubMed Central

    Bader, C R; Macleish, P R; Schwartz, E A

    1979-01-01

    1. Single, isolated, rod photoreceptors were obtained by enzymatic dissociation of the tiger salamander (Ambystoma tigrinum) retina. These solitary cells retained the morphological features of rods of the intact retina and could be maintained in culture for several days. Solitary cells were penetrated with one or two micropipettes and their electrophysiology was studied by the voltage-clamp technique. 2. Intracellular recording with two micropipettes demonstrated that the inner segment of a solitary rod was effectively isopotential with the outer segment. 3. The time course of the voltage response to a flash resembled that of responses observed in rods in the intact retina. At low light intensities the response reached a peak in approximately 0.7 sec and then slowly declined. At high light intensities the time to peak response decreased and an initial transient arose as the response, after reaching the peak, quickly decreased to a less polarized plateau. 4. The normal voltage response could be compared with the current observed during a voltage clamp. At low light intensities the time course of the current response resembled the time course of the voltage response. When light intensity was increased the time course of the current response differed from the voltage response in that the time to peak amplitude remained relatively constant and an initial transient did not occur. It was possible to predict the current response produced by any intensity of light by using (i) an empirical equation which reproduced the time course of a dim response and (ii) the Michaelis-Menten equation. 5. The time course of the voltage-clamp current produced by a flash was the same at different values of maintained voltage. 6. The maximum amplitude of the voltage-clamp current produced by a flash or step of light was a non-linear function of membrane potential. It was relatively constant within the physiological range, decreased as the membrane potential was moved toward 0 mV, reversed

  1. An improved double vaseline gap voltage clamp to study electroporated skeletal muscle fibers.

    PubMed Central

    Chen, W; Lee, R C

    1994-01-01

    An improved voltage clamp with a double vaseline gap chamber was designed to study electroporated skeletal muscle fibers. The new clamp eliminated spike overshock of membrane potential when applying step stimulation occurring in the traditional configuration. It allowed greater consistency in membrane potential distribution. After the intracellular resistances of the fiber segment at the vaseline gap area were compensated, it was possible to change membrane potential more quickly. Using this technique, strong electrical pulses used to mimic the situation of electrical shock can be delivered to the cell membrane by voltage clamp. Transmembrane currents of skeletal muscle cell were simultaneously measured during a high pulsed shock and resolved into different components. Distinct transient changes of the transmembrane current, involving the time courses of the formation of electroporation and their recovery time constants, can be recorded. Because of more even membrane potential distribution and faster response to pulsed membrane potential change, this technique is also suitable for membrane study under physiological conditions. PMID:8011901

  2. Additional efficient computation of branched nerve equations: adaptive time step and ideal voltage clamp.

    PubMed

    Borg-Graham, L J

    2000-01-01

    Various improvements are described for the simulation of biophysically and anatomically detailed compartmental models of single neurons and networks of neurons. These include adaptive time-step integration and a reordering of the circuit matrix to allow ideal voltage clamp of arbitrary nodes. We demonstrate how the adaptive time-step method can give equivalent accuracy as a fixed time-step method for typical current clamp simulation protocols, with about a 2.5 reduction in runtime. The ideal voltage clamp method is shown to be more stable than the nonideal case, in particular when used with the adaptive time-step method. Simulation results are presented using the Surf-Hippo Neuron Simulation System, a public domain object-oriented simulator written in Lisp. PMID:10809013

  3. Studies of calcium channels in rat clonal pituitary cells with patch electrode voltage clamp

    PubMed Central

    Hagiwara, Susumu; Ohmori, Harunori

    1982-01-01

    1. The properties of the Ca channel in tissue cultured clonal cells (GH3) isolated from a rat anterior pituitary tumour were studied with the patch electrode voltage-clamp technique. 2. To isolate the current through the Ca channel, the currents through the Na channel, the delayed K channel and the Ca2+ induced K channel were minimized by replacing the external Na+ with TEA+ and adding EGTA to the K-free solution inside the patch electrode. 3. The selectivity ratios through the Ca channel with different cations were 2·7 (Ba2+):1·6 (Sr2+):1·0 (Ca2+) and the m2 form of the activation kinetics and the relationships between the time constant and the membrane potential were common to the three divalent cations. 4. The amplitude of the Ba2+ current increased linearly with [Ba2+]o up to 25 mM and thereafter tended to show saturation. 5. The current—voltage relation showed a positive shift along the voltage axis as [Ba2+]o increased, probably due to the screening effect of Ba2+ on the negative surface charges. 6. The time constant of activation as a function of the membrane potential showed a parallel shift as [Ba2+]o was increased, suggesting that the activation kinetics were independent of the permeant ion concentration. 7. The time constant of the tail current was consistent with m2 kinetics for opening and closing of the Ca channel. 8. The extrapolated `instantaneous' tail current rapidly increased as the activating membrane potential became more positive and reached an apparent saturation at membrane potentials substantially more positive than the potential that gave the maximum peak inward current, and suggested that the single channel has a sigmoidal current—voltage relationship. 9. The power density spectrum obtained during the steady-state inward Ba2+ current had a cut-off frequency which was nearly voltage independent; this is expected if the fluctuation of the current originates from m2 activation kinetics. 10. The results of noise analysis suggest that

  4. The ventral photoreceptor cells of Limulus. 3. A voltage-clamp study.

    PubMed

    Millecchia, R; Mauro, A

    1969-09-01

    In the dark, the ventral photoreceptor of Limulus exhibits time-variant currents under voltage-clamp conditions; that is, if the membrane potential of the cell is clamped to a depolarized value there is an initial large outward current which slowly declines to a steady level. The current-voltage relation of the cell in the dark is nonlinear. The only ion tested which has any effect on the current-voltage relation is potassium; high potassium shifts the reversal potential towards zero and introduces a negative slope-conductance region. When the cell is illuminated under voltage-clamp conditions, an additional current, the light-induced current, flows across the cell membrane. The time course of this current mimics the time course of the light response (receptor potential) in the unclamped cell; namely, an initial transient phase is followed by a steady-state phase. The amplitude of the peak transient current can be as large as 60 times the amplitude of the steady-state current, while in the unclamped cell the amplitude of the peak transient voltage never exceeds 4 times the amplitude of the steady-state voltage. The current-voltage relations of the additional light-induced current obtained for different instants of time are also nonlinear, but differ from the current-voltage relations of the dark current. The ions tested which have the greatest effect on the light-induced current are sodium and calcium; low sodium decreases the current, while low calcium increases the current. The data strongly support the hypothesis that two systems of electric current exist in the membrane. Thus the total ionic current which flows in the membrane is accounted for as the sum of a dark current and a light-induced current. PMID:5806593

  5. 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.

  6. Rapid sodium channel conductance changes during voltage clamp steps in squid giant axons.

    PubMed Central

    Fohlmeister, J F; Adelman, W J

    1984-01-01

    The sodium conductance of the membrane of the giant axon of squid was isolated by the use of potassium-free solutions and voltage-clamped with pulses containing three levels of depolarization. The conductance appears to undergo rapid changes during certain repolarizing clamp steps whose voltage reach at least partially overlaps the gating range. The percentage change in conductance increases with time of depolarization from approximately 0 to approximately 25-30% at 7 ms for a potential step from +70 to -30 mV. Conductance steps were also observed for voltage steps from various depolarized levels to -70 mV. All observed shifts were in the direction of a decreased conductance. The conductance steps appear to be a weak function of the concentration of external calcium, which also acts as a voltage-dependent channel blocker for inwardly directed sodium currents. A number of possible mechanisms are suggested. One of these is discussed in some detail and postulates a voltage- and time-dependent molecular process that does not itself yield open or closed channel conformations, but that affects the magnitude of the rate constants that do connect open and closed state conformations. PMID:6324915

  7. A simple hardware model for the direct observation of voltage-clamp performance under realistic conditions.

    PubMed

    Draguhn, A; Pfeiffer, M; Heinemann, U; Polder, R

    1997-12-30

    A new hardware cell model for electrophysiological recording has been constructed which allows for the assessment of voltage clamp accuracy in different recording situations. Each compartment consists of a capacitor in parallel with a variable resistor and can be connected to other compartments by a variable axial resistance. The simulated membrane resistance can be changed extrinsically by a command voltage input which is optically coupled to the cell without any direct galvanic contact. Each compartment possesses a buffer amplifier which reads out the potential at the simulated membrane element, (e.g. 'somatic' or 'dendritic' potential). The model allows for the direct observation of typical situations and problems arising in electrophysiological experiments. We used the model to monitor deviations between the 'intracellular' and the command voltage, e.g. due to series resistance errors. We also used the model to simulate synaptic currents which were generated by triangular membrane conductance changes. The results demonstrate the strong influence of synaptic location and series resistance on voltage clamp fidelity. The cell model is a new and easy-to-handle tool for the observation of voltage control under realistic experimental conditions. PMID:9497006

  8. Crayfish stretch receptor: an investigation with voltage-clamp and ion-sensitive electrodes.

    PubMed Central

    Brown, H M; Ottoson, D; Rydqvist, B

    1978-01-01

    1. The membrane characteristics of the slowly adapting stretch receptor from the crayfish, Astacus fluviatilis, were examined with electrophysiological techniques consisting of membrane potential recording, voltage clamp and ion-sensitive microelectrodes. 2. The passive membrane current (Ip) following step changes of the membrane potential to levels above 0 mV required more than a minute to decay to a steady-state level. 3. The stretch-induced current (SIC, where SIC = Itotal--Ipassive) was not fully developed until the Ip had decayed to a steady state. 4. With Ip at the steady state and the stretch-induced current at the O-current potential, a slow stretch-induced inward current was isolated. The latter reaches a maximum after 1 sec of stretch and declines even more slowly after stretch. The I-V relation of the slow current had a negative slope and reversed sign near the resting potential. It is suggested that this current is due to a Cl- conductance change. 5. The stretch-induced current, consisting of a rapid transient phase and a steady component can be isolated from the slow stretch-induced current at a holding potential corresponding to the resting potential. 6. The SIC-Em relation is non-linear and reverses sign at about +15 mV. 7. In a given cell, the reversal potential of the stretch-induced potential change obtained with current clamp coincided with the 0-current potential of the stretch-induced current obtained by voltage clamp. The average value from twenty-six cells was +13 +/- 6.5 mV; cell to cell variability seemed to be correlated with dendrite length. 8. Tris (mol. wt. 121) or arginine (mol. wt. 174) susbstituted for Na+ reduces but does not abolish the stretch-induced current. 9. The permeability ratios of Tris:Na and arginine:Na were estimated from changes in the 0-current potential as these cations replaced Na+ in the external medium. The PTris:PNa was somewhat higher (0.31) than the Parginine:PNa ratio (0.25). 10. Changes in the external Ca2

  9. Voltage-clamp predictions by gompertz kinetics model relating squid-axon Na+-gating and ionic currents.

    PubMed

    Easton, Dexter M

    2005-10-01

    Gompertz kinetics is a simple, realistic, accurate, and computationally parsimonious alternative for prediction of macroscopic changes in Na+ conductance during voltage clamp. Conductance delay and time course depend on initial amplitudes and decay rates of surrogates for the macroscopic gating currents. The model is tested by the fit to published data of other authors. The proposed physical basis for the model is that membrane potential perturbation triggers motion of charged "gating" components of the axon membrane at rapid (activating) and at slow (inactivating) rates. The resulting distortion increases and more slowly diminishes the probability that conduction channels will be open.

  10. Patch clamp combined with voltage/concentration clamp to determine the kinetics and voltage dependency of N-methyl-D-aspartate (NMDA) receptor open channel blockers.

    PubMed

    Parsons, Chris G; Gilling, Kate E

    2014-01-01

    Electrophysiological techniques can be used to great effect to help determine the mechanism of action of a compound. However, many factors can compromise the resulting data and their analysis, such as the speed of solution exchange, expression of additional ion channel populations including other ligand-gated receptors and voltage-gated channels, compounds having multiple binding sites, and current desensitization and rundown. In this chapter, such problems and their solutions are discussed and illustrated using data from experiments involving the uncompetitive NMDA receptor antagonist memantine. Memantine differs from many other NMDA receptor channel blockers in that it is well tolerated and does not cause psychotomimetic effects at therapeutic doses. Various electrophysiological parameters of NMDA-induced current blockade by memantine have been proposed to be important in determining therapeutic tolerability; potency, onset and offset kinetics, and voltage dependency. These were all measured using whole cell patch clamp techniques using hippocampal neurons. Full results are shown here for memantine, and these are summarized and compared to those from similar experiments with other NMDA channel blockers. The interpretation of these results is discussed, as are theories concerning the tolerability of NMDA channel blockers, with the aim of illustrating how electrophysiological data can be used to form and support a physiological hypothesis.

  11. Membrane properties of a barnacle photoreceptor examined by the voltage clamp technique

    PubMed Central

    Brown, H. Mack; Hagiwara, S.; Koike, H.; Meech, R. M.

    1970-01-01

    1. Electrical properties of the membrane of photoreceptor cells in the lateral ocelli of barnacles, Balanus amphitrite and B. eburneus were investigated by intracellular recording, polarization and voltage-clamp techniques. 2. The resting potential of a dark adapted cell was 36·3 ± 6·6 mV (S.D.) and depended mainly on the external K+ concentration. 3. Current—voltage relations obtained from voltage—clamp experiments in the absence of light were non-linear and varied with time after the onset of a step change in membrane potential; the steady state was reached after about 0·5 sec. 4. Illumination resulted in a membrane potential change under current clamp and in a change of membrane current (light-initiated membrane current (L.I.C.): total membrane current with illumination minus current without illumination) under voltage—clamp conditions. Amplitudes and time course of L.I.C. depended on the light intensity as well as membrane potential. 5. The L.I.C.-voltage relation was non-linear and corresponded with a slope conductance increase with increasing positive membrane potential. 6. The reversal potential of L.I.C. was independent of the light intensity and the time after onset of illumination; the average value obtained in normal saline was +26·9 ± 5·0 mV. 7. The membrane conductance estimated from instantaneous L.I.C.-voltage relations agreed with the chord conductance of the non-linear L.I.C.-voltage relation. 8. Decreasing external Na+ concentration decreased the inward component of L.I.C. but not the outward component. 9. Decreasing external Ca2+ concentration increased the inward as well as the outward component of L.I.C. 10. The reversal potential shifted in the negative direction with decreasing external Na+ concentration (the rate was 10-15 mV for a tenfold change in concentration) and the rate was augmented in the absence of Ca2+ but did not exceed 21 mV. 11. The change of reversal potential with changes of external Ca2+ concentration was

  12. Simultaneous measurement of changes in current and tracer flux in voltage-clamped squid giant axon.

    PubMed Central

    Rakowski, R F

    1989-01-01

    A method is described for the simultaneous measurement of changes in membrane current and unidirectional radiotracer flux in internally dialyzed voltage-clamped squid giant axons. The small currents that are produced by electrogenic transport processes or steady-state ionic currents can be resolved using this method. Because the use of grounded guard electrodes in the end pools is not, by itself, an adequate means of eliminating end-effects, two ancillary end pool clamp circuits are described to eliminate extraneous current flow from the ends of the axon. The end pool voltage-clamp circuits serve to minimize net current flow between the end pools and center pool, and employ stable, low-impedance calomel electrodes to monitor the potentials of the end and center pools. The adequacy of the method is demonstrated by experiments in which unidirectional 22Na efflux and current, flowing through tetrodotoxin (TTX)-sensitive Na channels into Na-free seawater, under K-free conditions, are shown to be equal. The equality of unidirectional TTX-sensitive flux and current is maintained over the entire range of membrane potentials examined (-60 to +20 mV). The method has been applied to a series of experiments in which the voltage dependence and stoichiometry of the Na/K pump have been measured (Rakowski et al., 1989), and can be applied in general to the simultaneous measurement of changes in current and flux of other electrogenic transport processes, and of currents through ionic channels that open under steady-state conditions. PMID:2720065

  13. Depolarization-contraction coupling in short frog muscle fibers. A voltage clamp study

    PubMed Central

    1984-01-01

    Short muscle fibers (1.5 mm) were dissected from hindlimb muscles of frogs and voltage clamped with two microelectrodes to study phenomena related to depolarization-contraction coupling. Isometric myograms obtained in response to depolarizing pulses of durations between 10 and 500 ms and amplitudes up to 140 mV had the following properties. For suprathreshold pulses of fixed duration (in the range of 20-100 ms), the peak tension achieved, the time to peak tension, and contraction duration increased as the internal potential was made progressively more positive. Peak tension eventually saturates with increasing internal potentials. For pulse durations of greater than or equal to 50 ms, the rate of tension development becomes constant for increasing internal potentials when peak tensions become greater than one-third of the maximum tension possible. Both threshold and maximum steepness of the relation between internal potential and peak tension depend on pulse duration. The relation between the tension-time integral and the stimulus amplitude-duration product was examined. The utility of this relation for excitation-contraction studies is based on the observation that once a depolarizing pulse configuration has elicited maximum tension, further increases in either stimulus duration or amplitude only prolong the contractile response, while the major portion of the relaxation phase after the end of a pulse is exponential, with a time constant that is not significantly affected by either the amplitude or the duration of the pulse. Hence, the area under the tension-response curve provides a measure of the availability to troponin of the calcium released from the sarcoplasmic reticulum in response to membrane depolarization. The results from this work complement those obtained in experiments in which intramembrane charge movements related to contractile activation were studied and those in which intracellular Ca++ transients were measured. PMID:6611386

  14. Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish

    PubMed Central

    1975-01-01

    Ionic mechanisms of excitation were studied in the immature egg cell membrane of a starfish, Mediaster aequalis, by analyzing membrane currents during voltage clamp. The cell membrane shows two different inward current mechanisms. One is activated at a membrane potential of - 55 approximately -50 mV and the other at -7 approximately -6 mV. They are referred to as channels I and II, respectively. A similar difference is also found in the membrane potential of half inactivation. Currents of the two channels can, therefore, be separated by selective inactivation. The currents of both channels depend on Ca++ (Sr++ or Ba++) but only the current of channel I depends on Na+. The time-course of current differs significantly between the two channels when compared at the same membrane potential. The relationship between the membrane current and the concentration of the permeant ions is also different between the two channels. The result suggests that channel II is a more saturable system. The sensitivity of the current to blocking cations such as Co++ or Mg++ is substantially greater in channel II than in channel I. Currents of both channels depend on the external pH with an apparent pK of 5.6. They are insensitive to 3 muM tetrodotoxin (TTX) but are eliminated totally by 7.3 mM procaine. The properties of channel II are similar to those of the Ca channel found in various adult tissues. The properties of channel I differ, however, from those of either the typical Ca or Na channels. Although the current of the channel depends on the external Na the amplitude of the Na current decreases not only with the Na concentration but also with the Ca concentration. No selectivity is found among Li+, Na+, Rb+, and Cs+. The experimental result suggests that Na+ does not carry current but modifies the current carried by Ca in channel I. PMID:240906

  15. Intracellular calcium and its sodium-independent regulation in voltage-clamped snail neurones.

    PubMed Central

    Kennedy, H J; Thomas, R C

    1995-01-01

    1. We have used both Ca(2+)-sensitive microelectrodes and fura-2 to measure the intracellular free calcium ion concentration ([Ca2+]i or its negative log, pCai) of snail neurones voltage clamped to -50 or -60 mV. Using Ca(2+)-sensitive microelectrodes, [Ca2+]i was found to be approximately 174 nM and pCai, 6.76 +/- 0.09 (mean +/- S.E.M.; n = 11); using fura-2, [Ca2+]i was approximately 40 nM and pCai, 7.44 +/- 0.06 (mean +/- S.E.M., n = 10). 2. Depolarizations (1-20 s) caused an increase in [Ca2+]i which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i was due to Ca2+ influx through voltage-activated Ca2+ channels. 3. Caffeine (10-20 mM) caused an increase in [Ca2+]i in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+]i could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a small increase in resting [Ca2+]i and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N-methyl-D-glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+]i or the rate of recovery of [Ca2+]i following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+]i. Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+]i. 7. Intracellular orthovanadate caused an increase in resting [Ca2+]i and prevented the full recovery of [Ca2+]i following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+]i. We conclude that there is no Na(+)-Ca2+ exchanger present in the cell body of these neurones and that [Ca2+]i is maintained by an ATP-dependent Ca2+ pump. Images Figure 1 PMID:7623274

  16. Voltage clamp limitations of dual whole-cell gap junction current and voltage recordings. I. Conductance measurements.

    PubMed

    Veenstra, R D

    2001-05-01

    Previous correction methods for series access resistance errors in the dual whole-cell configuration did not take into account the effect of nonzero resting potentials (E(rest)) and junctional reversal potentials (E(rev)). Dual whole-cell currents were modeled according to resistor-circuit analysis and two correction formulas for the measurement of junctional currents (I(j)) were assessed. The equations for I(j), derived from Kirchoff's law before and after baseline subtraction of the nonjunctional current, were assessed for accuracy under a variety of whole-cell patch-clamp recording conditions. Both equations accurately correct for dual whole-cell voltage-clamp errors provided that the cellular parameters are included in the nonbaseline subtracted I(j) derivations. Junctional conductance (g(j)) estimates are most reliable at high junctional resistance (R(j)) values and minimize the need for corrective methods based on electrode series and cellular input resistances (R(el) and R(in)). In the "open-cell" configuration, low R(j) values relative to R(in) are required for accurate g(j) estimates. These methods provide the basis for accurate quantitative measurements of junctional resistance (or conductance) of gap junction channels or connexin hemichannels in the dual whole-cell or open-cell configurations. Revaluation of V(j)-dependent gating of rat connexin40 g(j) produced nearly identical Boltzmann fits to previously published data. Continuous g(j)-V(j) curves generated by variable slope V(j) ramps provide for more accurate fits and assessment of the time-dependence of the half-inactivation voltage and net gating charge movement. PMID:11325726

  17. X-ray microanalysis of single cardiac myocytes frozen under voltage-clamp conditions

    SciTech Connect

    Wendt-Gallitelli, M.F.; Isenberg, G.

    1989-02-01

    By means of a patch pipette, an isolated ventricular myocyte was transferred into the taper of a silver holder covered by pioloform film. Once the cell was on the film, the cell was voltage clamped (pulses from -45 to +5 mV at 0.5 Hz). The amount of Ca entry was estimated from the Ca current. When contractility (cell shortening) was potentiated with either five pulses of 0.2 s or four pulses of 1 s, shock freezing was timed 116 or 816 ms after start of the clamp pulse. Electron micrographs from freeze-substituted cells revealed the good preservation of the intracellular compartments. The myocytes were cut at -150 degrees C, and the cryosections were freeze dried. In representative examples, the amount of Ca entry is compared with the subcellular Ca distribution as it is analyzed with energy dispersive X-ray microprobe analysis in cytoplasm, junctional sarcoplasmic reticulum (SR), mitochondria, and the subsarcolemmal space (sarcolemma, peripheral SR, fringe of cytosol).

  18. Substance P-mediated membrane currents in voltage-clamped guinea pig inferior mesenteric ganglion cells.

    PubMed

    Griffith, W H; Hills, J M; Brown, D A

    1988-01-01

    Responses to substance P (SP) and to hypogastric nerve stimulation were recorded from voltage-clamped guinea pig inferior mesenteric ganglion (IMG) neurons, and compared with those to muscarine. Muscarine produced a voltage-dependent inward current accompanied by a reduced input conductance and inhibition of IM a time- and voltage-dependent K+-current (Brown and Adams: Nature 283:673-676, 1980). SP also produced an inward current, accompanied by a fall in input conductance (20 out of 31 cells) or a rise in input conductance (7 out of 31 cells). The fall in input conductance was not accompanied by an inhibition of M-current (unlike frog ganglia: Adams et al.: British Journal of Pharmacology 79:330-333, 1983) or an inhibition of the inward rectifier current (unlike globus pallidus neurons: Stanfield et al.: Nature 315:498-501, 1985). Repetitive hypogastric nerve stimulation (10-20 Hz, 2-10 s) produced a slow inward postsynaptic current lasting 1-3 min, with decreases or increases of input conductance matching those produced by SP. The postsynaptic current did not show a consistent or reproducible change in amplitude on varying the holding potential between -90 and -25 mV. It is concluded that SP and hypogastric stimulation produce complex and variable changes in ionic conductance in IMG neurons.

  19. A novel latch-up free SCR-LDMOS with high holding voltage for a power-rail ESD clamp

    NASA Astrophysics Data System (ADS)

    Hongwei, Pan; Siyang, Liu; Weifeng, Sun

    2013-01-01

    The low snapback holding voltage of the SCR-LDMOS device makes it susceptible to latch-up failure, when used in power-rail ESD (electro-static discharge) clamp circuits. In order to eliminate latch-up risk, this work presents a novel SCR-LDMOS structure with an N-type implantation layer to achieve a 17 V holding voltage and a 5.2 A second breakdown current. The device has been validated using TLP measurement analysis and is applied to a power-rail ESD clamp in half-bridge driver ICs.

  20. How cesium dialysis affects the passive properties of pyramidal neurons: implications for voltage clamp studies of persistent sodium current

    NASA Astrophysics Data System (ADS)

    Fleidervish, Ilya A.; Libman, Lior

    2008-03-01

    In order to accurately understand and model neuronal integration in the brain, we must know the biophysical properties of channels that are located far from the soma, in the axonal and dendritic membranes of central nerve cells. Reliable electrophysiological measurements in these regions are difficult to obtain, because the processes are too tiny to directly study with an electrode. One common strategy is to record with a somatic electrode that contains Cs+, to dialyze the intracellular space with this K+ channel blocker, and thereby to render the neuron electrotonically compact. Does this work? Here, we combine the experimental and modeling techniques to determine the extent to which a whole-cell voltage clamp, established with a Cs+-containing pipette in the soma of a cortical pyramidal cell, attains adequate voltage control of distal neuronal processes. We focus on the low-voltage-activated, slowly inactivating 'persistent' Na+ current (INaP), that plays a crucial role in determining neuronal excitability and synaptic integration.

  1. Action potential waveform voltage clamp shows significance of different Ca2+ channel types in developing ascidian muscle

    PubMed Central

    Dallman, Julia E; Dorman, Jennie B; Moody, William J

    2000-01-01

    Early in development, ascidian muscle cells generate spontaneous, long-duration action potentials that are mediated by a high-threshold, inactivating Ca2+ current. This spontaneous activity is required for appropriate physiological development.Mature muscle cells generate brief action potentials only in response to motor neuron input. The mature action potential is mediated by a high-threshold sustained Ca2+ current.Action potentials recorded from these two stages were imposed as voltage-clamp commands on cells of the same and different stages from which they were recorded. This strategy allowed us to study how immature and mature Ca2+ currents are optimized to their particular functions.Total Ca2+ entry during an action potential did not change during development. The developmental increase in Ca2+ current density exactly compensated for decreased spike duration. This compensation was a function purely of Ca2+ current density, not of the transition from immature to mature Ca2+ current types.In immature cells, Ca2+ entry was spread out over the entire waveform of spontaneous activity, including the interspike voltage trajectory. This almost continuous Ca2+ entry may be important in triggering Ca2+-dependent developmental programmes, and is a function of the slightly more negative voltage dependence of the immature Ca2+ current.In contrast, Ca2+ entry in mature cells was confined to the action potential itself, because of the slightly more positive voltage dependence of the mature Ca2+ current. This may be important in permitting rapid contraction-relaxation cycles during larval swimming.The inactivation of the immature Ca2+ current serves to limit the frequency and burst duration of spontaneous activity. The sustained kinetics of the mature Ca2+ current permit high-frequency firing during larval swimming. PMID:10766919

  2. Supercharging accelerates T-tubule membrane potential changes in voltage clamped frog skeletal muscle fibers.

    PubMed Central

    Kim, A M; Vergara, J L

    1998-01-01

    In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl] vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110-150 Omega.cm2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule. PMID:9746552

  3. An accurate online calibration system based on combined clamp-shape coil for high voltage electronic current transformers

    NASA Astrophysics Data System (ADS)

    Li, Zhen-hua; Li, Hong-bin; Zhang, Zhi

    2013-07-01

    Electronic transformers are widely used in power systems because of their wide bandwidth and good transient performance. However, as an emerging technology, the failure rate of electronic transformers is higher than that of traditional transformers. As a result, the calibration period needs to be shortened. Traditional calibration methods require the power of transmission line be cut off, which results in complicated operation and power off loss. This paper proposes an online calibration system which can calibrate electronic current transformers without power off. In this work, the high accuracy standard current transformer and online operation method are the key techniques. Based on the clamp-shape iron-core coil and clamp-shape air-core coil, a combined clamp-shape coil is designed as the standard current transformer. By analyzing the output characteristics of the two coils, the combined clamp-shape coil can achieve verification of the accuracy. So the accuracy of the online calibration system can be guaranteed. Moreover, by employing the earth potential working method and using two insulating rods to connect the combined clamp-shape coil to the high voltage bus, the operation becomes simple and safe. Tests in China National Center for High Voltage Measurement and field experiments show that the proposed system has a high accuracy of up to 0.05 class.

  4. An accurate online calibration system based on combined clamp-shape coil for high voltage electronic current transformers

    SciTech Connect

    Li, Zhen-hua; Li, Hong-bin; Zhang, Zhi

    2013-07-15

    Electronic transformers are widely used in power systems because of their wide bandwidth and good transient performance. However, as an emerging technology, the failure rate of electronic transformers is higher than that of traditional transformers. As a result, the calibration period needs to be shortened. Traditional calibration methods require the power of transmission line be cut off, which results in complicated operation and power off loss. This paper proposes an online calibration system which can calibrate electronic current transformers without power off. In this work, the high accuracy standard current transformer and online operation method are the key techniques. Based on the clamp-shape iron-core coil and clamp-shape air-core coil, a combined clamp-shape coil is designed as the standard current transformer. By analyzing the output characteristics of the two coils, the combined clamp-shape coil can achieve verification of the accuracy. So the accuracy of the online calibration system can be guaranteed. Moreover, by employing the earth potential working method and using two insulating rods to connect the combined clamp-shape coil to the high voltage bus, the operation becomes simple and safe. Tests in China National Center for High Voltage Measurement and field experiments show that the proposed system has a high accuracy of up to 0.05 class.

  5. An accurate online calibration system based on combined clamp-shape coil for high voltage electronic current transformers.

    PubMed

    Li, Zhen-hua; Li, Hong-bin; Zhang, Zhi

    2013-07-01

    Electronic transformers are widely used in power systems because of their wide bandwidth and good transient performance. However, as an emerging technology, the failure rate of electronic transformers is higher than that of traditional transformers. As a result, the calibration period needs to be shortened. Traditional calibration methods require the power of transmission line be cut off, which results in complicated operation and power off loss. This paper proposes an online calibration system which can calibrate electronic current transformers without power off. In this work, the high accuracy standard current transformer and online operation method are the key techniques. Based on the clamp-shape iron-core coil and clamp-shape air-core coil, a combined clamp-shape coil is designed as the standard current transformer. By analyzing the output characteristics of the two coils, the combined clamp-shape coil can achieve verification of the accuracy. So the accuracy of the online calibration system can be guaranteed. Moreover, by employing the earth potential working method and using two insulating rods to connect the combined clamp-shape coil to the high voltage bus, the operation becomes simple and safe. Tests in China National Center for High Voltage Measurement and field experiments show that the proposed system has a high accuracy of up to 0.05 class. PMID:23902112

  6. Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction

    PubMed Central

    Anderson, C. R.; Stevens, C. F.

    1973-01-01

    1. Acetylcholine produced end-plate current (e.p.c.) noise is shown to be the results of statistical fluctuations in the ionic conductance of voltage clamped end-plates of Rana pipiens. 2. These e.p.c. fluctuations are characterized by their e.p.c. spectra which conform to a relation predicted from a simple model of end-plate channel gating behaviour. 3. The rate constant of channel closing α is determined from e.p.c. spectra and is found to depend on membrane potential V according to the relation α = BeAV (B = 0·17 msec-1±0·04 S.E., A = 0·0058 mV-1±0·0009 S.E. at 8° C) and to vary with temperature T with a Q10 = 2·77, at -70 mV. A and B in this expression both vary with T and therefore produce a membrane potential dependent Q10 for α. 4. Nerve-evoked e.p.c.s and spontaneous miniature e.p.c.s decay exponentially in time with a rate constant which depends exponentially on V. The magnitude and voltage dependence of this decay constant is exactly that found from e.p.c. spectra for the channel closing rate α. 5. The conductance γ of a single open end-plate channel has been estimated from e.p.c. spectra and is found not to be detectibly dependent on membrane potential, temperature and mean end-plate current. γ = 0·32±0·0045 (S.E.) × 10-10 mhos. Some variation in values for γ occurs from muscle to muscle. 6. It is concluded that the relaxation kinetics of open ACh sensitive ionic channels is the rate limiting step in the decay of synaptic current and that this channel closing has a single time constant. The relaxation rate is independent of how it is estimated (ACh produced e.p.c. fluctuations, e.p.c., m.e.p.c.), and is consistent with the hypothesis that individual ionic channels open rapidly to a specific conductance which remains constant for an exponentially distributed duration. 7. The voltage and temperature dependence of the channel closing rate constant agree with the predictions of a simple dipole-conformation change model. PMID:4543940

  7. Calcium-activated conductance in skate electroreceptors: current clamp experiments

    PubMed Central

    1977-01-01

    When current clamped, skate electroreceptor epithelium produces large action potentials in response to stimuli that depolarize the lumenal faces of the receptor cells. With increasing stimulus strength these action potentials become prolonged. When the peak voltage exceeds about 140 mV the repolarizing phase is blocked until the end of the stimulus. Perfusion experiments show that the rising phase of the action potential results from an increase in calcium permeability in the lumenal membranes. Perfusion of the lumen with cobalt or with a zero calcium solution containing EGTA blocks the action potential. Perfusion of the lumen with a solution containing 10 mM Ca and 20 mM EGTA initially slows the repolarizing process at all voltages and lowers the potential at which it is blocked. With prolonged perfusion, repolarization is blocked at all voltages. When excitability is abolished by perfusion with cobalt, or with a zero calcium solution containing EGTA, no delayed rectification occurs. We suggest that repolarization during the action potential depends on an influx of calcium into the cytoplasm, and that the rate of repolarization depends on the magnitude of the inward calcium current. Increasingly large stimuli reduce the rate of repolarization by reducing the driving force for calcium, and then block repolarization by causing the lumenal membrane potential to exceed ECa. Changes in extracellular calcium affect repolarization in a manner consistent with the resulting change in ECa. PMID:190338

  8. Determination of ionic permeability coefficients of the plasma membrane of Xenopus laevis oocytes under voltage clamp.

    PubMed

    Costa, P F; Emilio, M G; Fernandes, P L; Ferreira, H G; Ferreira, K G

    1989-06-01

    1. A method of estimating absolute ionic permeability coefficients which does not depend on the use of impermeant substitutes is reported. 2. The method is based on a pump leak model of the Xenopus laevis oocyte membrane. The procedure consists of measuring, in the same experiment, the pump current and the currents generated under voltage clamp by the partial substitution of one or two ions at a time. For each experimental condition, the measured currents are substituted in a Goldman-Hodgkin-Katz type equation with two unknowns (the permeability coefficients). The set of equations thus generated enables the computation of all the ionic permeability coefficients. 3. The Xenopus oocyte membrane (stages IV and V, Dumont, 1972) has been found to be permeable to conventional ion substitutes such as N-methyl-D-glucamine (NMG), sulphate, isethionate and gluconate. 4. The values for sodium, potassium and chloride permeability coefficients obtained from sixty-eight pooled experiments were, respectively, 5.44, 17.41 and 1.49 x 10(-8) cm s-1. 5. The diffusional currents for sodium, potassium and chloride computed from the experiments referred to above were, respectively, -1.16, 0.69 and -0.038 microA cm-2. 6. A stoichiometry of the Na+-K+ pump exchange of 3/1.8 was computed. 7. The intracellular concentrations of sodium, potassium and chloride ions, as determined by ion-selective microelectrodes, were, respectively, 10.1 +/- 0.66 mM (n = 12), 109.5 +/- 3.3 mM (n = 13) and 37.7 +/- 1.18 mM (n = 19), corresponding to equilibrium potentials of 61, -95 and -28 mV. 8. Since chloride is not at equilibrium across the membrane, we propose that there is an inward uphill Cl- transport. PMID:2600847

  9. Transmission at voltage-clamped giant synapse of the squid: evidence for cooperativity of presynaptic calcium action.

    PubMed Central

    Smith, S J; Augustine, G J; Charlton, M P

    1985-01-01

    Synaptic transmission was studied at the squid giant synapse with voltage clamp control of both presynaptic and postsynaptic cells. Because presynaptic voltage gradients can complicate interpretation of electrophysiological data obtained from this preparation, we used local Ca application to restrict Ca influx and transmitter release to a short and relatively isopotential portion of the elongated presynaptic terminal. Under these conditions, we found that postsynaptic current varies approximately as the third power of presynaptic Ca current. This finding is consistent with the hypothesis that several Ca ions cooperate in triggering secretion of a single transmitter quantum. PMID:2982166

  10. Depletion and accumulation of potassium in the extracellular clefts of cardiac Purkinje fibers during voltage clamp hyperpolarization and depolarization.

    PubMed

    Baumgarten, C M; Isenberg, G

    1977-03-11

    1. Voltage clamp hyperpolarization and depolarization elicited current records consistent with depletion and accumulation, respectively, of potassium in the extracellular clefts of cardiac Purkinje fibers. Hyperpolarization was shown to shift the reversal potential for the pacemaker current, ik2, a measure of Ek, to more negative potentials. Upon depolarization, a slowly increasing outward current was observed. Analysis of the tail currents elicited by hyperpolarization revealed that a time-dependent change in gx could not explain the time-dependent outward current. However, the tail currents were consistent with a shift of Ek to more positive potentials during the depolarization. 2. Alteration in potassium driving force over time results in a time-dependent ik1 even though the underlying conductance is time-independent [29]. This time-dependent current may contribute to the currents usually identified as ik2 and ix. 3. The potential at which ik2 reverses direction is altered by the clamp program used to elicit it and is obscured by the superimposition of a time-dependent current due to depletion. 4. Records consistent with the extracellular cleft potassium concentration being less than that of the bulk phase in the quiescent fiber were obtained. However, an unequivocal interpretation of these current reocrds could not be made. 5. These results suggest that conclusions based on the assumption that potassium driving force remains constant during a voltage clamp pulse may be in error. Thus, time-dependent currents cannot be assumed to result solely from time-dependent conductance changes.

  11. Curvature-Driven Pore Growth in Charged Membranes during Charge-Pulse and Voltage-Clamp Experiments

    PubMed Central

    Kroeger, Jens H.; Vernon, Dan; Grant, Martin

    2009-01-01

    We find that curvature-driven growth of pores in electrically charged membranes correctly reproduces charge-pulse experiments. Our model, consisting of a Langevin equation for the time dependence of the pore radius coupled to an ordinary differential equation for the number of pores, captures the statistics of the pore population and its effect on the membrane conductance. The calculated pore radius is a linear, and not an exponential, function of time, as observed experimentally. Two other important features of charge-pulse experiments are recovered: pores reseal for low and high voltages but grow irreversibly for intermediate values of the voltage. Our set of coupled ordinary differential equations is equivalent to the partial differential equation used previously to study pore dynamics, but permits the study of longer timescales necessary for the simulations of voltage-clamp experiments. An effective phase diagram for such experiments is obtained. PMID:19186129

  12. The nature of the acetylcholine receptor in a Buccinum proboscis muscle examined by the sucrose-gap voltage clamp technique.

    PubMed

    Nelson, I D; Huddart, H

    1992-05-01

    1. ACh dose-response curves for the radicular retractor muscle of Buccinum showed maximum force and membrane depolarisation of 3.3 mV at 50 mumol l-1 ACh. 2. PCh was found to be almost a full agonist for force and induced higher membrane depolarisations than ACh while BCh was only a partial agonist of very low potency. This suggests an AChR neither muscarinic nor nicotinic in mammalian terminology. 3. Neither muscarine nor nicotine had any direct agonistic effects on the muscle but pre-exposure to nicotine inhibited both force and membrane depolarisation induced by a subsequent dose of ACh. 4. The specific muscarinic and nicotinic antagonists atropine, d-tubocurarine and gallamine all inhibited ACh responses in a dose-dependent manner. 5. Single sucrose-gap recording showed that ACh induced a depolarisation resulting in a contracture. Double sucrose-gap voltage clamp recording showed that 10 mumol l-1 ACh induced an inward transmembrane current of ca 2 microA. Both ACh-induced depolarisation and inward current were abolished in Na-free media. 6. When clamped at a series of membrane voltages between natural Em and positive potentials the ACh-induced Na-dependent inward current declined as Em was reduced and was abolished at -10 mV. This current showed no reversal even at strong positive membrane voltages. 7. The AChR of this muscle appears to be neither exclusively nicotinic nor muscarinic but a hybrid and shows characteristics of voltage inactivation.

  13. A digital feedback controller application for studying photoreceptor adaptation by 'voltage clamp by light'.

    PubMed

    Djupsund, K; Kouvalainen, E; Järvilehto, M; Weckström, M

    1995-11-01

    We present a new digital feedback application for the study of the sensitivity characteristics of photoreceptors. The amplitude of the recorded membrane voltage of a cell is steered by changing the incoming light intensity with a motor-driven circular, linear neutral-density wedge (CFW). The voltage response is sampled and fed to a software position controller of the CFW. The controller determines the position of the wedge according to the desired (command) value of the response. The light intensity changes during steady-state represent the sensitivity change, the time-course of adaptation.

  14. Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp.

    PubMed

    Wagner, Larry E; Groom, Linda A; Dirksen, Robert T; Yule, David I

    2014-08-01

    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 Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] 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 ∼750pS or 450pS in symmetrical 250mM 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 Ca(2+), 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.

  15. Correlation of 125I-LSD autoradiographic labeling with serotonin voltage clamp responses in Aplysia neurons

    SciTech Connect

    Evans, M.L.; Kadan, M.J.; Hartig, P.R.; Carpenter, D.O. )

    1991-05-01

    Autoradiographic receptor binding studies using 125I-LSD (2-(125I)lysergic acid diethyamide) revealed intense labelling on the soma of a symmetrically located pair of cells in the abdominal ganglion of Aplysia californica. This binding was blocked by micromolar concentrations of serotonin and lower concentrations of the serotonergic antagonists, cyproheptadine and mianserin. Electrophysiological investigation of responses to serotonin of neurons in the left upper quadrant, where one of the labeled neurons is located, revealed a range of serotonin responses. Cells L3 and L6 have a K+ conductance increase in response to serotonin that is not blocked by cyproheptadine or mianserin. Cells L2 and L4 have a biphasic response to serotonin: a Na+ conductance increase, which can be blocked by cyproheptadine and mianserin, followed by a voltage dependent Ca2+ conductance which is blocked by Co2+ but not the serotonergic antagonists. Cell L1, and its symmetrical pair, R1, have in addition to the Na+ and Ca2+ responses observed in L2 and L4, a Cl- conductance increase blocked by LSD, cyproheptadine and mianserin. LSD had little effect on the other responses. The authors conclude that the symmetrically located cells L1 and R1 have a Cl- channel linked to a cyproheptadine- and mianserin-sensitive serotonin receptor that is selectively labelled by 125I-LSD. This receptor has many properties in common with the mammalian serotonin 1C receptor.

  16. Determination and compensation of series resistances during whole-cell patch-clamp recordings using an active bridge circuit and the phase-sensitive technique.

    PubMed

    Riedemann, Therese; Polder, Hans Reiner; Sutor, Bernd

    2016-10-01

    We present a technique which combines two methods in order to measure the series resistance (R S) during whole-cell patch-clamp recordings from excitable and non-excitable cells. R S is determined in the amplifier's current-clamp mode by means of an active bridge circuit. The correct neutralization of the electrode capacitance and the adjustment of the bridge circuit is achieved by the so-called phase-sensitive method: Short sine wave currents with frequencies between 3 and 7 kHz are injected into the cells. Complete capacitance neutralization is indicated by the disappearance of the phase lag between current and voltage, and correct bridge balance is indicated by a minimized voltage response to the sine wave current. The R S value determined in the current-clamp mode then provides the basis for R S compensation in the voltage-clamp recording mode. The accuracy of the procedure has been confirmed on single-compartment cell models where the error amounted to 2-3 %. Similar errors were observed during dual patch clamp recordings from single neocortical layer 5 pyramidal cells where one electrode was connected to the bridge amplifier and the other one to a time-sharing, single-electrode current- and voltage-clamp amplifier with negligible R S. The technique presented here allows R S compensation for up to 80-90 %, even in cells with low input resistances (e.g., astrocytes). In addition, the present study underlines the importance of correct R S compensation by showing that significant series resistances directly affect the determination of membrane conductances as well as the kinetic properties of spontaneous synaptic currents with small amplitudes. PMID:27539299

  17. Current-fed Step-up DC/DC Converter for Fuel Cell Applications with Active Overvoltage Clamping

    NASA Astrophysics Data System (ADS)

    Andreiciks, Aleksandrs; Steiks, Ingars; Krievs, Oskars

    2010-01-01

    In order to use hydrogen fuel cells in domestic applications either as main power supply or backup source, their low DC output voltage has to be matched to the level and frequency of the utility grid AC voltage. Such power converter systems usually consist of a DC-DC converter and a DC-AC inverter. A double inductor step-up push-pull converter is investigated in this paper, presenting simulation and experimental results for passive and active overvoltage clamping. The prototype of the investigated converter is elaborated for 1200 W power to match the rated power of the proton exchange membrane (PEM) fuel cell located in hydrogen fuel cell research laboratory.

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

    PubMed

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

    2011-06-01

    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

  19. State-of-the-art automated patch clamp: heat activation, action potentials, and high throughput in ion channel screening.

    PubMed

    Stoelzle-Feix, Sonja

    2014-01-01

    A successful robotic approach of the patch clamp technique is based on planar patch clamp chips where a glass pipette, as used in conventional patch clamping, is replaced by a thin planar glass sheet with a small hole in the middle. Automated patch clamp (APC) systems utilizing this chip design offer higher throughput capabilities and ease of use and thus have become common in basic research, drug development, and safety screening. Further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. Here, two features with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike are described. The utilized system is a medium throughput APC device capable of recording up to eight cells simultaneously. The temperature control capability and the possibility to perform recordings not only in the voltage clamp but also in the current clamp mode are described in detail. Since eight recordings can be generated in parallel without compromising data quality, reliable and cost-effective and time-effective screening of compounds against ion channels using voltage clamp and current clamp electrophysiology can be performed. PMID:25023302

  20. Effects of injecting calcium-buffer solution on [Ca2+]i in voltage-clamped snail neurons.

    PubMed Central

    Kennedy, H J; Thomas, R C

    1996-01-01

    We have investigated why fura-2 and Ca(2+)-sensitive microelectrodes report different values for the intracellular free calcium ion concentration ([Ca(2+)]i or its negative log, pCa(i)) of snail neurons voltage-clamped to -50 or -60 mV. Both techniques were initially calibrated in vitro, using calcium calibration solutions that had ionic concentrations similar to those of snail neuron cytoplasm. Pressure injections of the same solutions at resting and elevated [Ca(2+)]i were used to calibrate both methods in vivo. In fura-2-loaded cells these pressure injections generated changes in [Ca(2+)]i that agreed well with those expected from the in vitro calibration. Thus, using fura-2 calibrated in vitro, the average resting [Ca(2+)]i was found to be 38 nM (pCa(i) 7.42 +/- 0.05). With Ca(2+)-sensitive microelectrodes, the first injection of calibration solutions always caused a negative shift in the recorded microelectrode potential, as if the injection lowered [Ca2+]i. No such effects were seen on the fura-2 ratio. When calibrated in vivo the Ca(2+)-sensitive microelectrode gave an average resting [Ca2+]i of approximately 25 nM (pCa(i) 7.6 +/- 0.1), much lower than when calibrated in vitro. We conclude that [Ca(2+)]i in snail neurons is approximately 40 nM and that Ca(2+)-sensitive microelectrodes usually cause a leak at the point of insertion. The effects of the leak were minimized by injection of a mobile calcium buffer. PMID:9172736

  1. Effects of low doses of caffeine on [Ca2+]i in voltage-clamped snail (Helix aspersa) neurones.

    PubMed Central

    Orkand, R K; Thomas, R C

    1995-01-01

    1. We have measured cytosolic free Ca2+ concentrations ([Ca2+]i) in voltage-clamped snail neurones using fura-2. Transient increases in [Ca2+]i were induced by depolarizing voltage steps of 20-60 mV for 0.1-10 s from a holding potential of -50 or -60 mV. 2. Low doses of caffeine, 0.2-1 mM, increased the size of the [Ca2+]i transients by both increasing the peak and producing an undershoot. 3. Ryanodine, an inhibitor of Ca2+ release from the intracellular Ca2+ stores, and cyclopiazonic acid (CPA), an inhibitor of the Ca(2+)-ATPase of the intracellular Ca2+ stores, both reduced the size of the [Ca2+]i transients and blocked the effects of caffeine on the transients. 4. The effects of caffeine and CPA were greater on transients produced by long, small, rather than short, large depolarizations. This suggests that calcium-induced calcium release (CICR) played a greater role in the [Ca2+]i increase resulting from longer, smaller depolarizations. 5. Increasing the extracellular pH from 7.5 to over 9, which inhibits the plasmalemmal Ca(2+)-H(+)-ATPase, increased the resting [Ca2+]i level. Depolarization-induced [Ca2+]i transients became much larger while the two effects of caffeine remained. CPA was ineffective at high pH. 6. In some experiments the increase in basal [Ca2+]i caused by alkaline pH was reduced by 0.2 or 0.5 mM caffeine. The increase in basal [Ca2+]i caused by maintained depolarization was reduced, after a transient increase, by 0.5 mM caffeine. Both reduction and increase were blocked by CPA. 7. We conclude that low doses of caffeine can increase uptake by intracellular Ca2+ stores. Caffeine could also release Ca2+ from ryanodine-insensitive Ca(2+)-ATPase-dependent stores as well as facilitating normal ryanodine-sensitive CICR. PMID:8583402

  2. Protein binding-dependent decreases in hERG channel blocker potency assessed by whole-cell voltage clamp in serum.

    PubMed

    Margulis, Michael; Sorota, Steve; Chu, Inhou; Soares, Anthony; Priestley, Tony; Nomeir, Amin A

    2010-04-01

    In vitro hERG blocking potency is measured in drug discovery as part of an integrated cardiovascular risk assessment. Typically, the concentrations producing 50% inhibition are measured in protein-free saline solutions and compared with calculated free therapeutic in vivo Cmax values to estimate a hERG safety multiple. The free/unbound fraction is believed responsible for activity. We tested the validity of this approach with 12 compounds by determining potencies in voltage clamp studies conducted in the absence and presence of 100% dialyzed fetal bovine serum (FBS). Bath drug concentrations in saline solutions were measured to account for loss of compounds due to solubility, stability, and/or adsorption. Protein binding in dialyzed FBS was measured to enable predictions of serum IC50s based on the unbound fraction and the saline IC50. For 11 of 12 compounds, the measured potency in the presence of dialyzed FBS was within 2-fold of the predicted potency. The predicted IC50 in dialyzed FBS for one highly bound compound, amiodarone, was 9-fold higher than the measured serum IC50. These data suggest that for highly bound compounds, direct measurement of IC50s in the presence of 100% serum may provide a more accurate estimate of in vivo potencies than the approach based on calculated serum shifts. PMID:20125032

  3. A voltage-activated proton current in human cardiac fibroblasts

    SciTech Connect

    El Chemaly, Antoun; Guinamard, Romain; Demion, Marie; Fares, Nassim; Jebara, Victor; Faivre, Jean-Francois; Bois, Patrick . E-mail: patrick.bois@univ-poitiers.fr

    2006-02-10

    A voltage-activated proton current in human cardiac fibroblasts, measured using the whole-cell recording configuration of the patch-clamp technique, is reported. Increasing the pH of the bathing solution shifted the current activation threshold to more negative potentials and increased both the current amplitude and its rate of activation. Changing the pH gradient by one unit caused a 51 mV shift in the reversal potential of the current, demonstrating a high selectivity for protons of the channel carrying the current. Extracellularly applied Zn{sup 2+} reversibly inhibited the current. Activation of the current contributes to the resting membrane conductance under conditions of intracellular acidosis. It is proposed that this current in cardiac fibroblasts is involved in the regulation of the intracellular pH and the membrane potential under physiological conditions as well as in response to pathological conditions such as ischemia.

  4. Recordings of cultured neurons and synaptic activity using patch-clamp chips

    NASA Astrophysics Data System (ADS)

    Martina, Marzia; Luk, Collin; Py, Christophe; Martinez, Dolores; Comas, Tanya; Monette, Robert; Denhoff, Mike; Syed, Naweed; Mealing, Geoffrey A. R.

    2011-06-01

    Planar patch-clamp chip technology has been developed to enhance the assessment of novel compounds for therapeutic efficacy and safety. However, this technology has been limited to recording ion channels expressed in isolated suspended cells, making the study of ion channel function in synaptic transmission impractical. Recently, we developed single- and dual-recording site planar patch-clamp chips and demonstrated their capacity to record ion channel activity from neurons established in culture. Such capacity provides the opportunity to record from synaptically connected neurons cultured on-chip. In this study we reconstructed, on-chip, a simple synaptic circuit between cultured pre-synaptic visceral dorsal 4 neurons and post-synaptic left pedal dorsal 1 neurons isolated from the mollusk Lymnaea stagnalis. Here we report the first planar patch-clamp chip recordings of synaptic phenomena from these paired neurons and pharmacologically demonstrate the cholinergic nature of this synapse. We also report simultaneous dual-site recordings from paired neurons, and demonstrate dedicated cytoplasmic perfusion of individual neurons via on-chip subterranean microfluidics. This is the first application of planar patch-clamp technology to examine synaptic communication.

  5. Characterization of active hair-bundle motility by a mechanical-load clamp

    NASA Astrophysics Data System (ADS)

    Salvi, Joshua D.; Maoiléidigh, Dáibhid Ó.; Fabella, Brian A.; Tobin, Mélanie; Hudspeth, A. J.

    2015-12-01

    Active hair-bundle motility endows hair cells with several traits that augment auditory stimuli. The activity of a hair bundle might be controlled by adjusting its mechanical properties. Indeed, the mechanical properties of bundles vary between different organisms and along the tonotopic axis of a single auditory organ. Motivated by these biological differences and a dynamical model of hair-bundle motility, we explore how adjusting the mass, drag, stiffness, and offset force applied to a bundle control its dynamics and response to external perturbations. Utilizing a mechanical-load clamp, we systematically mapped the two-dimensional state diagram of a hair bundle. The clamp system used a real-time processor to tightly control each of the virtual mechanical elements. Increasing the stiffness of a hair bundle advances its operating point from a spontaneously oscillating regime into a quiescent regime. As predicted by a dynamical model of hair-bundle mechanics, this boundary constitutes a Hopf bifurcation.

  6. State-of-the-Art Automated Patch Clamp Devices: Heat Activation, Action Potentials, and High Throughput in Ion Channel Screening

    PubMed Central

    Stoelzle, Sonja; Obergrussberger, Alison; Brüggemann, Andrea; Haarmann, Claudia; George, Michael; Kettenhofen, Ralf; Fertig, Niels

    2011-01-01

    Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. The availability of standardized cell lines expressing ion channels of interest lead to the development of diverse automated patch clamp (APC) systems with high-throughput capabilities. These systems are now available for drug screening, but there are limitations in the application range. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. The addition of well controlled and fast solution exchange, temperature control and the availability of the current clamp mode are required to analyze standard cell lines and excitable cells such as stem cell-derived cardiomyocytes in a more physiologically relevant environment. Here we describe two systems with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike. The here utilized medium throughput APC device is a planar patch clamp system capable of recording up to eight cells simultaneously. Features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The high-throughput system used here is a planar patch clamp screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The data provided reveals the suitability and relevance of both APC platforms for drug discovery, ion channel research, and safety testing. PMID:22131976

  7. State-of-the-Art Automated Patch Clamp Devices: Heat Activation, Action Potentials, and High Throughput in Ion Channel Screening.

    PubMed

    Stoelzle, Sonja; Obergrussberger, Alison; Brüggemann, Andrea; Haarmann, Claudia; George, Michael; Kettenhofen, Ralf; Fertig, Niels

    2011-01-01

    Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. The availability of standardized cell lines expressing ion channels of interest lead to the development of diverse automated patch clamp (APC) systems with high-throughput capabilities. These systems are now available for drug screening, but there are limitations in the application range. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. The addition of well controlled and fast solution exchange, temperature control and the availability of the current clamp mode are required to analyze standard cell lines and excitable cells such as stem cell-derived cardiomyocytes in a more physiologically relevant environment. Here we describe two systems with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike. The here utilized medium throughput APC device is a planar patch clamp system capable of recording up to eight cells simultaneously. Features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The high-throughput system used here is a planar patch clamp screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The data provided reveals the suitability and relevance of both APC platforms for drug discovery, ion channel research, and safety testing. PMID:22131976

  8. Adhesive curing through low-voltage activation

    NASA Astrophysics Data System (ADS)

    Ping, Jianfeng; Gao, Feng; Chen, Jian Lin; Webster, Richard D.; Steele, Terry W. J.

    2015-08-01

    Instant curing adhesives typically fall within three categories, being activated by either light (photocuring), heat (thermocuring) or chemical means. These curing strategies limit applications to specific substrates and can only be activated under certain conditions. Here we present the development of an instant curing adhesive through low-voltage activation. The electrocuring adhesive is synthesized by grafting carbene precursors on polyamidoamine dendrimers and dissolving in aqueous solvents to form viscous gels. The electrocuring adhesives are activated at -2 V versus Ag/AgCl, allowing tunable crosslinking within the dendrimer matrix and on both electrode surfaces. As the applied voltage discontinued, crosslinking immediately terminated. Thus, crosslinking initiation and propagation are observed to be voltage and time dependent, enabling tuning of both material properties and adhesive strength. The electrocuring adhesive has immediate implications in manufacturing and development of implantable bioadhesives.

  9. Adhesive curing through low-voltage activation

    PubMed Central

    Ping, Jianfeng; Gao, Feng; Chen, Jian Lin; Webster, Richard D.; Steele, Terry W. J.

    2015-01-01

    Instant curing adhesives typically fall within three categories, being activated by either light (photocuring), heat (thermocuring) or chemical means. These curing strategies limit applications to specific substrates and can only be activated under certain conditions. Here we present the development of an instant curing adhesive through low-voltage activation. The electrocuring adhesive is synthesized by grafting carbene precursors on polyamidoamine dendrimers and dissolving in aqueous solvents to form viscous gels. The electrocuring adhesives are activated at −2 V versus Ag/AgCl, allowing tunable crosslinking within the dendrimer matrix and on both electrode surfaces. As the applied voltage discontinued, crosslinking immediately terminated. Thus, crosslinking initiation and propagation are observed to be voltage and time dependent, enabling tuning of both material properties and adhesive strength. The electrocuring adhesive has immediate implications in manufacturing and development of implantable bioadhesives. PMID:26282730

  10. Fast and slow activation kinetics of voltage-gated sodium channels in molluscan neurons.

    PubMed

    Gilly, W F; Gillette, R; McFarlane, M

    1997-05-01

    Whole cell patch-clamp recordings of Na current (I(Na)) were made under identical experimental conditions from isolated neurons from cephalopod (Loligo, Octopus) and gastropod (Aplysia, Pleurobranchaea, Doriopsilla) species to compare properties of activation gating. Voltage dependence of peak Na conductance (gNa) is very similar in all cases, but activation kinetics in the gastropod neurons studied are markedly slower. Kinetic differences are very pronounced only over the voltage range spanned by the gNa-voltage relation. At positive and negative extremes of voltage, activation and deactivation kinetics of I(Na) are practically indistinguishable in all species studied. Voltage-dependent rate constants underlying activation of the slow type of Na channel found in gastropods thus appear to be much more voltage dependent than are the equivalent rates in the universally fast type of channel that predominates in cephalopods. Voltage dependence of inactivation kinetics shows a similar pattern and is representative of activation kinetics for the two types of Na channels. Neurons with fast Na channels can thus make much more rapid adjustments in the number of open Na channels at physiologically relevant voltages than would be possible with only slow Na channels. This capability appears to be an adaptation that is highly evolved in cephalopods, which are well known for their high-speed swimming behaviors. Similarities in slow and fast Na channel subtypes in molluscan and mammalian neurons are discussed. PMID:9163364

  11. Hippocampal glucocorticoid receptor activation enhances voltage-dependent Ca2+ conductances: relevance to brain aging.

    PubMed Central

    Kerr, D S; Campbell, L W; Thibault, O; Landfield, P W

    1992-01-01

    Glucocorticoids (GCs) activate several biochemical/molecular processes in the hippocampus through two receptor types. In addition, GCs influence cognitive behaviors and hippocampal neural activity and can also increase the rate of aging-dependent cell loss in the hippocampus. However, the ionic mechanisms through which GCs modulate hippocampal neuronal function are not well understood. We report here direct evidence that activation of cytosolic steroid receptors, specifically of the type II GC receptor, can enhance voltage-dependent Ca2+ conductances in brain neurons. Ca2+ current was assessed by current-clamp measures of Ca2+ action potentials and by sharp electrode voltage-clamp analyses of voltage-sensitive currents in cesium-, tetrodotoxin-, and tetraethylammonium-treated CA1 neurons in hippocampal slices. Both Ca2+ action potentials and voltage-activated Ca2+ currents (N- and L-like) were increased by 2-hr exposure to the synthetic GC receptor agonist, RU 28362. This effect of RU 28362 was blocked by coincubation with cycloheximide, indicating that the GC receptor-Ca2+ channel interaction depends on de novo protein synthesis. Dysregulated calcium homeostasis is also viewed as a candidate mechanism in brain aging. Thus, present results are consistent with the hypothesis that excessive GC-receptor activation and resultant increased Ca2+ influx may be two sequential phases of a brain-aging process that results initially in impairment of function and eventually in neuronal loss. PMID:1528857

  12. Rigid clamp

    DOEpatents

    Benavides, Gilbert L.; Burt, Jack D.

    1994-01-01

    The invention relates to a clamp mechanism that can be used to attach or temporarily support objects inside of tubular goods. The clamp mechanism can also be modified so that it grips objects. The clamp has a self-centering feature to accommodate out-of-roundness or other internal defections in tubular objects such as pipe. A plurality of clamping shoes are expanded by a linkage which is preferably powered by a motor to contact the inside of a pipe. The motion can be reversed and jaw elements can be connected to the linkage so as to bring the jaws together to grab an object.

  13. Rigid clamp

    DOEpatents

    Benavides, G.L.; Burt, J.D.

    1994-07-12

    The invention relates to a clamp mechanism that can be used to attach or temporarily support objects inside of tubular goods. The clamp mechanism can also be modified so that it grips objects. The clamp has a self-centering feature to accommodate out-of-roundness or other internal defections in tubular objects such as pipe. A plurality of clamping shoes are expanded by a linkage which is preferably powered by a motor to contact the inside of a pipe. The motion can be reversed and jaw elements can be connected to the linkage so as to bring the jaws together to grab an object. 12 figs.

  14. Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording

    PubMed Central

    Kolb, Ilya; Kodandaramaiah, Suhasa B.; Chubykin, Alexander A.; Yang, Aimei; Bear, Mark F.; Boyden, Edward S.; Forest, Craig R.

    2014-01-01

    Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation. PMID:25429119

  15. Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording.

    PubMed

    Harrison, Reid R; Kolb, Ilya; Kodandaramaiah, Suhasa B; Chubykin, Alexander A; Yang, Aimei; Bear, Mark F; Boyden, Edward S; Forest, Craig R

    2015-02-15

    Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation.

  16. Activation of Ih and TTX-sensitive sodium current at subthreshold voltages during CA1 pyramidal neuron firing.

    PubMed

    Yamada-Hanff, Jason; Bean, Bruce P

    2015-10-01

    We used dynamic clamp and action potential clamp techniques to explore how currents carried by tetrodotoxin-sensitive sodium channels and HCN channels (Ih) regulate the behavior of CA1 pyramidal neurons at resting and subthreshold voltages. Recording from rat CA1 pyramidal neurons in hippocampal slices, we found that the apparent input resistance and membrane time constant were strongly affected by both conductances, with Ih acting to decrease apparent input resistance and time constant and sodium current acting to increase both. We found that both Ih and sodium current were active during subthreshold summation of artificial excitatory postsynaptic potentials (EPSPs) generated by dynamic clamp, with Ih dominating at less depolarized voltages and sodium current at more depolarized voltages. Subthreshold sodium current-which amplifies EPSPs-was most effectively recruited by rapid voltage changes, while Ih-which blunts EPSPs-was maximal for slow voltage changes. The combined effect is to selectively amplify rapid EPSPs. We did similar experiments in mouse CA1 pyramidal neurons, doing voltage-clamp experiments using experimental records of action potential firing of CA1 neurons previously recorded in awake, behaving animals as command voltages to quantify flow of Ih and sodium current at subthreshold voltages. Subthreshold sodium current was larger and subthreshold Ih was smaller in mouse neurons than in rat neurons. Overall, the results show opposing effects of subthreshold sodium current and Ih in regulating subthreshold behavior of CA1 neurons, with subthreshold sodium current prominent in both rat and mouse CA1 pyramidal neurons and additional regulation by Ih in rat neurons.

  17. Energy harvesting under excitation of clamped-clamped beam

    NASA Astrophysics Data System (ADS)

    Batra, Ashok; Alomari, Almuatasim; Aggarwal, Mohan; Bandyopadhyay, Alak

    2016-04-01

    In this article, a piezoelectric energy harvesting has been developed experimentally and theoretically based on Euler- Bernoulli Theory. A PVDF piezoelectric thick film has attached along of clamped-clamped beam under sinusoidal base excitation of shaker. The results showed a good agreement between the experimental and simulation of suggested model. The voltage output frequency response function (FRF), current FRF, and output power has been studied under short and open circuit conditions at first vibration mode. The mode shape of the clamped-clamped beam for first three resonance frequency has been modeled and investigated using COMSOL Multiphysics and MATLAB.

  18. Depression of voltage-activated Ca2+ release in skeletal muscle by activation of a voltage-sensing phosphatase

    PubMed Central

    Berthier, Christine; Kutchukian, Candice; Bouvard, Clément; Okamura, Yasushi

    2015-01-01

    Phosphoinositides act as signaling molecules in numerous cellular transduction processes, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) regulates the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P2 in Ca2+ homeostasis and excitation–contraction (E-C) coupling of mouse muscle fibers using in vivo expression of the voltage-sensing phosphatases (VSPs) Ciona intestinalis VSP (Ci-VSP) or Danio rerio VSP (Dr-VSP). Confocal images of enhanced green fluorescent protein–tagged Dr-VSP revealed a banded pattern consistent with VSP localization within the transverse tubule membrane. Rhod-2 Ca2+ transients generated by 0.5-s-long voltage-clamp depolarizing pulses sufficient to elicit Ca2+ release from the sarcoplasmic reticulum (SR) but below the range at which VSPs are activated were unaffected by the presence of the VSPs. However, in Ci-VSP–expressing fibers challenged by 5-s-long depolarizing pulses, the Ca2+ level late in the pulse (3 s after initiation) was significantly lower at 120 mV than at 20 mV. Furthermore, Ci-VSP–expressing fibers showed a reversible depression of Ca2+ release during trains, with the peak Ca2+ transient being reduced by ∼30% after the application of 10 200-ms-long pulses to 100 mV. A similar depression was observed in Dr-VSP–expressing fibers. Cav1.1 Ca2+ channel–mediated current was unaffected by Ci-VSP activation. In fibers expressing Ci-VSP and a pleckstrin homology domain fused with monomeric red fluorescent protein (PLCδ1PH-mRFP), depolarizing pulses elicited transient changes in mRFP fluorescence consistent with release of transverse tubule–bound PLCδ1PH domain into the cytosol; the voltage sensitivity of these changes was consistent with that of Ci-VSP activation, and recovery occurred with a time constant in the 10-s range. Our results indicate that the PtdIns(4,5)P2 level is tightly maintained in the transverse tubule membrane of the muscle fibers

  19. Effects of deuterium oxide on the rate and dissociation constants for saxitoxin and tetrodotoxin action. Voltage-clamp studies on frog myelinated nerve

    SciTech Connect

    Hahin, R.; Strichartz, G.

    1981-08-01

    The actions of tetrodotoxin (TTX) and saxitoxin (STX) in normal water and in deuterium oxide (D/sub 20/) have been studied in frog myelinated nerve. Substitution of D/sub 20/ for H/sub 20/ in normal Ringer's solution has no effect on the potency of TTX in blocking action potentials but increases the potency of STX by approximately 50%. Under voltage clamp, the steady-state inhibition of sodium currents by 1 nM STX is doubled in D/sub 20/ as a result of a halving of the rate of dissociation of STX from the sodium channel; the rate of block by STX is not measurably changed by D/sub 20/. Neither steady-state inhibition nor the on- or off-rate constants of TTX are changed by D/sub 20/ substitution. The isotopic effects on STX binding are observed less than 10 min after the toxin has been added to D/sub 20/, thus eliminating the possibility that slow-exchange (t 1/2 greater than 10 h) hydrogen-binding sites on STX are involved. The results are consistent with a hypothesis that attributes receptor-toxin stabilization to isotopic changes of hydrogen bonding; this interpretation suggests that hydrogen bonds contribute more to the binding of STX than to that of TTX at the sodium channel.

  20. Ionic dependence of active Na-K transport: "clamping" of cellular Na+ with monensin.

    PubMed

    Haber, R S; Pressley, T A; Loeb, J N; Ismail-Beigi, F

    1987-07-01

    The Na+ ionophore monensin was used to study the Na+- and K+-dependence of ouabain-inhibitable 86Rb+ uptake in ARL 15 cells, a rat liver cell line. Graded concentrations of monensin rapidly induced incremental elevations of cellular Na+ that were stable for up to 2 h. In experiments in which cellular Na+ was thus "clamped" at various levels, the activation curve for ouabain-inhibitable 86Rb+ uptake as a function of intracellular Na+ was found to be steepest near basal Na+ levels (Hill coefficient approximately equal to 2.4), indicating that these cells can respond to relatively large changes in passive Na+ entry by increasing the race of Na-K pump function with only minimal increases in cellular Na+. Exposure of cells to monensin also permitted examination of the extracellular-K+ dependence of ouabain-inhibitable 86Rb+ uptake in the presence of saturating intracellular Na+ and yielded a Hill coefficient of approximately 1.5. The rate of ATP hydrolysis calculated from measurements of the maximal rate of ouabain-inhibitable 86Rb+ uptake in intact cells was similar to the enzymatic Vmax of the Na+-K+-ATPase in cell lysates, suggesting that the Na+-K+-ATPase activity in these broken-cell preparations closely reflects the functional transport capacity of the Na-K pump.

  1. 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

  2. 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.

  3. Force-controlled patch clamp of beating cardiac cells.

    PubMed

    Ossola, Dario; Amarouch, Mohamed-Yassine; Behr, Pascal; Vörös, János; Abriel, Hugues; Zambelli, Tomaso

    2015-03-11

    From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage. PMID:25639960

  4. Immediate or early cord clamping vs delayed clamping.

    PubMed

    Hutchon, D J R

    2012-11-01

    Over the past 40 years, there have been a number of review articles attempting to rationalise cord clamping practice. Early cord clamping was originally thought to be important in active management of the third stage of labour, but this was never evidence based. Without an evidence base to justify it, early cord clamping in clinical practice has remained very variable. There is good evidence that early cord clamping leads to hypovolaemia, anaemia and low iron stores in the neonate. We review all the evidence and discuss possible reasons why some obstetricians and midwives persevere with early clamping. We explain how a variable definition, defective education, deferred responsibility between obstetrician and paediatrician, variable guidelines and a lack of appreciation for the potential harm of the intervention, have all contributed. This study describes how the need for early cord clamping can be avoided in practically all clinical complications of birth.

  5. 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

  6. Voltage-dependent drug blockade of L-glutamate activated channels of the crayfish.

    PubMed Central

    Dekin, M S; Edwards, C

    1983-01-01

    The actions of d-tubocurarine (d-TC) and local anaesthetics on the L-glutamate activated channel at the voltage-clamped crayfish neuromuscular junction were studied. The effect of d-TC and local anaesthetics on the dose-response relationship between ionophoretically applied L-glutamate and synaptic current suggested that both acted as non-competitive inhibitors. The amount of inhibition was voltage dependent, and increased as the membrane potential was hyperpolarized. This voltage-dependent block was also manifest in a flattening of the I-V relationship between L-glutamate induced current and membrane potential in the presence of d-TC. However, the reversal potential for the L-glutamate activated channel was not affected; it was about +7 mV in both the presence and absence of d-TC. The neurally evoked excitatory post-synaptic current (e.p.s.c.) was depressed in the presence of these drugs and this effect was also voltage dependent. The time course of the e.p.s.c. was affected, but less so than expected if the L-glutamate activated channel were identical to the channel opened by acetylcholine at the vertebrate neuromuscular junction. Possible reasons for this discrepancy are discussed. PMID:6312026

  7. Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture.

    PubMed Central

    Fain, G L; Farahbakhsh, N A

    1989-01-01

    1. The whole-cell recording mode of the patch-clamp technique was used to investigate the presence of voltage-activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization-activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization-activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)-independent. 5. The hyperpolarization-activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below -120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage-dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)-dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials. Images Fig. 2 PMID:2621623

  8. Voltage Dependence of a Neuromodulator-Activated Ionic Current123

    PubMed Central

    2016-01-01

    Abstract The neuromodulatory inward current (IMI) generated by crab Cancer borealis stomatogastric ganglion neurons is an inward current whose voltage dependence has been shown to be crucial in the activation of oscillatory activity of the pyloric network of this system. It has been previously shown that IMI loses its voltage dependence in conditions of low extracellular calcium, but that this effect appears to be regulated by intracellular calmodulin. Voltage dependence is only rarely regulated by intracellular signaling mechanisms. Here we address the hypothesis that the voltage dependence of IMI is mediated by intracellular signaling pathways activated by extracellular calcium. We demonstrate that calmodulin inhibitors and a ryanodine antagonist can reduce IMI voltage dependence in normal Ca2+, but that, in conditions of low Ca2+, calmodulin activators do not restore IMI voltage dependence. Further, we show evidence that CaMKII alters IMI voltage dependence. These results suggest that calmodulin is necessary but not sufficient for IMI voltage dependence. We therefore hypothesize that the Ca2+/calmodulin requirement for IMI voltage dependence is due to an active sensing of extracellular calcium by a GPCR family calcium-sensing receptor (CaSR) and that the reduction in IMI voltage dependence by a calmodulin inhibitor is due to CaSR endocytosis. Supporting this, preincubation with an endocytosis inhibitor prevented W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride)-induced loss of IMI voltage dependence, and a CaSR antagonist reduced IMI voltage dependence. Additionally, myosin light chain kinase, which is known to act downstream of the CaSR, seems to play a role in regulating IMI voltage dependence. Finally, a Gβγ-subunit inhibitor also affects IMI voltage dependence, in support of the hypothesis that this process is regulated by a G-protein-coupled CaSR. PMID:27257619

  9. Loading clamps for DNA replication and repair.

    PubMed

    Bloom, Linda B

    2009-05-01

    Sliding clamps and clamp loaders were initially identified as DNA polymerase processivity factors. Sliding clamps are ring-shaped protein complexes that encircle and slide along duplex DNA, and clamp loaders are enzymes that load these clamps onto DNA. When bound to a sliding clamp, DNA polymerases remain tightly associated with the template being copied, but are able to translocate along DNA at rates limited by rates of nucleotide incorporation. Many different enzymes required for DNA replication and repair use sliding clamps. Clamps not only increase the processivity of these enzymes, but may also serve as an attachment point to coordinate the activities of enzymes required for a given process. Clamp loaders are members of the AAA+ family of ATPases and use energy from ATP binding and hydrolysis to catalyze the mechanical reaction of loading clamps onto DNA. Many structural and functional features of clamps and clamp loaders are conserved across all domains of life. Here, the mechanism of clamp loading is reviewed by comparing features of prokaryotic and eukaryotic clamps and clamp loaders.

  10. 200-kV active optical fiber voltage transformer

    NASA Astrophysics Data System (ADS)

    Xu, Yan; Luo, Sunan; Ye, Miaoyuan

    1999-02-01

    The report describes a 220kV Active Optical Fiber Voltage Transformer (AOVT). The transformer is different from the passive optical fiber voltage transformer, for no optical crystal is used in the 220kV AOVT. Its principle is that a low voltage is divided for the 220kV high voltage by a capacitive divider and then is converted into a digital signal. The optical fiber is used to transfer the measured digital signal and control signal. The 220kV AOVT consists of an outdoors-high voltage measurement unit and an indoors low voltage metering and controlling unit. The optical fiber connects these units. The low voltage is effectively isolated from the high voltage by means of the optical fiber and a special power supply method which is specially designed for the outdoor high voltage unit. As a result, the safe protection is reliable for the indoor low voltage equipment and the operation staff. Compared to the conventional voltage transformer, the advantages of the 220kV AOVT are high accuracy, low cost, excellent dynamic characteristics and immunity from electromagnetic interference. The 220kV AOVT has been tested, and its accuracy could achieve +/- 0.2 percent.

  11. The slow and the quick anion conductance in whole guard cells: their voltage-dependent alternation, and the modulation of their activities by abscisic acid and CO2.

    PubMed

    Raschke, Klaus; Shabahang, Mahbobeh; Wolf, Rupert

    2003-08-01

    We explored the functioning of the slowly activating anion conductance, S-type or SLAC, and of the quickly activating anion conductance, R-type or QUAC, in whole guard cells of Vicia faba L.; details of QUAC activity had not previously been demonstrated in guard cells possessing their walls. The discontinuous single-electrode voltage-clamp method was used to record current responses to voltage pulses and voltage ramps as well as the free-running membrane voltage. At all voltages tested between -200 and 60 mV, SLAC activated with two components, one had a time constant similar to 7 s, the other similar to 40 s. The current-voltage relationship resembled that obtained by patch-clamp experiments. In pulse experiments and 1-s ramps, QUAC activity appeared with half-maximum activation near -50 mV and full activation above -10 mV; it inactivated with a half-time of approximately 10 s. Inactivation of QUAC at -40 mV led to the appearance of SLAC. After deactivation of SLAC at -200 mV, QUAC could be activated again. We concluded that voltage-dependent interchanges between SLAC and QUAC had occurred. Frequently, SLAC and QUAC were active simultaneously in the same cell. Abscisic acid (ABA, 20 microM) activated SLAC as well as QUAC. External Ca2+ was not required, but enhanced the activation of QUAC. Rises in the partial pressure of CO2, in the range between 0 and 700 microbar, caused rapid and reversible increases in the activity of SLAC (and outward currents of K+). QUAC also responded to CO2, however in an unpredictable manner (either by increased or by decreased activity). Oscillations in the free-running membrane voltage arose either spontaneously or after changes in CO2. They were correlated with periodic activations and inactivations of QUAC and required the simultaneous activity of an electrogenic pump.

  12. Role of high-voltage activated potassium currents in high-frequency neuronal firing: evidence from a basal metazoan.

    PubMed

    Buckingham, Steven D; Spencer, Andrew N

    2002-08-01

    Certain neurons of vertebrates are specialized for high-frequency firing. Interestingly, high-frequency firing is also seen in central neurons in basal bilateral metazoans. Recently, the role of potassium currents with rightward-shifted activation curves in producing high-frequency firing has come under scrutiny. We apply intracellular recording, patch-clamp techniques, and compartmental modeling to examine the roles of rightward-shifted potassium currents in repetitive firing and shaping of action potentials in central neurons of the flatworm, Notoplana atomata (Phylum Platyhelminthes). The kinetic properties of potassium and sodium currents were determined from patch-clamp experiments on dissociated brain cells. To predict the effects of changing the steady-state and kinetic properties of these potassium currents, these data were incorporated into a computer model of a 30-microm spherical cell with the levels of current adjusted to approximate the values recorded in voltage-clamp experiments. The model was able to support regenerative spikes at high frequencies in response to injected current. Current-clamp recordings of cultured cells and of neurons in situ also showed evidence of very-high-frequency firing. Adjusting the ratio of inactivating to non-inactivating potassium currents had little effect upon the firing pattern of the cell or its ability to fire at high frequencies, whereas the presence of the non-inactivating current was necessary for repetitive firing. Computer simulations suggested that the rightward shift in voltage sensitivity confers a raised firing threshold, while rapid channel kinetics underlie high frequency firing, and the large activation range enhances the coding range of the cell.

  13. Ethanol inhibition of N-methyl-D-aspartate-activated current in mouse hippocampal neurones: whole-cell patch-clamp analysis

    PubMed Central

    Peoples, Robert W; White, Geoffrey; Lovinger, David M; Weight, Forrest F

    1997-01-01

    The action of ethanol on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. Ethanol inhibited NMDA-activated current in a voltage-independent manner, and did not alter the reversal potential of NMDA-activated current. Concentration–response analysis of NMDA- and glycine-activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. The polyamine spermine (1 μM) increased amplitude of NMDA-activated current but did not alter the percentage inhibition of ethanol. Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA-activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. Mg2+ (10, 100, 500 μM), Zn2+ (5, 20 μM) or ketamine (2, 10 μM) decreased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non-competitive mechanism that does not involve several modulatory sites on the NMDA receptor–ionophore complex. PMID:9401766

  14. Clamp usable as jig and lifting clamp

    DOEpatents

    Tsuyama, Yoshizo

    1976-01-01

    There is provided a clamp which is well suited for use as a lifting clamp for lifting and moving materials of assembly in a shipyard, etc. and as a pulling jig in welding and other operations. The clamp comprises a clamp body including a shackle for engagement with a pulling device and a slot for receiving an article, and a pair of jaws provided on the leg portions of the clamp body on the opposite sides of the slot to grip the article in the slot, one of said jaws consisting of a screw rod and the other jaw consisting of a swivel jaw with a spherical surface, whereby when the article clamped in the slot by the pair of jaws tends to slide in any direction with respect to the clamp body, the article is more positively gripped by the pair of jaws.

  15. Patch clamp studies on root cell vacuoles of a salt-tolerant and a salt-sensitive plantago species : regulation of channel activity by salt stress.

    PubMed

    Maathuis, F J; Prins, H B

    1990-01-01

    Plantago media L. and Plantago maritima L. differ in their strategy toward salt stress, a major difference being the uptake and distribution of ions. Patch clamp techniques were applied to root cell vacuoles to study the tonoplast channel characteristics. In both species the major channel found was a 60 to 70 picosiemens channel with a low ion selectivity. The conductance of this channel for Na(+) was the same as for K(+), P(K) (+)/P(Na) (+) = 1, whereas the cation/anion selectivity (P(K) (+)/P(c1) (-)) was about 5. Gating characteristics were voltage and calcium dependent. An additional smaller channel of 25 picosiemens was present in P. maritima. In the whole vacuole configuration, the summation of the single channel currents resulted in slowly activated inward currents (t((1/2)) = 1.2 second). Inwardly directed, ATP-dependent currents could be measured against a DeltapH gradient of 1.5 units over the tonoplast. This observation strongly indicated the physiological intactness of the used vacuoles. The open probability of the tonoplast channels dramatically decreased when plants were grown on NaCl, although single channel conductance and selectivity were not altered.

  16. Planar patch clamp for neuronal networks--considerations and future perspectives.

    PubMed

    Bosca, Alessandro; Martina, Marzia; Py, Christophe

    2014-01-01

    The patch-clamp technique is generally accepted as the gold standard for studying ion channel activity allowing investigators to either "clamp" membrane voltage and directly measure transmembrane currents through ion channels, or to passively monitor spontaneously occurring intracellular voltage oscillations. However, this resulting high information content comes at a price. The technique is labor-intensive and requires highly trained personnel and expensive equipment. This seriously limits its application as an interrogation tool for drug development. Patch-clamp chips have been developed in the last decade to overcome the tedious manipulations associated with the use of glass pipettes in conventional patch-clamp experiments. In this chapter, we describe some of the main materials and fabrication protocols that have been developed to date for the production of patch-clamp chips. We also present the concept of a patch-clamp chip array providing high resolution patch-clamp recordings from individual cells at multiple sites in a network of communicating neurons. On this chip, the neurons are aligned with the aperture-probes using chemical patterning. In the discussion we review the potential use of this technology for pharmaceutical assays, neuronal physiology and synaptic plasticity studies. PMID:25023304

  17. Voltage-activated sodium current is inhibited by capsaicin in rat atrial myocytes.

    PubMed

    Milesi, V; Rebolledo, A; Alvis, A G; Raingo, J; Grassi de Gende, A O

    2001-04-13

    The effects of capsaicin, the active principle of hot pepper genus Capsicum, were studied on voltage-activated, tetrodotoxin-sensitive Na+ currents in isolated rat atrial cells using the patch clamp technique in the whole-cell configuration. 0.4 and 4 microM of capsaicin produced a significant tonic block on voltage-activated Na+ current (I(Na)) evoked by a depolarizing step to -40 mV from a holding potential of -100 mV (49 +/- 7% n = 11, P < 0.05 and 72 +/- 13% n = 4, P < 0.05 respectively). We didn't observe any use-dependent block of capsaicin in our experimental conditions. Capsaicin slowed the time decay of inactivation of I(Na), and increased the time constant of the recovery of inactivation. Capsaicin and tetrodotoxin (TTX) depressed contractility of isolated electrically driven left rat atria, being the depression of maximal velocity of force development (dF/dt(max)) with respect to control values of 19 +/- 3% at 1 microM of capsaicin and 22 +/- 2% at 1 microM of TTX. These results show an inhibitory effect of capsaicin on I(Na) in isolated atrial cells that may modify the electrical and contractile function of the rat heart. PMID:11352646

  18. Implementing dynamic clamp with synaptic and artificial conductances in mouse retinal ganglion cells.

    PubMed

    Huang, Jin Y; Stiefel, Klaus M; Protti, Dario A

    2013-05-16

    Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp (1, 2, 3) and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

  19. Optogenetic Monitoring of Synaptic Activity with Genetically Encoded Voltage Indicators

    PubMed Central

    Nakajima, Ryuichi; Jung, Arong; Yoon, Bong-June; Baker, Bradley J.

    2016-01-01

    The age of genetically encoded voltage indicators (GEVIs) has matured to the point that changes in membrane potential can now be observed optically in vivo. Improving the signal size and speed of these voltage sensors has been the primary driving forces during this maturation process. As a result, there is a wide range of probes using different voltage detecting mechanisms and fluorescent reporters. As the use of these probes transitions from optically reporting membrane potential in single, cultured cells to imaging populations of cells in slice and/or in vivo, a new challenge emerges—optically resolving the different types of neuronal activity. While improvements in speed and signal size are still needed, optimizing the voltage range and the subcellular expression (i.e., soma only) of the probe are becoming more important. In this review, we will examine the ability of recently developed probes to report synaptic activity in slice and in vivo. The voltage-sensing fluorescent protein (VSFP) family of voltage sensors, ArcLight, ASAP-1, and the rhodopsin family of probes are all good at reporting changes in membrane potential, but all have difficulty distinguishing subthreshold depolarizations from action potentials and detecting neuronal inhibition when imaging populations of cells. Finally, we will offer a few possible ways to improve the optical resolution of the various types of neuronal activities. PMID:27547183

  20. Low voltage electrical activity preceding right atrial depolarisation in man.

    PubMed Central

    Mackintosh, A F; English, M J; Vincent, R; Woollons, D J; Chamberlain, D A

    1979-01-01

    Electrical recordings were made in the high right atrium in 28 patients undergoing cardiac catheterisation and in 3 healthy volunteers. After filtering and amplification by 3 to 10 million times, the signals were passed through a signal averaging process in a digital computer. Of the 28 subjects who had technically satisfactory recordings, 23 showed low voltage electrical activity preceding the conventionally-recorded atrial depolarisation. The low voltage activity started 50 to 200 ms before the atrial deflection and was variable in shape. These early signals may be the result of activity in the region of the sinus node. PMID:486271

  1. 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

  2. Voltage-activated K+ conductances in freshly isolated embryonic chicken osteoclasts.

    PubMed

    Ravesloot, J H; Ypey, D L; Vrijheid-Lammers, T; Nijweide, P J

    1989-09-01

    Patch-clamp measurements on freshly isolated embryonic chicken osteoclasts revealed three distinct types of voltage-dependent K+ conductance. The first type of conductance, present in 72% of the cells, activated at membrane potentials less negative than -30 to -20 mV and reached full activation at +40 mV. It activated with a delay, reached a peak value, and then inactivated with a time constant of approximately 1.5 s. Inactivation was complete or almost so. Recovery from inactivation, at -70 mV, had a time constant of roughly 1 s. The conductance could be blocked, at least partly, by 4 mM 4-aminopyridine. The second type of conductance (present in all cells) activated at membrane potentials more negative than -40 to -80 mV and reached full activation at -130 mV. Activation potential and maximal conductance were dependent on the extracellular K+ concentration. Inactivation of the conductance first became apparent at membrane potentials more negative than -100 mV and was a two-exponential process. The conductance could be blocked by external 5 mM Cs+ ions. The third type of conductance (present in all cells) activated at membrane potentials more positive than +30 mV. Generally, the conductance did not inactivate.

  3. Force-Measuring Clamps

    NASA Technical Reports Server (NTRS)

    Nunnelee, Mark

    2003-01-01

    Force-measuring clamps have been invented to facilitate and simplify the task of measuring the forces or pressures applied to clamped parts. There is a critical need to measure clamping forces or pressures in some applications for example, while bonding sensors to substrates or while clamping any sensitive or delicate parts. Many manufacturers of adhesives and sensors recommend clamping at specific pressures while bonding sensors or during adhesive bonding between parts in general. In the absence of a force-measuring clamp, measurement of clamping force can be cumbersome at best because of the need for additional load sensors and load-indicating equipment. One prior method of measuring clamping force involved the use of load washers or miniature load cells in combination with external power sources and load-indicating equipment. Calibrated spring clamps have also been used. Load washers and miniature load cells constitute additional clamped parts in load paths and can add to the destabilizing effects of loading mechanisms. Spring clamps can lose calibration quickly through weakening of the springs and are limited to the maximum forces that the springs can apply. The basic principle of a force-measuring clamp can be implemented on a clamp of almost any size and can enable measurement of a force of almost any magnitude. No external equipment is needed because the component(s) for transducing the clamping force and the circuitry for supplying power, conditioning the output of the transducers, and displaying the measurement value are all housed on the clamp. In other words, a force-measuring clamp is a complete force-application and force-measurement system all in one package. The advantage of unitary packaging of such a system is that it becomes possible to apply the desired clamping force or pressure with precision and ease.

  4. Voltage dependence of the Ca(2+)-activated K(+) channel K(Ca)3.1 in human erythroleukemia cells.

    PubMed

    Stoneking, Colin J; Shivakumar, Oshini; Thomas, David Nicholson; Colledge, William H; Mason, Michael J

    2013-05-01

    We have isolated a K(+)-selective, Ca(2+)-dependent whole cell current and single-channel correlate in the human erythroleukemia (HEL) cell line. The whole cell current was inhibited by the intermediate-conductance KCa3.1 inhibitors clotrimazole, TRAM-34, and charybdotoxin, unaffected by the small-conductance KCa2 family inhibitor apamin and the large-conductance KCa1.1 inhibitors paxilline and iberiotoxin, and augmented by NS309. The single-channel correlate of the whole cell current was blocked by TRAM-34 and clotrimazole, insensitive to paxilline, and augmented by NS309 and had a single-channel conductance in physiological K(+) gradients of ~9 pS. RT-PCR revealed that the KCa3.1 gene, but not the KCa1.1 gene, was expressed in HEL cells. The KCa3.1 current, isolated in HEL cells under whole cell patch-clamp conditions, displayed an activated current component during depolarizing voltage steps from hyperpolarized holding potentials and tail currents upon repolarization, consistent with voltage-dependent modulation. This activated current increased with increasing voltage steps above -40 mV and was sensitive to inhibition by clotrimazole, TRAM-34, and charybdotoxin and insensitive to apamin, paxilline, and iberiotoxin. In single-channel experiments, depolarization resulted in an increase in open channel probability (Po) of KCa3.1, with no increase in channel number. The voltage modulation of Po was an increasing monotonic function of voltage. In the absence of elevated Ca(2+), voltage was ineffective at inducing channel activity in whole cell and single-channel experiments. These data indicate that KCa3.1 in HEL cells displays a unique form of voltage dependence modulating Po.

  5. Voltage dependence of the Ca(2+)-activated K(+) channel K(Ca)3.1 in human erythroleukemia cells.

    PubMed

    Stoneking, Colin J; Shivakumar, Oshini; Thomas, David Nicholson; Colledge, William H; Mason, Michael J

    2013-05-01

    We have isolated a K(+)-selective, Ca(2+)-dependent whole cell current and single-channel correlate in the human erythroleukemia (HEL) cell line. The whole cell current was inhibited by the intermediate-conductance KCa3.1 inhibitors clotrimazole, TRAM-34, and charybdotoxin, unaffected by the small-conductance KCa2 family inhibitor apamin and the large-conductance KCa1.1 inhibitors paxilline and iberiotoxin, and augmented by NS309. The single-channel correlate of the whole cell current was blocked by TRAM-34 and clotrimazole, insensitive to paxilline, and augmented by NS309 and had a single-channel conductance in physiological K(+) gradients of ~9 pS. RT-PCR revealed that the KCa3.1 gene, but not the KCa1.1 gene, was expressed in HEL cells. The KCa3.1 current, isolated in HEL cells under whole cell patch-clamp conditions, displayed an activated current component during depolarizing voltage steps from hyperpolarized holding potentials and tail currents upon repolarization, consistent with voltage-dependent modulation. This activated current increased with increasing voltage steps above -40 mV and was sensitive to inhibition by clotrimazole, TRAM-34, and charybdotoxin and insensitive to apamin, paxilline, and iberiotoxin. In single-channel experiments, depolarization resulted in an increase in open channel probability (Po) of KCa3.1, with no increase in channel number. The voltage modulation of Po was an increasing monotonic function of voltage. In the absence of elevated Ca(2+), voltage was ineffective at inducing channel activity in whole cell and single-channel experiments. These data indicate that KCa3.1 in HEL cells displays a unique form of voltage dependence modulating Po. PMID:23407879

  6. Making an Adjustable C-Clamp. Kit No. 603. Instructor's Manual [and] Student Learning Activity Manual. [Revised.] T & I--Metalwork.

    ERIC Educational Resources Information Center

    White, Jim; Alexander, Larry

    This student activity kit consists of a programmed, self-instructional learning guide and an accompanying instructor's manual for use in teaching trade and industrial education students how to make an adjustable C-clamp. The student guide contains step-by-step instructions in the following areas: basic layout principles; use of a hack saw, file,…

  7. Force-Measuring Clamp

    NASA Technical Reports Server (NTRS)

    Nunnelee, Mark (Inventor)

    2004-01-01

    A precision clamp that accurately measures force over a wide range of conditions is described. Using a full bridge or other strain gage configuration. the elastic deformation of the clamp is measured or detected by the strain gages. Thc strain gages transmit a signal that corresponds to the degree of stress upon the clamp. Thc strain gage signal is converted to a numeric display. Calibration is achieved by ero and span potentiometers which enable accurate measurements by the force-measuring clamp.

  8. 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.

  9. 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. PMID:27018144

  10. Quick-attach clamp

    NASA Technical Reports Server (NTRS)

    Vano, A. E.

    1968-01-01

    Clamp of the slideable jaw type can be applied to moving lines such as cables or ropes. The clamp has a trigger-operated jaw that can be attached to a redrop parachute on a moving tow cable. The trigger mechanism maintains the jaws retracted in the housing until they are released for clamping.

  11. Preliminary characterization of voltage-activated whole-cell currents in developing human vestibular hair cells and calyx afferent terminals.

    PubMed

    Lim, Rebecca; Drury, Hannah R; Camp, Aaron J; Tadros, Melissa A; Callister, Robert J; Brichta, Alan M

    2014-10-01

    We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11-14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15-18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a "collapsing" tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15-18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.

  12. Photovoltaic panel clamp

    SciTech Connect

    Brown, Malcolm P.; Mittan, Margaret Birmingham; Miros, Robert H. J.; Stancel, Robert

    2013-03-19

    A photovoltaic panel clamp includes an upper and lower section. The interface between the assembled clamp halves and the module edge is filled by a flexible gasket material, such as EPDM rubber. The gasket preferably has small, finger like protrusions that allow for easy insertion onto the module edge while being reversed makes it more difficult to remove them from the module once installed. The clamp includes mounting posts or an integral axle to engage a bracket. The clamp also may include a locking tongue to secure the clamp to a bracket.

  13. Photovoltaic panel clamp

    DOEpatents

    Mittan, Margaret Birmingham; Miros, Robert H. J.; Brown, Malcolm P.; Stancel, Robert

    2012-06-05

    A photovoltaic panel clamp includes an upper and lower section. The interface between the assembled clamp halves and the module edge is filled by a flexible gasket material, such as EPDM rubber. The gasket preferably has small, finger like protrusions that allow for easy insertion onto the module edge while being reversed makes it more difficult to remove them from the module once installed. The clamp includes mounting posts or an integral axle to engage a bracket. The clamp also may include a locking tongue to secure the clamp to a bracket.

  14. Compact, Stiff, Remotely-Actuable Quick-Release Clamp

    NASA Technical Reports Server (NTRS)

    Tsai, Ted W. (Inventor)

    2000-01-01

    The present invention provides a clamp that is compact and lightweight, yet provides high holding strength and stiffness or rigidity. The clamp uses a unique double slant interface design which provides mechanical advantages to resist forces applied to the clamp member as the load increases. The clamp allows for rapid and remote-activated release of the clamp jaws by applying only a small operating force to an over-center lock/release mechanism, such as by pulling a manual tether.

  15. Voltage-activated currents in somatic muscle of the nematode parasite Ascaris suum.

    PubMed

    Martin, R J; Thorn, P; Gration, K A; Harrow, I D

    1992-12-01

    1. Voltage-activated currents in cell bodies of the somatic muscle cells of Ascaris suum were studied using a two-microelectrode voltage-clamp technique. Cells recorded from had resting membrane potentials around -35 mV and had input conductances in the range 1-10 microS. 2. In cells bathed in artificial perienteric fluid, depolarizing steps from a holding potential of -35 mV elicited outward currents at a threshold of -15 mV. These currents had inwardly directed inflections on the rising phase, suggesting the presence of more than one current. Hyperpolarizing steps did not activate current. 3. Tetraethylammonium (TEA+, 69 mmol l-1) blocked the outward currents and allowed a voltage-dependent inactivating Ca2+ current to be observed. The peak current-voltage relationship was U-shaped with a threshold around -15 mV and peak at +5 mV. The reversal potential of the Ca2+ current was estimated by extrapolation to be +45 mV. 4. The permeability of the voltage-activated outward currents was studied by examining reversal potentials of tail currents. The reversal potentials were linearly dependent on the logarithm of the extracellular potassium concentration if extracellular [K+] was greater than 10 mmol l-1. The Na+/K+ permeability ratio of the currents was 0.04. 5. Inactivation, seen as a decline following the peak of the K+ current, was produced by maintained depolarization. The recovery from inactivation was complex and could be described by the sum of two exponentials with time constants of 0.67 s and 20.1 s. Steady-state inactivation of the K+ currents was observed at a range of holding potentials. Only a proportion (34%) of the total K+ current was inactivated by holding potentials more positive than -20 mV. 6. Extracellular application of 5 mmol l-1 4-aminopyridine (4-AP) selectively abolished an early fast component of the K+ current (the peak). The 4-AP-sensitive current decayed quickly with a time constant of around 10 ms; a Boltzmann fit to its activation curve

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

    PubMed

    Hristov, Kiril L; Smith, Amy C; Parajuli, Shankar P; Malysz, John; Petkov, Georgi V

    2014-03-01

    Large-conductance voltage- and Ca(2+)-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 Ca(2+) 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 Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) 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 Ca(2+)-dependent mechanism, thus increasing DSM contractility.

  17. Expression of voltage-activated chloride currents in acute slices of human gliomas.

    PubMed

    Ullrich, N; Bordey, A; Gillespie, G Y; Sontheimer, H

    1998-04-01

    Using whole-cell patch-clamp recordings, we identified a novel voltage-activated chloride current that was selectively expressed in glioma cells from 23 patient biopsies. Chloride currents were identified in 64% of glioma cells studied in acute slices of nine patient biopsies. These derived from gliomas of various pathological grades. In addition, 98% of cells acutely isolated or in short-term culture from 23 patients diagnosed with gliomas showed chloride current expression. These currents, which we termed glioma chloride currents activated at potentials >45 mV, showed pronounced outward rectification, and were sensitive to bath application of the presumed Cl- channel specific peptide chlorotoxin (approximately 600 nM) derived from Leiurus scorpion venom. Interestingly, low grade tumours (e.g., pilocytic astrocytomas), containing more differentiated, astrocyte-like cells showed expression of glioma chloride currents in concert with voltage-activated sodium and potassium currents also seen in normal astrocytes. By contrast, high grade tumours (e.g., glioblastoma multiforme) expressed almost exclusively chloride currents, suggesting a gradual loss of Na+ currents and gain of Cl- currents with increasing pathological tumour grade. To expand on the observation that these chloride currents are glioma-specific, we introduced experimental tumours in scid mice by intracranial injection of D54MG glioma cells and subsequently recorded from tumour cells and adjacent normal glial cells in acute slices. We consistently observed expression of chlorotoxin-sensitive chloride channels in implanted glioma cells, but without evidence for expression of chloride channels in surrounding "normal" host glial cells, suggesting that these chloride channels are probably a glioma-specific feature. Finding of this novel glioma specific Cl- channel in gliomas in situ and it's selective binding of chlorotoxin may provide a way to identify or target glioma cells in the future.

  18. KCNE3 acts by promoting voltage sensor activation in KCNQ1

    PubMed Central

    Barro-Soria, Rene; Perez, Marta E.; Larsson, H. Peter

    2015-01-01

    KCNE β-subunits assemble with and modulate the properties of voltage-gated K+ channels. In the colon, stomach, and kidney, KCNE3 coassembles with the α-subunit KCNQ1 to form K+ channels important for K+ and Cl− secretion that appear to be voltage-independent. How KCNE3 subunits turn voltage-gated KCNQ1 channels into apparent voltage-independent KCNQ1/KCNE3 channels is not completely understood. Different mechanisms have been proposed to explain the effect of KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometry to determine how KCNE3 affects the voltage sensor S4 and the gate of KCNQ1. We find that S4 moves in KCNQ1/KCNE3 channels, and that inward S4 movement closes the channel gate. However, KCNE3 shifts the voltage dependence of S4 movement to extreme hyperpolarized potentials, such that in the physiological voltage range, the channel is constitutively conducting. By separating S4 movement and gate opening, either by a mutation or PIP2 depletion, we show that KCNE3 directly affects the S4 movement in KCNQ1. Two negatively charged residues of KCNE3 (D54 and D55) are found essential for the effect of KCNE3 on KCNQ1 channels, mainly exerting their effects by an electrostatic interaction with R228 in S4. Our results suggest that KCNE3 primarily affects the voltage-sensing domain and only indirectly affects the gate. PMID:26668384

  19. Insulated pipe clamp design

    SciTech Connect

    Anderson, M.J.; Hyde, L.L.; Wagner, S.E.; Severud, L.K.

    1980-01-01

    Thin wall large diameter piping for breeder reactor plants can be subjected to significant thermal shocks during reactor scrams and other upset events. On the Fast Flux Test Facility, the addition of thick clamps directly on the piping was undesired because the differential metal temperatures between the pipe wall and the clamp could have significantly reduced the pipe thermal fatigue life cycle capabilities. Accordingly, an insulated pipe clamp design concept was developed. The design considerations and methods along with the development tests are presented. Special considerations to guard against adverse cracking of the insulation material, to maintain the clamp-pipe stiffness desired during a seismic event, to minimize clamp restraint on the pipe during normal pipe heatup, and to resist clamp rotation or spinning on the pipe are emphasized.

  20. Cumulative Activation of Voltage-Dependent KVS-1 Potassium Channels

    PubMed Central

    Rojas, Patricio; Garst-Orozco, Jonathan; Baban, Beravan; de Santiago-Castillo, Jose Antonio; Covarrubias, Manuel; Salkoff, Lawrence

    2008-01-01

    In this study, we reveal the existence of a novel use-dependent phenomenon in potassium channels, which we refer to as cumulative activation (CA). CA consists of an increase in current amplitude in response to repetitive depolarizing step pulses to the same potential. CA persists for up to 20 s and is similar to a phenomenon called “voltage-dependent facilitation” observed in some calcium channels. The KVS-1 K+ channel, which exhibits CA, is a rapidly activating and inactivating voltage-dependent potassium channel expressed in chemosensory and other neurons of Caenorhabditis elegans. It is unusual in being most closely related to the Shab (Kv2) family of potassium channels, which typically behave like delayed rectifier K+ channels in other species. The magnitude of CA depends on the frequency, voltage, and duration of the depolarizing step pulse. CA also radically changes the activation and inactivation kinetics of the channel, suggesting that the channel may undergo a physical modification in a use-dependent manner; thus, a model that closely simulates the behavior of the channel postulates the existence of two populations of channels, unmodified and modified. Use-dependent changes in the behavior of potassium channels, such as CA observed in KVS-1, could be involved in functional mechanisms of cellular plasticity such as synaptic depression that represent the cellular basis of learning and memory. PMID:18199775

  1. Radial wedge flange clamp

    DOEpatents

    Smith, Karl H.

    2002-01-01

    A radial wedge flange clamp comprising a pair of flanges each comprising a plurality of peripheral flat wedge facets having flat wedge surfaces and opposed and mating flat surfaces attached to or otherwise engaged with two elements to be joined and including a series of generally U-shaped wedge clamps each having flat wedge interior surfaces and engaging one pair of said peripheral flat wedge facets. Each of said generally U-shaped wedge clamps has in its opposing extremities apertures for the tangential insertion of bolts to apply uniform radial force to said wedge clamps when assembled about said wedge segments.

  2. Monitoring Brain Activity with Protein Voltage and Calcium Sensors

    PubMed Central

    Storace, Douglas A.; Braubach, Oliver R.; Jin, Lei; Cohen, Lawrence B.; Sung, Uhna

    2015-01-01

    Understanding the roles of different cell types in the behaviors generated by neural circuits requires protein indicators that report neural activity with high spatio-temporal resolution. Genetically encoded fluorescent protein (FP) voltage sensors, which optically report the electrical activity in distinct cell populations, are, in principle, ideal candidates. Here we demonstrate that the FP voltage sensor ArcLight reports odor-evoked electrical activity in the in vivo mammalian olfactory bulb in single trials using both wide-field and 2-photon imaging. ArcLight resolved fast odorant-responses in individual glomeruli, and distributed odorant responses across a population of glomeruli. Comparisons between ArcLight and the protein calcium sensors GCaMP3 and GCaMP6f revealed that ArcLight had faster temporal kinetics that more clearly distinguished activity elicited by individual odorant inspirations. In contrast, the signals from both GCaMPs were a saturating integral of activity that returned relatively slowly to the baseline. ArcLight enables optical electrophysiology of mammalian neuronal population activity in vivo. PMID:25970202

  3. Monitoring brain activity with protein voltage and calcium sensors.

    PubMed

    Storace, Douglas A; Braubach, Oliver R; Jin, Lei; Cohen, Lawrence B; Sung, Uhna

    2015-05-13

    Understanding the roles of different cell types in the behaviors generated by neural circuits requires protein indicators that report neural activity with high spatio-temporal resolution. Genetically encoded fluorescent protein (FP) voltage sensors, which optically report the electrical activity in distinct cell populations, are, in principle, ideal candidates. Here we demonstrate that the FP voltage sensor ArcLight reports odor-evoked electrical activity in the in vivo mammalian olfactory bulb in single trials using both wide-field and 2-photon imaging. ArcLight resolved fast odorant-responses in individual glomeruli, and distributed odorant responses across a population of glomeruli. Comparisons between ArcLight and the protein calcium sensors GCaMP3 and GCaMP6f revealed that ArcLight had faster temporal kinetics that more clearly distinguished activity elicited by individual odorant inspirations. In contrast, the signals from both GCaMPs were a saturating integral of activity that returned relatively slowly to the baseline. ArcLight enables optical electrophysiology of mammalian neuronal population activity in vivo.

  4. Ligand and coactivator identity determines the requirement of the charge clamp for coactivation of the peroxisome proliferator-activated receptor gamma.

    PubMed

    Wu, Yifei; Chin, William W; Wang, Yong; Burris, Thomas P

    2003-03-01

    The activation function 2 (AF-2)-dependent recruitment of coactivator is essential for gene activation by nuclear receptors. We show that the peroxisome proliferator-activated receptor gamma (PPARgamma) (NR1C3) coactivator-1 (PGC-1) requires both the intact AF-2 domain of PPARgamma and the LXXLL domain of PGC-1 for ligand-dependent and ligand-independent interaction and coactivation. Although the AF-2 domain of PPARgamma is absolutely required for PGC-1-mediated coactivation, this coactivator displayed a unique lack of requirement for the charge clamp of the ligand-binding domain of the receptor that is thought to be essential for LXXLL motif recognition. The mutation of a single serine residue adjacent to the core LXXLL motif of PGC-1 led to restoration of the typical charge clamp requirement. Thus, the unique structural features of the PGC-1 LXXLL motif appear to mediate an atypical mode of interaction with PPARgamma. Unexpectedly, we discovered that various ligands display variability in terms of their requirement for the charge clamp of PPARgamma for coactivation by PGC-1. This ligand-selective variable requirement for the charge clamp was coactivator-specific. Thus, distinct structural determinants, which may be unique for a particular ligand, are utilized by the receptor to recognize the coactivator. Our data suggest that even subtle differences in ligand structure are perceived by the receptor and translated into a unique display of the coactivator-binding surface of the ligand-binding domain, allowing for differential recognition of coactivators that may underlie distinct pharmacological profiles observed for ligands of a particular nuclear receptor.

  5. MATLAB implementation of a dynamic clamp with bandwidth of >125 kHz capable of generating I Na at 37 °C.

    PubMed

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

    2013-04-01

    We describe the construction of a dynamic clamp with a bandwidth of >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 and 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 (I Na) 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: I Na, the hyperpolarizing-activated inward pacemaker current (I f) and an inward-rectifier K(+) current (I K1). 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.

  6. Reusable thermal cycling clamp

    NASA Technical Reports Server (NTRS)

    Debnam, W. J., Jr.; Fripp, A. L.; Crouch, R. K. (Inventor)

    1985-01-01

    A reusable metal clamp for retaining a fused quartz ampoule during temperature cycling in the range of 20 deg C to 1000 deg C is described. A compressible graphite foil having a high radial coefficient of thermal expansion is interposed between the fused quartz ampoule and metal clamp to maintain a snug fit between these components at all temperature levels in the cycle.

  7. Mechanisms of Activation of Voltage-Gated Potassium Channels

    PubMed Central

    Grizel, A. V.; Glukhov, G. S.; Sokolova, O. S.

    2014-01-01

    Voltage-gated potassium ion channels (Kv) play an important role in a variety of cellular processes, including the functioning of excitable cells, regulation of apoptosis, cell growth and differentiation, the release of neurotransmitters and hormones, maintenance of cardiac activity, etc. Failure in the functioning of Kv channels leads to severe genetic disorders and the development of tumors, including malignant ones. Understanding the mechanisms underlying Kv channels functioning is a key factor in determining the cause of the diseases associated with mutations in the channels, and in the search for new drugs. The mechanism of activation of the channels is a topic of ongoing debate, and a consensus on the issue has not yet been reached. This review discusses the key stages in studying the mechanisms of functioning of Kv channels and describes the basic models of their activation known to date. PMID:25558391

  8. Improved Active Harmonic Current Elimination Based on Voltage Detection

    PubMed Central

    Tan, Tianyuan; Dong, Shuan; Huang, Yingwei; Liu, Jian; Le, Jian; Liu, Kaipei

    2016-01-01

    With the increasing penetration of power electronic equipment in modern residential distribution systems, harmonics mitigation through the distributed generation (DG) interfacing converters has received significant attention. Among recently proposed methods, the so-called active resonance damper (ARD) and harmonic voltage compensator (HVC) based on voltage detection can effectively reduce the harmonic distortions in selected areas of distribution systems. However, it is found out that when traditional ARD algorithm is used to eliminate harmonic current injected by non-linear loads, its performance is constrained by stability problems and can at most eliminate half of the load harmonic currents. Thus, inspired by the duality between ARD and HVC, this paper presents a novel improved resistive active power filter (R-APF) algorithm based on integral-decoupling control. The design guideline for its parameters is then investigated through carefully analyzing the closed-loop poles’ trajectory. Computer studies demonstrate that the proposed algorithm can effectively mitigate the load harmonic currents and its performance is much better than traditional ARD based on proportional control. PMID:27295213

  9. Improved Active Harmonic Current Elimination Based on Voltage Detection.

    PubMed

    Tan, Tianyuan; Dong, Shuan; Huang, Yingwei; Liu, Jian; Le, Jian; Liu, Kaipei

    2016-01-01

    With the increasing penetration of power electronic equipment in modern residential distribution systems, harmonics mitigation through the distributed generation (DG) interfacing converters has received significant attention. Among recently proposed methods, the so-called active resonance damper (ARD) and harmonic voltage compensator (HVC) based on voltage detection can effectively reduce the harmonic distortions in selected areas of distribution systems. However, it is found out that when traditional ARD algorithm is used to eliminate harmonic current injected by non-linear loads, its performance is constrained by stability problems and can at most eliminate half of the load harmonic currents. Thus, inspired by the duality between ARD and HVC, this paper presents a novel improved resistive active power filter (R-APF) algorithm based on integral-decoupling control. The design guideline for its parameters is then investigated through carefully analyzing the closed-loop poles' trajectory. Computer studies demonstrate that the proposed algorithm can effectively mitigate the load harmonic currents and its performance is much better than traditional ARD based on proportional control. PMID:27295213

  10. Estimating the voltage-dependent free energy change of ion channels using the median voltage for activation.

    PubMed

    Chowdhury, Sandipan; Chanda, Baron

    2012-01-01

    Voltage-gated ion channels are crucial for electrical activity and chemical signaling in a variety of cell types. Structure-activity studies involving electrophysiological characterization of mutants are widely used and allow us to quickly realize the energetic effects of a mutation by measuring macroscopic currents and fitting the observed voltage dependence of conductance to a Boltzmann equation. However, such an approach is somewhat limiting, principally because of the inherent assumption that the channel activation is a two-state process. In this analysis, we show that the area delineated by the gating charge displacement curve and its ordinate axis is related to the free energy of activation of a voltage-gated ion channel. We derive a parameter, the median voltage of charge transfer (V(m)), which is proportional to this area, and prove that the chemical component of free energy change of a system can be obtained from the knowledge of V(m) and the maximum number of charges transferred. Our method is not constrained by the number or connectivity of intermediate states and is applicable to instances in which the observed responses show a multiphasic behavior. We consider various models of ion channel gating with voltage-dependent steps, latent charge movement, inactivation, etc. and discuss the applicability of this approach in each case. Notably, our method estimates a net free energy change of approximately -14 kcal/mol associated with the full-scale activation of the Shaker potassium channel, in contrast to -2 to -3 kcal/mol estimated from a single Boltzmann fit. Our estimate of the net free energy change in the system is consistent with those derived from detailed kinetic models (Zagotta et al. 1994. J. Gen. Physiol. doi:10.1085/jgp.103.2.321). The median voltage method can reliably quantify the magnitude of free energy change associated with activation of a voltage-dependent system from macroscopic equilibrium measurements. This will be particularly useful

  11. Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence.

    PubMed

    Keum, Dongil; Kruse, Martin; Kim, Dong-Il; Hille, Bertil; Suh, Byung-Chang

    2016-06-28

    Voltage-sensing phosphatases (VSPs) are homologs of phosphatase and tensin homolog (PTEN), a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] 3-phosphatase. However, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P2 and probably PI(3,4,5)P3 as well as 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 in response to membrane depolarization. Recent proposals say these reactions have differing voltage dependence. Using Förster resonance energy transfer probes specific for different PIs in living cells with zebrafish VSP, we quantitate both voltage-dependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3-phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather, the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling revealed that 3-phosphatase activity against PI(3,4,5)P3 is 55-fold slower than 5-phosphatase activity against PI(4,5)P2; thus, PI(4,5)P2 generated more slowly from dephosphorylating PI(3,4,5)P3 might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4-phosphate 5-kinase, there was accumulation of PI(4,5)P2 in parallel to PI(3,4,5)P3 dephosphorylation

  12. Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli.

    PubMed

    Reusch, R N; Huang, R; Bramble, L L

    1995-09-01

    The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi). The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels. Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E. coli. Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine. The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order. The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi. This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E. coli. This is the first report of a biological non-proteinaceous calcium channel. We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels. PMID:8519976

  13. Laser beam guard clamps

    DOEpatents

    Dickson, Richard K.

    2010-09-07

    A quick insert and release laser beam guard panel clamping apparatus having a base plate mountable on an optical table, a first jaw affixed to the base plate, and a spring-loaded second jaw slidably carried by the base plate to exert a clamping force. The first and second jaws each having a face acutely angled relative to the other face to form a V-shaped, open channel mouth, which enables wedge-action jaw separation by and subsequent clamping of a laser beam guard panel inserted through the open channel mouth. Preferably, the clamping apparatus also includes a support structure having an open slot aperture which is positioned over and parallel with the open channel mouth.

  14. A monogenean without clamps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ectoparasites face a daily challenge: to remain attached to their host. Polyopisthocotylean monogeneans attach to the surface of fish gills by highly specialized structures, the sclerotized clamps. In the original description of the protomicrocotylid species Lethacotyle fijiensis, described 50 years...

  15. The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient

    PubMed Central

    1995-01-01

    Voltage-activated H+ currents were studied in rat alveolar epithelial cells using tight-seal whole-cell voltage clamp recording and highly buffered, EGTA-containing solutions. Under these conditions, the tail current reversal potential, Vrev, was close to the Nernst potential, EH, varying 52 mV/U pH over four delta pH units (delta pH = pHo - pHi). This result indicates that H+ channels are extremely selective, PH/PTMA > 10(7), and that both internal and external pH, pHi, and pHo, were well controlled. The H+ current amplitude was practically constant at any fixed delta pH, in spite of up to 100-fold symmetrical changes in H+ concentration. Thus, the rate-limiting step in H+ permeation is pH independent, must be localized to the channel (entry, permeation, or exit), and is not bulk diffusion limitation. The instantaneous current- voltage relationship exhibited distinct outward rectification at symmetrical pH, suggesting asymmetry in the permeation pathway. Sigmoid activation kinetics and biexponential decay of tail currents near threshold potentials indicate that H+ channels pass through at least two closed states before opening. The steady state H+ conductance, gH, as well as activation and deactivation kinetic parameters were all shifted along the voltage axis by approximately 40 mV/U pH by changes in pHi or pHo, with the exception of the fast component of tail currents which was shifted less if at all. The threshold potential at which H+ currents were detectably activated can be described empirically as approximately 20-40(pHo-pHi) mV. If internal and external protons regulate the voltage dependence of gH gating at separate sites, then they must be equally effective. A simpler interpretation is that gating is controlled by the pH gradient, delta pH. We propose a simple general model to account for the observed delta pH dependence. Protonation at an externally accessible site stabilizes the closed channel conformation. Deprotonation of this site permits a

  16. Proton currents constrain structural models of voltage sensor activation

    PubMed Central

    Randolph, Aaron L; Mokrab, Younes; Bennett, Ashley L; Sansom, Mark SP; Ramsey, Ian Scott

    2016-01-01

    The Hv1 proton channel is evidently unique among voltage sensor domain proteins in mediating an intrinsic ‘aqueous’ H+ conductance (GAQ). Mutation of a highly conserved ‘gating charge’ residue in the S4 helix (R1H) confers a resting-state H+ ‘shuttle’ conductance (GSH) in VGCs and Ci VSP, and we now report that R1H is sufficient to reconstitute GSH in Hv1 without abrogating GAQ. Second-site mutations in S3 (D185A/H) and S4 (N4R) experimentally separate GSH and GAQ gating, which report thermodynamically distinct initial and final steps, respectively, in the Hv1 activation pathway. The effects of Hv1 mutations on GSH and GAQ are used to constrain the positions of key side chains in resting- and activated-state VS model structures, providing new insights into the structural basis of VS activation and H+ transfer mechanisms in Hv1. DOI: http://dx.doi.org/10.7554/eLife.18017.001 PMID:27572256

  17. Perisomatic voltage-gated sodium channels actively maintain linear synaptic integration in principal neurons of the medial superior olive

    PubMed Central

    Scott, Luisa L.; Mathews, Paul J.; Golding, Nace L.

    2010-01-01

    Principal neurons of the medial superior olive (MSO) compute azimuthal sound location by integrating phase-locked inputs from each ear. While previous experimental and modeling studies have proposed that voltage-gated sodium channels (VGSCs) play an important role in synaptic integration in the MSO, these studies appear at odds with the unusually weak active backpropagation of action potentials into the soma and dendrites. To understand the spatial localization and biophysical properties of VGSCs, we isolated sodium currents in MSO principal neurons in gerbil brainstem slices. Nucleated and cell-attached patches revealed that VGSC density at the soma is comparable to that of many other neuron types, but channel expression is largely absent from the dendrites. Further, while somatic VGSCs activated with conventional voltage dependence (V1/2 = −30 mV), they exhibited an unusually negative range of steady-state inactivation (V1/2 = − 77 mV), leaving ~92% of VGSCs inactivated at the resting potential (~ −58 mV). In current-clamp experiments, non-inactivated VGSCs were sufficient to amplify subthreshold EPSPs near action potential threshold, counterbalancing the suppression of EPSP peaks by low voltage-activated potassium channels. EPSP amplification was restricted to the perisomatic region of the neuron, and relatively insensitive to preceding inhibition. Finally, computational modeling showed that the exclusion of VGSCs from the dendrites equalizes somatic EPSP amplification across synaptic locations and lowered the threshold for bilateral vs. unilateral excitatory synaptic inputs. Taken together, these findings suggest that the pattern of sodium channel expression in MSO neurons contributes to these neurons’ selectivity for coincident binaural inputs. PMID:20147532

  18. Piezoresistive cantilever force-clamp system

    NASA Astrophysics Data System (ADS)

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

    2011-04-01

    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.

  19. Piezoresistive cantilever force-clamp system

    SciTech Connect

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

    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 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 {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.

  20. The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity

    PubMed Central

    Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Yang, Weiqiang; Wang, Shihua; Wei, Wenjing; Zhou, Jie; Zhu, Guofeng; Deng, Jiaoyu; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun

    2016-01-01

    DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an αβ2ε polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its α subunit, Mtb DNA pol III has two potential proofreading subunits; the α and ε subunits. During DNA replication, the presence of the β2 clamp strongly promotes the polymerization of the αβ2ε replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb. PMID:26822057

  1. Clamping characteristics study on different types of clamping unit

    SciTech Connect

    Jiao, Zhiwei; Liu, Haichao; Xie, Pengcheng; Yang, Weimin

    2015-05-22

    Plastic products are becoming more and more widely used in aerospace, IT, digital electronics and many other fields. With the development of technology, the requirement of product precision is getting higher and higher. However, type and working performance of clamping unit play a decisive role in product precision. Clamping characteristics of different types of clamping unit are discussed in this article, which use finite element numerical analysis method through the software ABAQUS to study the clamping uniformity, and detect the clamping force repeatability precision. The result shows that compared with toggled three-platen clamping unit, clamping characteristics of internal circulation two-platen clamping unit are better, for instance, its mold cavity deformation and force that bars and mold parting surface suffered are more uniform, and its clamping uniformity and repeatability precision is also better.

  2. Activation of protein kinase C and inositol 1,4,5-triphosphate receptors antagonistically modulate voltage-gated sodium channels in striatal neurons.

    PubMed

    Hourez, Raphaël; Azdad, Karima; Vanwalleghem, Gilles; Roussel, Céline; Gall, David; Schiffmann, Serge N

    2005-10-19

    Regulation of voltage-gated sodium channels is crucial to firing patterns that constitute the output of medium spiny neurons (MSN), projecting neurons of the striatum. This modulation is thus critical for the final integration of information processed within the striatum. It has been shown that the adenylate cyclase pathway reduces sodium currents in MSN through channel phosphorylation by cAMP-dependent protein kinase. However, it is unknown whether a phospholipase C (PLC)-mediated signaling cascade could also modulate voltage-gated sodium channels within MSN. Using the whole-cell patch clamp technique, we investigated the effects of activation of two key components in PLC-mediated signaling cascades: protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP(3)) receptors on voltage-dependent sodium current. Cellular dialysis with phorbol 12-myristate 13-acetate, an activator of PKC, significantly reduced peak sodium current amplitude, while adenophostin A, an activator of IP(3) receptors, significantly increased peak sodium current amplitude. This effect of adenophostin was abolished by calcium chelation or by FK506, an inhibitor of calcineurin. These results suggest an antagonistic role of PKC and IP(3) in the modulation of striatal voltage-gated sodium channels, peak current amplitude being decreased through phosphorylation by PKC and increased through dephosphorylation by calcineurin.

  3. Clamping down on mammalian meiosis

    PubMed Central

    Lyndaker, Amy M; Vasileva, Ana; Wolgemuth, Debra J; Weiss, Robert S; Lieberman, Howard B

    2013-01-01

    The RAD9A-RAD1-HUS1 (9-1-1) complex is a PCNA-like heterotrimeric clamp that binds damaged DNA to promote cell cycle checkpoint signaling and DNA repair. While various 9-1-1 functions in mammalian somatic cells have been established, mounting evidence from lower eukaryotes predicts critical roles in meiotic germ cells as well. This was investigated in 2 recent studies in which the 9-1-1 complex was disrupted specifically in the mouse male germline through conditional deletion of Rad9a or Hus1. Loss of these clamp subunits led to severely impaired fertility and meiotic defects, including faulty DNA double-strand break repair. While 9-1-1 is critical for ATR kinase activation in somatic cells, these studies did not reveal major defects in ATR checkpoint pathway signaling in meiotic cells. Intriguingly, this new work identified separable roles for 9-1-1 subunits, namely RAD9A- and HUS1-independent roles for RAD1. Based on these studies and the high-level expression of the paralogous proteins RAD9B and HUS1B in testis, we propose a model in which multiple alternative 9-1-1 clamps function during mammalian meiosis to ensure genome maintenance in the germline. PMID:24013428

  4. Phenolic acids isolated from the fungus Schizophyllum commune exert analgesic activity by inhibiting voltage-gated sodium channels.

    PubMed

    Yao, Hui-Min; Wang, Gan; Liu, Ya-Ping; Rong, Ming-Qiang; Shen, Chuan-Bin; Yan, Xiu-Wen; Luo, Xiao-Dong; Lai, Ren

    2016-09-01

    The present study was designed to search for compounds with analgesic activity from the Schizophyllum commune (SC), which is widely consumed as edible and medicinal mushroom world. Thin layer chromatography (TLC), tosilica gel column chromatography, sephadex LH 20, and reverse-phase high performance liquid chromatography (RP-HPLC) were used to isolate and purify compounds from SC. Structural analysis of the isolated compounds was based on nuclear magnetic resonance (NMR). The effects of these compounds on voltage-gated sodium (NaV) channels were evaluated using patch clamp. The analgesic activity of these compounds was tested in two types of mouse pain models induced by noxious chemicals. Five phenolic acids identified from SC extracts in the present study included vanillic acid, m-hydroxybenzoic acid, o-hydroxybenzeneacetic acid, 3-hydroxy-5-methybenzoic acid, and p-hydroxybenzoic acid. They inhibited the activity of both tetrodotoxin-resistant (TTX-r) and tetrodotoxin-sensitive (TTX-s) NaV channels. All the compounds showed low selectivity on NaV channel subtypes. After intraperitoneal injection, three compounds of these compounds exerted analgesic activity in mice. In conclusion, phenolic acids identified in SC demonstrated analgesic activity, facilitating the mechanistic studies of SC in the treatment of neurasthenia. PMID:27667511

  5. Mask side wall clamping

    NASA Astrophysics Data System (ADS)

    Naaijkens, G. J. P.; Rosielle, P. C. J. N.; Steinbuch, M.

    2013-04-01

    Current state-of-the-art optical lithography scanners using 193nm wavelength lasers and numerical apertures of 1.35 have reached fundamental printing limits. Yet, consumer demands and device trends continue to drive smaller feature sizes, and most IC manufacturers have already navigated beyond the lithographic printing limits by turning to double patterning techniques.1 Requiring an extra lithography step for these techniques, it is essential to keep costs down by e.g. increasing wafer throughput. Currently, leading edge immersion scanners consistently produce over 190 wafers per hour (wph). However, to keep decreasing the cost per transistor, higher throughputs of 250 wph are key targets for the year 20132. Amongst others, higher throughput can be acquired by increasing acceleration of the positioning stages. One of the constraining technologies is the current mask or reticle clamping concept due to its friction based acceleration. While current reticle accelerations amount to 150 m/s2, some research3 has already been performed to reticle stage accelerations of 400 m/s2. In this paper, a novel reticle clamping concept is presented. The concept is shown to be suitable for accelerations larger than 400 m/s2 entirely eliminating reticle slip, whilst meeting specifications for clamping induced error with a pattern deformation of < 0.12 nm on wafer stage level (WS) and comprising high clamp stiffness.

  6. Clamp for detonating fuze

    NASA Technical Reports Server (NTRS)

    Holderman, E. J.

    1968-01-01

    Quick acting clamp provides physical support for a closely confined detonating fuse in an application requiring removal and replacement at frequent intervals during test. It can be designed with a base of any required strength and configuration to permit the insertion of an object.

  7. Planar patch-clamp force microscopy on living cells.

    PubMed

    Pamir, Evren; George, Michael; Fertig, Niels; Benoit, Martin

    2008-05-01

    Here we report a new combination of the patch-clamp technique with the atomic force microscope (AFM). A planar patch-clamp chip microstructured from borosilicate glass was used as a support for mechanical probing of living cells. The setup not only allows for immobilizing even a non-adherent cell for measurements of its mechanical properties, but also for simultaneously measuring the electrophysiological properties of a single cell. As a proof of principle experiment we measured the voltage-induced membrane movement of HEK293 and Jurkat cells in the whole-cell voltage clamp configuration. The results of these measurements are in good agreement with previous studies. By using the planar patch-clamp chip for immobilization, the AFM not only can image non-adhering cells, but also gets easily access to an electrophysiologically controlled cellular probe at low vibrational noise. PMID:17933465

  8. A PLL Scheme for Synchronization with Grid Voltage Phasor in Active Power Filter Systems

    NASA Astrophysics Data System (ADS)

    Krievs, Oskars; Steiks, Ingars; Ribickis, Leonids

    2010-01-01

    Voltage source inverters connected to the grid in applications such as active power filters require synchronization with the grid voltage. Since in practice the grid voltage can be unbalanced and distorted, but the operation of the whole active filter control system is strongly dependant on precise estimation of grid voltage phase, the fundamental positive sequence phasor of the grid voltage has to be extracted. In this paper a system for smooth estimation of the position of the voltage phasor at the point of common coupling of a parallel active filter system is presented using a sinusoidal signal integrator and a simple software PLL. The performance of the proposed system is verified by simulation and experimental results. The proposed PLL scheme can also be used in other vector oriented control systems.

  9. [Clamping procedures in hepatic surgery].

    PubMed

    Frangov, T; Dimitrova, V; Kasten, D; Bismiut, A

    2005-01-01

    The advance of liver surgery and transplantation offers a new procedures--vascular clamping. Results of hepatic resections depends essentially on proper control of intraoperative hemorrhage. We present here the different procedures for vascular clamping and discussing their indications. Four parametres can be used to define the type of clamping: 1) place of application--control of arterial or glisson pedicles and portal veins (pedicles, selective hilar, suprahilar and intrahepatic clamps), suprahepatic veins or vena cava; 2) selectivity--partial or total clamp of hepatic blood supply; 3) duration--continuous or intermittent; 4) association measures to favor tolerence to ischemia (cooling, preservation fluids) or to limit downstream consequences (extracorporal circulaton, derivation). The clamping procedures depends on the localisation of the lesion and its relationships with the great vessels, presence of liver desease and the patients general and cardiovascular status. The aim is to use clamp moderate, favoring selective clamps to avoid ischemia. PMID:18693516

  10. Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes.

    PubMed Central

    Farley, J.; Rudy, B.

    1988-01-01

    K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could

  11. Functional coupling between sodium-activated potassium channels and voltage-dependent persistent sodium currents in cricket Kenyon cells

    PubMed Central

    Takahashi, Izumi

    2015-01-01

    In this study, we examined the functional coupling between Na+-activated potassium (KNa) channels and Na+ influx through voltage-dependent Na+ channels in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Single-channel activity of KNa channels was recorded with the cell-attached patch configuration. The open probability (Po) of KNa channels increased with increasing Na+ concentration in a bath solution, whereas it decreased by the substitution of Na+ with an equimolar concentration of Li+. The Po of KNa channels was also found to be reduced by bath application of a high concentration of TTX (1 μM) and riluzole (100 μM), which inhibits both fast (INaf) and persistent (INaP) Na+ currents, whereas it was unaffected by a low concentration of TTX (10 nM), which selectively blocks INaf. Bath application of Cd2+ at a low concentration (50 μM), as an inhibitor of INaP, also decreased the Po of KNa channels. Conversely, bath application of the inorganic Ca2+-channel blockers Co2+ and Ni2+ at high concentrations (500 μM) had little effect on the Po of KNa channels, although Cd2+ (500 μM) reduced the Po of KNa channels. Perforated whole cell clamp analysis further indicated the presence of sustained outward currents for which amplitude was dependent on the amount of Na+ influx. Taken together, these results indicate that KNa channels could be activated by Na+ influx passing through voltage-dependent persistent Na+ channels. The functional significance of this coupling mechanism was discussed in relation to the membrane excitability of Kenyon cells and its possible role in the formation of long-term memory. PMID:26269549

  12. Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells

    PubMed Central

    Atia, Jolene; McCloskey, Conor; Shmygol, Anatoly S.; Rand, David A.; van den Berg, Hugo A.; Blanks, Andrew M.

    2016-01-01

    Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the ‘conductance repertoire’ being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors) consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations. PMID:27105427

  13. Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

    PubMed

    Atia, Jolene; McCloskey, Conor; Shmygol, Anatoly S; Rand, David A; van den Berg, Hugo A; Blanks, Andrew M

    2016-04-01

    Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors) consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations. PMID:27105427

  14. Discrete Waves and Phototransduction in Voltage-damped Ventral Photoreceptors

    PubMed Central

    Behbehani, Michael; Srebro, Richard

    1974-01-01

    Discrete waves in the voltage-clamped photoreceptor of Limulus are remarkably similar in all essential properties to those found in an unclamped cell. The latency distribution of discrete waves is not affected by considerable changes in the holding potential in a voltage-clamped cell. Both large and small waves occur in voltage-clamped and unclamped cells and in approximately the same proportion. Large and small waves also share the same latency distributions and spectral sensitivity. We suggest that small waves may result from the activation of damaged membrane areas. Large waves have an average amplitude of approximately 5 nA in voltage-clamped photoreceptors. It probably requires several square microns of cell membrane to support this much photo-current. Thus the amplification inherent in the discrete wave process may involve spatial spread of activation from unimolecular dimensions to several square microns of cell membrane surface. Neither local current flow, nor pre-packaging of any transmitter substance appears to be involved in the amplification process. The possible mechanisms of the amplification are evaluated with relationship to the properties of discrete waves. PMID:4846766

  15. 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

  16. Patch-Clamp Technologies for Ion Channel Research

    NASA Astrophysics Data System (ADS)

    Sigworth, Fred J.; Klemic, Kathryn G.

    The electrical activity of living cells can be monitored in various ways, but for the study of ion channels and the drugs that affect them, the patch-clamp techniques are the most sensitive. In this chapter the principles of patch-clamp recording are reviewed, and recent developments in microfabricated patch-clamp electrodes are described.Technical challenges and prospects for the future are discussed.

  17. Diverless pipeline repair clamp, Phase 3

    SciTech Connect

    Miller, J.E.

    1993-08-01

    The objective of this project is to develop a system suitable for repairing small leaks in deep water pipelines. It is assumed that leak repair operations at the water depths in question will be performed by Remotely Operated Vehicles (ROV`s). This report summarizes the results of the third and final phase of this project. Phase 3 work included design, manufacture, and dry testing of (1) a one-half scale model of a 12 inch repair clamp, (2) a full-scale bolt test fixture to demonstrate bolt containment and startup under realistic misalignment of the clamp halves, and (3) a full-scale one-way cylinder for end seal activation. Phase 3 also included a study commissioned from Oceaneering directed at defining the interfaces of the clamp package and the ROV, including suggested procedures for deployment and positioning of the clamp package on the pipeline. Issues regarding bolt make-up by the ROV were also studied in detail and limitations in bolting capability were outlined. The conclusion of this work is that the clamping system described herein may be implemented in a direct manner. The design issues causing the most concern have been resolved through laboratory tests. Note however that all testing performed was mechanical in nature and performed in a dry environment. The recommended next development step, prior to declaring the system operational, is to manufacture a fully outfitted clamp package and to perform installation tests in a controlled underwater environment using a typical deepwater ROV. Wet tests are required in order to demonstrate ROV interfaces and installation procedures, however, the major mechanical features represented by the clamp design as well as its operation have been proven.

  18. Dopamine D2 receptor stimulation differentially affects voltage-activated calcium channels in rat pituitary melanotropic cells.

    PubMed

    Keja, J A; Stoof, J C; Kits, K S

    1992-05-01

    1. Whole-cell voltage clamp recordings were made from 141 rat pituitary melanotropic cells in short-term, serum-free, primary culture. The effects of the dopamine D2 receptor agonist, LY 171555, on sodium, potassium and barium currents were investigated. 2. Application of 1 microM-LY 171555 did not affect the inward sodium and outward potassium currents. 3. Application of LY 171555 reversibly inhibited barium currents, with the strongest inhibition on the early inward current. The effect was dose dependent (IC50 = 4 x 10(-8) M), maximal inhibition of the total current was 30% and the LY 171555-induced block (1 microM) was reversibly antagonized by (+/-)sulpiride (4 microM). 4. Using barium-selective saline solutions, different types of barium current (T, N, and two L components) were identified on the basis of their voltage-dependent kinetics. Their relative amplitudes differed between cells. 5. The T-type current activated at potentials positive to -60 mV, reaching peak amplitude between -20 and -10 mV. At -30 mV, this current was inhibited up to 30% by 1 microM-LY 171555. The time constants of activation (10-3 ms) and inactivation (50-20 ms) as well as the voltage dependence of inactivation (potential of half-maximal inactivation (H), -61 mV; slope factor (S), 4.9 mV) were not affected by LY 171555 application. 6. A rapidly inactivating (time constants 100-50 ms), high threshold current component was identified as an N-type current. This current activated at command potentials positive to -30 mV and reached a maximal amplitude at +10 mV. The steady-state inactivation was described by a single Boltzmann equation with H = -65 mV and S = 11.7 mV. Application of 1 microM-LY 171555 completely suppressed this current. 7. The slowly inactivating (time constants > 1500 ms), high-threshold, L-type current displayed the same voltage dependence of activation as the N current. The voltage dependence of inactivation was modelled by the sum of two Boltzmann equations (L1: H1

  19. Reduced low-voltage activated K+ conductances and enhanced central excitability in a congenitally deaf (dn/dn) mouse

    PubMed Central

    Leao, Richardson N; Berntson, Amy; Forsythe, Ian D; Walmsley, Bruce

    2004-01-01

    We have investigated changes in the neuronal excitability of the auditory brainstem in a congenitally deaf mouse (deafness dn/dn). Whole cell patch recordings from principal neurones of the medial nucleus of the trapezoid body (MNTB) showed strikingly enhanced excitability in the deaf mice when compared to control CBA mice at 12–14 days postnatal. MNTB neurones in normal CBA mice showed the phenotypic single action potential response on depolarization in current clamp; however, recordings from CBA mice carrying the homozygous deafness mutation fired trains of action potentials on depolarization. We show here that these changes are associated with reduced functional expression of dendrotoxin-sensitive Kv1 potassium channels. In contrast, no differences were found in voltage-gated calcium currents between control and deaf mice. These results reveal that loss of hair cell function in the cochlea leads to changes in ion channel expression in the central nervous system and suggests that this deafness model will be an important tool in understanding central changes occurring in human congenital deafness and in exploring activity-dependent regulation of ion channel expression. PMID:15235085

  20. MATLAB implementation of a dynamic clamp with bandwidth of >125 kHz capable of generating I Na at 37 °C.

    PubMed

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

    2013-04-01

    We describe the construction of a dynamic clamp with a bandwidth of >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 and 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 (I Na) 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: I Na, the hyperpolarizing-activated inward pacemaker current (I f) and an inward-rectifier K(+) current (I K1). 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

  1. Effect of trimebutine on voltage-activated calcium current in rabbit ileal smooth muscle cells.

    PubMed

    Nagasaki, M; Komori, S; Ohashi, H

    1993-09-01

    1. The effect of trimebutine on the voltage-dependent inward Ca2+ current was investigated by the whole-cell voltage-clamp technique in single smooth muscle cells from rabbit ileum. 2. Trimebutine (3-100 microM) reduced the Ca2+ current in a concentration-dependent manner. The inhibitory effect on the Ca2+ current was also dependent on the holding potential. The Ca2+ current after a low holding potential was inhibited to a greater extent than that after a high membrane potential: the IC50 values were 7 microM and 36 microM at holding potentials of -40 mV and -60 mV, respectively. The Ca2+ current elicited from a holding potential of -80 mV could not be reduced by as much as 50% of the control by trimebutine at concentrations as high as 100 microM. 3. Trimebutine (30 microM) shifted the voltage-dependent inactivation curve for the Ca2+ current by 18 mV in the negative direction. The affinity of the drug for Ca2+ channels was calculated to be 36 times higher in the inactivated state than in the closed-available state. 4. Blockade of the Ca2+ current by trimebutine, unlike verapamil, was not use-dependent. 5. The results suggest that trimebutine inhibits the voltage-dependent inward Ca2+ current through a preferential binding to Ca2+ channels in the inactivated state in the smooth muscle cell from rabbit ileum. The inhibitory effect of trimebutine on gastrointestinal motility is discussed in the light of the present findings. PMID:8220900

  2. Nonlinear modal interactions in clamped-clamped mechanical resonators.

    PubMed

    Westra, H J R; Poot, M; van der Zant, H S J; Venstra, W J

    2010-09-10

    A theoretical and experimental investigation is presented on the intermodal coupling between the flexural vibration modes of a single clamped-clamped beam. Nonlinear coupling allows an arbitrary flexural mode to be used as a self-detector for the amplitude of another mode, presenting a method to measure the energy stored in a specific resonance mode. The observed complex nonlinear dynamics are quantitatively captured by a model based on coupling of the modes via the beam extension; the same mechanism is responsible for the well-known Duffing nonlinearity in clamped-clamped beams. PMID:20867605

  3. Cantilever clamp fitting

    NASA Technical Reports Server (NTRS)

    Melton, Patrick B. (Inventor)

    1989-01-01

    A device is disclosed for sealing and clamping a cylindrical element which is to be attached to an object such as a wall, a pressurized vessel or another cylindrical element. The device includes a gland having an inner cylindrical wall, which is threaded at one end and is attached at a bendable end to a deformable portion, which in turn is attached to one end of a conical cantilever structure. The other end of the cantilever structure connects at a bendable area to one end of an outer cylindrical wall. The opposite end of cylindrical wall terminates in a thickened portion, the radially outer surface of which is adapted to accommodate a tool for rotating the gland. The terminal end of cylindrical wall also includes an abutment surface, which is adapted to engage a seal, which in turn engages a surface of a receiver. The receiver further includes a threaded portion for engagement with the threaded portion of gland whereby a tightening rotation of gland relative to receiver will cause relative movement between cylindrical walls and of gland. This movement causes a rotation of the conical structure and thus a bending action at bending area and at the bending end of the upper end of inner cylindrical wall. These rotational and bending actions result in a forcing of the deformable portion radially inwardly so as to contact and deform a pipe. This forcible contact creates a seal between gland and pipe, and simultaneously clamps the pipe in position.

  4. 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

  5. Strong cooperativity between subunits in voltage-gated proton channels

    PubMed Central

    Gonzalez, Carlos; Koch, Hans P.; Drum, Ben M.; Larsson, H. Peter

    2010-01-01

    Voltage-activated proton (HV) channels are essential components in the innate immune response. HV channels are dimeric proteins with one proton permeation pathway per subunit. It is not known how HV channels are activated by voltage and whether there is any cooperativity between subunits during voltage activation. Using cysteine accessibility measurements and voltage clamp fluorometry, we show data that are consistent with that the fourth transmembrane segment S4 functions as the voltage sensor in HV channels from Ciona intestinalis. Surprisingly, in a dimeric HV channel, S4 in both subunits have to move to activate the two proton permeation pathways. In contrast, if HV subunits are prevented from dimerizing, then the movement of a single S4 is sufficient to activate the proton permeation pathway in a subunit. These results suggest a strong cooperativity between subunits in dimeric HV channels. PMID:20023639

  6. 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

  7. Hybrid voltage sensor imaging of electrical activity from neurons in hippocampal slices from transgenic mice

    PubMed Central

    Wang, Dongsheng; McMahon, Shane; Zhang, Zhen

    2012-01-01

    Gene targeting with genetically encoded optical voltage sensors brings the methods of voltage imaging to genetically defined neurons and offers a method of studying circuit activity in these selected populations. The present study reports the targeting of genetically encoded hybrid voltage sensors (hVOS) to neurons in transgenic mice. The hVOS family of probes employs a membrane-targeted fluorescent protein, which generates voltage-dependent fluorescence changes in the presence of dipicrylamine (DPA) as the result of a voltage-dependent optical interaction between the two molecules. We generated transgenic mice with two different high-performance hVOS probes under control of a neuron-specific thy-1 promoter. Hippocampal slices from these animals present distinct spatial patterns of expression, and electrical stimulation evoked fluorescence changes as high as 3%. Glutamate receptor and Na+ channel antagonists blocked these responses. One hVOS probe tested here harbors an axonal targeting motif (from GAP-43) and shows preferential expression in axons; this probe can thus report axonal voltage changes. Voltage imaging in transgenic mice expressing hVOS probes opens the door to the study of functional activity in genetically defined populations of neurons in intact neural circuits. PMID:22993267

  8. A pyridinium derivative from Red Sea soft corals inhibited voltage-activated potassium conductances and increased excitability of rat cultured sensory neurones

    PubMed Central

    Temraz, Tarek A; Houssen, Wael E; Jaspars, Marcel; Woolley, David R; Wease, Kerrie N; Davies, Steven N; Scott, Roderick H

    2006-01-01

    Background Whole cell patch clamp recording and intracellular Ca2+ imaging were carried out on rat cultured dorsal root ganglion (DRG) neurones to characterize the actions of crude extracts and purified samples from Red Sea soft corals. The aim of the project was to identify compounds that would alter the excitability of DRG neurones. Results Crude extracts of Sarcophyton glaucum and Lobophyton crassum attenuated spike frequency adaptation causing DRG neurones to switch from firing single action potentials to multiple firing. The increase in excitability was associated with enhanced KCl-evoked Ca2+ influx. The mechanism of action of the natural products in the samples from the soft corals involved inhibition of voltage-activated K+ currents. An active component of the crude marine samples was identified as 3-carboxy-1-methyl pyridinium (trigonelline). Application of synthetic 3-carboxy-1-methyl pyridinium at high concentration (0.1 mM) also induced multiple firing and reduced voltage-activated K+ current. The changes in excitability of DRG neurones induced by 3-carboxy-1-methyl pyridinium suggest that this compound contributes to the bioactivity produced by the crude extracts from two soft corals. Conclusion Sarcophyton glaucum and Lobophyton crassum contain natural products including 3-carboxy-1-methyl pyridinium that increase the excitability of DRG neurones. We speculate that in addition to developmental control and osmoregulation these compounds may contribute to chemical defenses. PMID:16824204

  9. Subthreshold voltage noise due to channel fluctuations in active neuronal membranes.

    PubMed

    Steinmetz, P N; Manwani, A; Koch, C; London, M; Segev, I

    2000-01-01

    Voltage-gated ion channels in neuronal membranes fluctuate randomly between different conformational states due to thermal agitation. Fluctuations between conducting and nonconducting states give rise to noisy membrane currents and subthreshold voltage fluctuations and may contribute to variability in spike timing. Here we study subthreshold voltage fluctuations due to active voltage-gated Na+ and K+ channels as predicted by two commonly used kinetic schemes: the Mainen et al. (1995) (MJHS) kinetic scheme, which has been used to model dendritic channels in cortical neurons, and the classical Hodgkin-Huxley (1952) (HH) kinetic scheme for the squid giant axon. We compute the magnitudes, amplitude distributions, and power spectral densities of the voltage noise in isopotential membrane patches predicted by these kinetic schemes. For both schemes, noise magnitudes increase rapidly with depolarization from rest. Noise is larger for smaller patch areas but is smaller for increased model temperatures. We contrast the results from Monte Carlo simulations of the stochastic nonlinear kinetic schemes with analytical, closed-form expressions derived using passive and quasi-active linear approximations to the kinetic schemes. For all subthreshold voltage ranges, the quasi-active linearized approximation is accurate within 8% and may thus be used in large-scale simulations of realistic neuronal geometries.

  10. An Active Substrate Driver for Enabling Mixed-Voltage SOI Systems-On-A-Chip

    NASA Technical Reports Server (NTRS)

    Jackson, S. A.; Blalock, B. J.; Mojarradi, M. M.; Li, H. W.

    2001-01-01

    The current trend for space application systems is towards fully integrated systems-on-a-chip. To facilitate this drive, high-voltage transistors must reside on the same substrate as low-voltage transistors. These systems must also be radiation tolerant, particularly for space missions such as the Europa Lander and Titan Explorer. SOI CMOS technology offers high levels of radiation hardness. As a result, a high-voltage lateral MOSFET has been developed in a partially-depleted (PD) SOI technology. Utilizing high voltages causes a parasitic transistor to have non-negligible effects on a circuit. Several circuit architectures have been used to compensate for the radiation induced threshold voltage shift of the parasitic back-channel transistor. However, a new architecture for high-voltage systems must be employed to bias the substrate to voltage levels insuring all parasitic transistors remain off. An active substrate driver has been developed to accomplish task. Additional information is contained in the original extended abstract.

  11. Estimation of neuronal activity based on voltage-sensitive dye imaging in a moving preparation.

    PubMed

    Fathiazar, Elham; Kretzberg, Jutta

    2015-01-01

    Voltage-sensitive dye imaging allows simultaneous recording of graded voltage changes of multiple neurons. While this experimental technique is a great tool to study neuronal network activity in neuroscience, the optical recording suffers from artifacts. In particular, bleaching of the dye and cell movement impede the analysis and interpretation of imaging results. In this paper, we present methods to tackle these two main artifacts. Cell movement during the experiment is corrected by an optical flow method. Bleaching decay is estimated based on a line fit of recordings without stimulus, which is subtracted from the rest of the recordings in the same experiment. Here, we use a leech ganglion as an example tissue to evaluate these processing procedures. This preparation allows simultaneous voltage-sensitive dye imaging of the entire neuronal network and intracellular recording of one cell's membrane voltage. Using the intracellularly recorded voltage as the ground truth reference, we show that our processing methods for the VSD imaging signal clearly improve the correlation between the real and the estimated voltage. Since other imaging techniques (e.g., calcium imaging) suffer from the same type of artifacts as voltage-sensitive dye imaging, our processing method might be useful for a wide range of biomedical imaging studies. PMID:26737729

  12. Planar patch clamp: advances in electrophysiology.

    PubMed

    Brüggemann, Andrea; Farre, Cecilia; Haarmann, Claudia; Haythornthwaite, Ali; Kreir, Mohamed; Stoelzle, Sonja; George, Michael; Fertig, Niels

    2008-01-01

    Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer. PMID:18998092

  13. A new dynamic voltage restorer with separating active and reactive power circuit design

    NASA Astrophysics Data System (ADS)

    Pai, Fu-Sheng

    2015-05-01

    Conventional dynamic voltage restorers (DVRs) install parallel battery and capacitor sets at the DC bus to supply the required power for voltage sag compensation. However, the reactive power output of a DVR may increase the ripple voltage at the inner DC bus, possibly resulting in a higher operating temperature of the battery and thus decreased battery life. This paper proposes a DVR system that uses a cascade power module to effectively compensate voltage sag. By separating the active and reactive compensation powers, the proposed DVR provides a lower ripple DC link for extending battery life and offers a flexible way to design the capacitor bank. To confirm the effectiveness of the proposed design, theoretical analysis and experimental validation were conducted under various scenarios. Test results confirm the feasibility and practicality of the proposed method.

  14. Interneuron activity leads to initiation of low-voltage fast-onset seizures.

    PubMed

    Shiri, Zahra; Manseau, Frédéric; Lévesque, Maxime; Williams, Sylvain; Avoli, Massimo

    2015-03-01

    Seizures in temporal lobe epilepsy can be classified as hypersynchronous and low-voltage fast according to their onset patterns. Experimental evidence suggests that low-voltage fast-onset seizures mainly result from the synchronous activity of γ-aminobutyric acid-releasing cells. In this study, we tested this hypothesis using the optogenetic control of parvalbumin-positive interneurons in the entorhinal cortex, in the in vitro 4-aminopyridine model. We found that both spontaneous and optogenetically induced seizures had similar low-voltage fast-onset patterns. In addition, both types of seizures presented with higher ripple than fast ripple rates. Our data demonstrate the involvement of interneuronal networks in the initiation of low-voltage fast-onset seizures.

  15. The effects of high-voltage pulse electric discharges on ion adsorption on activated carbons

    NASA Astrophysics Data System (ADS)

    Gafurov, M. M.; Sveshnikova, D. A.; Larin, S. V.; Rabadanov, K. Sh.; Shabanova, Z. E.; Yusupova, A. A.; Ramazanov, A. Sh.

    2008-07-01

    The effects of high-voltage pulse electric discharges (HPED) on sorption of boron and sulfate ions on activated carbons of different kinds (KM-2, BAU, DAK) were investigated. The effect of HPED activation on the sorption characteristics of the systems was found to be similar to the temperature effect.

  16. Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells.

    PubMed

    Tilley, Drew C; Eum, Kenneth S; Fletcher-Taylor, Sebastian; Austin, Daniel C; Dupré, Christophe; Patrón, Lilian A; Garcia, Rita L; Lam, Kit; Yarov-Yarovoy, Vladimir; Cohen, Bruce E; Sack, Jon T

    2014-11-01

    Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX-fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling. PMID:25331865

  17. A conserved threonine in the S1-S2 loop of KV7.2 and K V7.3 channels regulates voltage-dependent activation.

    PubMed

    Füll, Yvonne; Seebohm, Guiscard; Lerche, Holger; Maljevic, Snezana

    2013-06-01

    The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.

  18. Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons

    PubMed Central

    Lin, Zhi-ying; Chen, Li-min; Zhang, Jing; Pan, Xiao-dong; Zhu, Yuan-gui; Ye, Qin-yong; Huang, Hua-pin; Chen, Xiao-chun

    2012-01-01

    Aim: To investigate the effect of ginsenoside Rb1 on voltage-gated calcium currents in cultured rat hippocampal neurons and the modulatory mechanism. Methods: Cultured hippocampal neurons were prepared from Sprague Dawley rat embryos. Whole-cell configuration of the patch-clamp technique was used to record the voltage-gated calcium currents (VGCCs) from the hippocampal neurons,and the effect of Rb1 was examined. Results: Rb1 (2–100 μmol/L) inhibited VGCCs in a concentration-dependent manner, and the current was mostly recovered upon wash-out. The specific L-type Ca2+ channel inhibitor nifedipine (10 μmol/L) occluded Rb1-induced inhibition on VGCCs. Neither the selective N-type Ca2+ channel blocker ω-conotoxin-GVIA (1 μmol/L), nor the selective P/Q-type Ca2+ channel blocker ω-agatoxin IVA (30 nmol/L) diminished Rb1-sensitive VGCCs. Rb1 induced a leftward shift of the steady-state inactivation curve of ICa to a negative potential without affecting its activation kinetics or reversal potential in the I–V curve. The inhibitory effect of Rb1 was neither abolished by the adenylyl cyclase activator forskolin (10 μmol/L), nor by the PKA inhibitor H-89 (10 μmol/L). Conclusion: Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels, without affecting the N-type or P/Q-type Ca2+ channels in hippocampal neurons. cAMP-PKA signaling pathway is not involved in this effect. PMID:22407229

  19. Internal V-Band Clamp

    DOEpatents

    Vaughn, Mark R.; Hafenrichter, Everett S.; Chapa, Agapito C.; Harris, Steven M.; Martinez, Marcus J.; Baty, Roy S.

    2006-02-28

    A system for clamping two tubular members together in an end-to-end relationship uses a split ring with a V-shaped outer rim that can engage a clamping surface on each member. The split ring has a relaxed closed state where the ends of the ring are adjacent and the outside diameter of the split ring is less than the minimum inside diameter of the members at their ends. The members are clamped when the split ring is spread into an elastically stretched position where the ring rim is pressed tightly against the interior surfaces of the members. Mechanisms are provided for removing the spreader so the split ring will return to the relaxed state, releasing the clamped members.

  20. Management of umbilical cord clamping.

    PubMed

    Webbon, Lucy

    2013-02-01

    The Royal College of Midwives (RCM) has updated its third stage of labour guidelines (RCM 2012) to be clearly supportive of a delay in umbilical cord clamping, although specific guidance on timing is yet to be announced. It is therefore imperative that both midwives and student midwives understand and are able to integrate delaying into their practice, as well as communicating to women the benefits; only in this way can we give women fully informed choices on this aspect of care. The main benefit of delayed cord clamping is the protection it can provide in reducing childhood anaemia, which is a major issue, especially in poorer countries. A review of the evidence found no risks linked to delayed clamping, and no evidence that it cannot be used in combination with the administration of uterotonic drugs. Delayed cord clamping can be especially beneficial for pre term and compromised babies.

  1. Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels

    PubMed Central

    Salari, Autoosa; Vega, Benjamin S.; Milescu, Lorin S.; Milescu, Mirela

    2016-01-01

    Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b–S4 “paddle motif,” which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3–S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning. PMID:27045173

  2. Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels.

    PubMed

    Salari, Autoosa; Vega, Benjamin S; Milescu, Lorin S; Milescu, Mirela

    2016-01-01

    Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b-S4 "paddle motif," which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3-S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning. PMID:27045173

  3. The venom of the fishing spider Dolomedes sulfurous contains various neurotoxins acting on voltage-activated ion channels in rat dorsal root ganglion neurons.

    PubMed

    Wang, Hengyun; Zhang, Fan; Li, Dan; Xu, Shiyan; He, Juan; Yu, Hai; Li, Jiayan; Liu, Zhonghua; Liang, Songping

    2013-04-01

    Dolomedes sulfurous is a venomous spider distributed in the south of China and characterized with feeding on fish. The venom exhibits great diversity and contains hundreds of peptides as revealed by off-line RP-HPLC/MALDI-TOF-MS analysis. The venom peptides followed a triple-modal distribution, with 40.7% of peptides falling in the mass range of 1000-3000 Da, 25.6% peptides in the 7000-9000 Da range and 23.5% peptides in the 3000-5000 Da range. This distribution modal is rather different from these of peptides from other spider venoms analyzed. The venom could inhibit voltage-activated Na(+), K(+) and Ca(2+) channels in rat DRG neurons as revealed by voltage-clamp analysis. Significantly, the venom exhibited inhibitory effects on TTX-R Na(+) and T-type Ca(2+) currents, suggesting that there exist both channel antagonists which might be valuable tools for investigation of both channels and drug development. Additionally, intrathoracically injection of venom could cause serve neurotoxic effects on zebrafish and death at higher concentrations. The LD50 value was calculated to be 28.8 μg/g body weight. Our results indicated that the venom of D. sulfurous contain diverse neurotoxins which serve to capture prey. Intensive studies will be necessary to investigate the structures and functions of specific peptides of the venom in the future.

  4. Calcium-Activated SK Channels Influence Voltage-Gated Ion Channels to Determine the Precision of Firing in Globus Pallidus Neurons

    PubMed Central

    Deister, Christopher A.; Chan, C. Savio; Surmeier, D. James; Wilson, Charles J.

    2012-01-01

    Globus pallidus (GP) neurons fire rhythmically in the absence of synaptic input, suggesting that they may encode their inputs as changes in the phase of their rhythmic firing. Action potential afterhyperpolarization (AHP) enhances precision of firing by ensuring that the ion channels recover from inactivation by the same amount on each cycle. Voltage-clamp experiments in slices showed that the longest component of the GP neuron’s AHP is blocked by apamin, a selective antagonist of calcium-activated SK channels. Application of 100 nm apamin also disrupted the precision of firing in perforated-patch and cell-attached recordings. SK channel blockade caused a small depolarization in spike threshold and made it more variable, but there was no reduction in the maximal rate of rise during an action potential. Thus, the firing irregularity was not caused solely by a reduction in voltage-gated Na+ channel availability. Subthreshold voltage ramps triggered a large outward current that was sensitive to the initial holding potential and had properties similar to the A-type K+ current in GP neurons. In numerical simulations, the availability of both Na+ and A-type K+ channels during autonomous firing were reduced when SK channels were removed, and a nearly equal reduction in Na+ and K+ subthreshold-activated ion channel availability produced a large decrease in the neuron’s slope conductance near threshold. This change made the neuron more sensitive to intrinsically generated noise. In vivo, this change would also enhance the sensitivity of GP neurons to small synaptic inputs. PMID:19571136

  5. Automated Patch Clamp Analysis of nAChα7 and Na(V)1.7 Channels.

    PubMed

    Obergrussberger, Alison; Haarmann, Claudia; Rinke, Ilka; Becker, Nadine; Guinot, David; Brueggemann, Andrea; Stoelzle-Feix, Sonja; George, Michael; Fertig, Niels

    2014-01-01

    Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (Na(V)1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of Na(V)1.7, and activation and allosteric modulation of nAChα7R. PMID:24934604

  6. Reevaluating the mechanisms of focal ictogenesis: The role of low-voltage fast activity.

    PubMed

    de Curtis, Marco; Gnatkovsky, Vadym

    2009-12-01

    The mechanisms that control the transition into a focal seizure are still uncertain. The introduction of presurgical intracranial recordings to localize the epileptogenic zone in patients with drug-resistant focal epilepsies opened a new window to the interpretation of seizure generation (ictogenesis). One of the most frequent focal patterns observed with intracranial electrodes at seizure onset is characterized by low-voltage fast activity in the beta-gamma range that may or may not be preceded by changes of ongoing interictal activities. In the present commentary, the mechanisms of generation of focal seizures are reconsidered, focusing on low-voltage fast activity patterns. Experimental findings on models of temporal lobe seizures support the view that the low-voltage fast activity observed at seizure onset is associated with reinforcement and synchronization of inhibitory networks. A minor role for the initiation of the ictal pattern is played by principal neurons that are progressively recruited with a delay, when inhibition declines and synchronous high-voltage discharges ensue. The transition from inhibition into excitatory recruitment is probably mediated by local increase in potassium concentration associated with synchronized interneuronal firing. These findings challenge the classical theory that proposes an increment of excitation and/or a reduction of inhibition as a cause for the transition to seizure in focal epilepsies. A new definition of ictogenesis mechanisms, as herewith hypothesized, might possibly help to develop new therapeutic strategies for focal epilepsies.

  7. Induced Voltage Linear Extraction Method Using an Active Kelvin Bridge for Disturbing Force Self-Sensing

    PubMed Central

    Yang, Yuanyuan; Wang, Lei; Tan, Jiubin; Zhao, Bo

    2016-01-01

    This paper presents an induced voltage linear extraction method for disturbing force self-sensing in the application of giant magnetostrictive actuators (GMAs). In this method, a Kelvin bridge combined with an active device is constructed instead of a conventional Wheatstone bridge for extraction of the induced voltage, and an additional GMA is adopted as a reference actuator in the self-sensing circuit in order to balance the circuit bridge. The linear fitting of the measurement data is done according to the linear relationship between the disturbing forces and the integral of the induced voltage. The experimental results confirm the good performance of the proposed method, and the self-sensitivity of the disturbing forces is better than 2.0 (mV·s)/N. PMID:27213399

  8. In Situ Measurement of Voltage-Induced Stress in Conducting Polymers with Redox-Active Dopants.

    PubMed

    Sen, Sujat; Kim, Sung Yeol; Palmore, Lia R; Jin, Shenghua; Jadhav, Nitin; Chason, Eric; Palmore, G Tayhas R

    2016-09-14

    Minimization of stress-induced mechanical rupture and delamination of conducting polymer (CP) films is desirable to prevent failure of devices based on these materials. Thus, precise in situ measurement of voltage-induced stress within these films should provide insight into the cause of these failure mechanisms. The evolution of stress in films of polypyrrole (pPy), doped with indigo carmine (IC), was measured in different electrochemical environments using the multibeam optical stress sensor (MOSS) technique. The stress in these films gradually increases to a constant value during voltage cycling, revealing an initial break-in period for CP films. The nature of the ions involved in charge compensation of pPy[IC] during voltage cycling was determined from electrochemical quartz crystal microbalance (EQCM) data. The magnitude of the voltage-induced stress within pPy[IC] at neutral pH correlated with the radius of the hydrated mobile ion in the order Li(+) > Na(+) > K(+). At acidic pH, the IC dopant in pPy[IC] undergoes reversible oxidation and reduction within the range of potentials investigated, providing a secondary contribution to the observed voltage-induced stress. We report on the novel stress response of these polymers due to the presence of pH-dependent redox-active dopants and how it can affect material performance.

  9. In Situ Measurement of Voltage-Induced Stress in Conducting Polymers with Redox-Active Dopants.

    PubMed

    Sen, Sujat; Kim, Sung Yeol; Palmore, Lia R; Jin, Shenghua; Jadhav, Nitin; Chason, Eric; Palmore, G Tayhas R

    2016-09-14

    Minimization of stress-induced mechanical rupture and delamination of conducting polymer (CP) films is desirable to prevent failure of devices based on these materials. Thus, precise in situ measurement of voltage-induced stress within these films should provide insight into the cause of these failure mechanisms. The evolution of stress in films of polypyrrole (pPy), doped with indigo carmine (IC), was measured in different electrochemical environments using the multibeam optical stress sensor (MOSS) technique. The stress in these films gradually increases to a constant value during voltage cycling, revealing an initial break-in period for CP films. The nature of the ions involved in charge compensation of pPy[IC] during voltage cycling was determined from electrochemical quartz crystal microbalance (EQCM) data. The magnitude of the voltage-induced stress within pPy[IC] at neutral pH correlated with the radius of the hydrated mobile ion in the order Li(+) > Na(+) > K(+). At acidic pH, the IC dopant in pPy[IC] undergoes reversible oxidation and reduction within the range of potentials investigated, providing a secondary contribution to the observed voltage-induced stress. We report on the novel stress response of these polymers due to the presence of pH-dependent redox-active dopants and how it can affect material performance. PMID:27579593

  10. 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

  11. Split-tapered joint clamping device

    DOEpatents

    Olsen, Max J.; Schwartz, Jr., John F.

    1988-01-01

    This invention relates to a clamping device for removably attaching a tool element to a bracket element wherein a bracket element is disposed in a groove in the tool and a clamping member is disposed in said groove and in engagement with a clamping face of the bracket and a wall of the groove and with the clamping member having pivot means engaging the bracket and about which the clamping member rotates.

  12. 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.

  13. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    PubMed

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

  14. A novel method for patch-clamp automation.

    PubMed

    Vasilyev, D; Merrill, T; Iwanow, A; Dunlop, J; Bowlby, M

    2006-05-01

    An increasing demand of the pharmaceutical industry for automated electrophysiological stations for ion channel drug discovery has recently resulted in the development of several commercial platforms for secondary and safety screening of ion channel modulators. These commercial systems have demonstrated an enhanced throughput, however, often at the expense of some quality-sensitive aspects of traditional patch-clamp recordings. To improve data quality and content, we have developed a patch-clamp robot that fully automates manual patch-clamp recordings, including patch pipette handling, gigaseal formation, obtaining whole-cell or perforated-cell configuration, drug application, and data acquisition. Utilization of glass micropipettes results in high-quality electrophysiological recordings with an overall success rate of about 30% in perforated-cell mode. A fast drug application system with low volume requirements (1-1.5 ml) allows the study of ligand-gated ion channels on a millisecond scale. As proof-of-concept, we present two assays developed for voltage-gated human ether-a-go-go-related and ligand-gated alpha(7) nicotinic receptor ion channels. The system throughput was a single concentration-response curve every 30-40 min or 12-17 6-point concentration-response curves daily, representing a significant improvement of typical manual patch-clamp throughput. This system represents an efficient method for patch-clamp automation without the need for a complex and expensive electrophysiological set-up for cell visualization.

  15. Local postsynaptic voltage-gated sodium channel activation in dendritic spines of olfactory bulb granule cells.

    PubMed

    Bywalez, Wolfgang G; Patirniche, Dinu; Rupprecht, Vanessa; Stemmler, Martin; Herz, Andreas V M; Pálfi, Dénes; Rózsa, Balázs; Egger, Veronica

    2015-02-01

    Neuronal dendritic spines have been speculated to function as independent computational units, yet evidence for active electrical computation in spines is scarce. Here we show that strictly local voltage-gated sodium channel (Nav) activation can occur during excitatory postsynaptic potentials in the spines of olfactory bulb granule cells, which we mimic and detect via combined two-photon uncaging of glutamate and calcium imaging in conjunction with whole-cell recordings. We find that local Nav activation boosts calcium entry into spines through high-voltage-activated calcium channels and accelerates postsynaptic somatic depolarization, without affecting NMDA receptor-mediated signaling. Hence, Nav-mediated boosting promotes rapid output from the reciprocal granule cell spine onto the lateral mitral cell dendrite and thus can speed up recurrent inhibition. This striking example of electrical compartmentalization both adds to the understanding of olfactory network processing and broadens the general view of spine function.

  16. Modulation of Voltage-Gated Sodium Channels by Activation of Tumor Necrosis Factor Receptor-1 and Receptor-2 in Small DRG Neurons of Rats.

    PubMed

    Leo, M; Argalski, S; Schäfers, M; Hagenacker, T

    2015-01-01

    Tumor necrosis factor- (TNF-) α is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain. Its effects are mediated by two receptors, TNF receptor-1 (TNFR-1) and TNF receptor-2 (TNFR-2). These receptors play a crucial role in the sensitization of voltage-gated sodium channels (VGSCs), a key mechanism in the pathogenesis of chronic pain. Using the whole-cell patch-clamp technique, we examined the influence of TNFR-1 and TNFR-2 on VGSCs and TTX-resistant NaV1.8 channels in isolated rat dorsal root ganglion neurons by using selective TNFR agonists. The TNFR-1 agonist R32W (10 pg/mL) caused an increase in the VGSC current (I(Na(V))) by 27.2 ± 5.1%, while the TNFR-2 agonist D145 (10 pg/mL) increased the current by 44.9 ± 2.6%. This effect was dose dependent. Treating isolated NaV1.8 with R32W (100 pg/mL) resulted in an increase in I(NaV(1.8)) by 18.9 ± 1.6%, while treatment with D145 (100 pg/mL) increased the current by 14.5 ± 3.7%. Based on the current-voltage relationship, 10 pg of R32W or D145 led to an increase in I(Na(V)) in a bell-shaped, voltage-dependent manner with a maximum effect at -30 mV. The effects of TNFR activation on VGSCs promote excitation in primary afferent neurons and this might explain the sensitization mechanisms associated with neuropathic and inflammatory pain. PMID:26504355

  17. Modulation of Voltage-Gated Sodium Channels by Activation of Tumor Necrosis Factor Receptor-1 and Receptor-2 in Small DRG Neurons of Rats

    PubMed Central

    Leo, M.; Argalski, S.; Schäfers, M.; Hagenacker, T.

    2015-01-01

    Tumor necrosis factor- (TNF-) α is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain. Its effects are mediated by two receptors, TNF receptor-1 (TNFR-1) and TNF receptor-2 (TNFR-2). These receptors play a crucial role in the sensitization of voltage-gated sodium channels (VGSCs), a key mechanism in the pathogenesis of chronic pain. Using the whole-cell patch-clamp technique, we examined the influence of TNFR-1 and TNFR-2 on VGSCs and TTX-resistant NaV1.8 channels in isolated rat dorsal root ganglion neurons by using selective TNFR agonists. The TNFR-1 agonist R32W (10 pg/mL) caused an increase in the VGSC current (INa(V)) by 27.2 ± 5.1%, while the TNFR-2 agonist D145 (10 pg/mL) increased the current by 44.9 ± 2.6%. This effect was dose dependent. Treating isolated NaV1.8 with R32W (100 pg/mL) resulted in an increase in INaV(1.8) by 18.9 ± 1.6%, while treatment with D145 (100 pg/mL) increased the current by 14.5 ± 3.7%. Based on the current-voltage relationship, 10 pg of R32W or D145 led to an increase in INa(V) in a bell-shaped, voltage-dependent manner with a maximum effect at −30 mV. The effects of TNFR activation on VGSCs promote excitation in primary afferent neurons and this might explain the sensitization mechanisms associated with neuropathic and inflammatory pain. PMID:26504355

  18. 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.

  19. Heterogeneity of Voltage- and Chemosignal-Activated Response Profiles in Vomeronasal Sensory Neurons

    PubMed Central

    Labra, Antonieta; Brann, Jessica H.; Fadool, Debra A.

    2009-01-01

    Liolaemus lizards were explored to ascertain whether they would make an amenable model to study single-cell electrophysiology of neurons in the vomeronasal organ (VNO). Despite a rich array of chemosensory-related behaviors chronicled for this genus, no anatomical or functional data exist for the VNO, the organ mediating these types of behaviors. Two Liolaemus species (L. bellii and L. nigroviridis) were collected in Central Chile in the Farellones Mountains and transported to the United States. Lizards were subjected to hypothermia and then a lethal injection of sodium pentabarbitol prior to all experiments described in the following text. Retrograde dye perfusion combined with histological techniques demonstrated a compartmentalization of the proportionally large VNO from the main olfactory epithelium (MOE) in cryosections of L. bellii. SDS-PAGE analysis of the VNO of both species demonstrated the expression of three G protein subunits, namely, Gαo, Gαi2, and Gβ, and the absence of Gαolf, Gα11, and Gq, the latter of which are traditionally found in the MOE. Vomeronasal (VN) neurons were enzymatically isolated for whole cell voltage-clamp electrophysiology of single neurons. Both species demonstrated a tetrodotoxin (TTX)-sensitive, rapidly inactivating sodium current and a tetraethylammonium (TEA)-sensitive potassium current that had a transient and sustained component. VN neurons were classified into two types dependent on the ratio of sodium over sustained potassium current. VN neurons exhibited outward and inward chemosignal-evoked currents when stimulated with pheromone-containing secretions taken from the feces, skin, and precloacal pores. Fifty-nine percent of the neurons were responsive to at least one compound when presented with a battery of five different secretions. The breadth of responsiveness (H metric) demonstrated a heterogeneous population of tuning with a mean of 0.29. PMID:15972830

  20. 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.

  1. 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. PMID:25491826

  2. Structure of a Sliding Clamp on DNA

    SciTech Connect

    Georgescu,R.; Kim, S.; Yurieva, O.; Kuriyan, J.; Kong, X.; O'Donnell, M.

    2008-01-01

    The structure of the E. coli {beta} clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the {beta} ring. The pronounced 22 angle of DNA through {beta} may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.

  3. A clamp-like biohybrid catalyst for DNA oxidation

    NASA Astrophysics Data System (ADS)

    van Dongen, Stijn F. M.; Clerx, Joost; Nørgaard, Kasper; Bloemberg, Tom G.; Cornelissen, Jeroen J. L. M.; Trakselis, Michael A.; Nelson, Scott W.; Benkovic, Stephen J.; Rowan, Alan E.; Nolte, Roeland J. M.

    2013-11-01

    In processive catalysis, a catalyst binds to a substrate and remains bound as it performs several consecutive reactions, as exemplified by DNA polymerases. Processivity is essential in nature and is often mediated by a clamp-like structure that physically tethers the catalyst to its (polymeric) template. In the case of the bacteriophage T4 replisome, a dedicated clamp protein acts as a processivity mediator by encircling DNA and subsequently recruiting its polymerase. Here we use this DNA-binding protein to construct a biohybrid catalyst. Conjugation of the clamp protein to a chemical catalyst with sequence-specific oxidation behaviour formed a catalytic clamp that can be loaded onto a DNA plasmid. The catalytic activity of the biohybrid catalyst was visualized using a procedure based on an atomic force microscopy method that detects and spatially locates oxidized sites in DNA. Varying the experimental conditions enabled switching between processive and distributive catalysis and influencing the sliding direction of this rotaxane-like catalyst.

  4. Atomic layer deposition encapsulated activated carbon electrodes for high voltage stable supercapacitors.

    PubMed

    Hong, Kijoo; Cho, Moonkyu; Kim, Sang Ouk

    2015-01-28

    Operating voltage enhancement is an effective route for high energy density supercapacitors. Unfortunately, widely used activated carbon electrode generally suffers from poor electrochemical stability over 2.5 V. Here we present atomic layer deposition (ALD) encapsulation of activated carbons for high voltage stable supercapacitors. Two-nanometer-thick Al2O3 dielectric layers are conformally coated at activated carbon surface by ALD, well-maintaining microporous morphology. Resultant electrodes exhibit excellent stability at 3 V operation with 39% energy density enhancement from 2.5 V operation. Because of the protection of surface functional groups and reduction of electrolyte degradation, 74% of initial voltage was maintained 50 h after full charge, and 88% of capacitance was retained after 5000 cycles at 70 °C accelerated test, which correspond to 31 and 17% improvements from bare activated carbon, respectively. This ALD-based surface modification offers a general method to enhance electrochemical stability of carbon materials for diverse energy and environmental applications.

  5. Automated planar patch-clamp.

    PubMed

    Milligan, Carol J; Möller, Clemens

    2013-01-01

    Ion channels are integral membrane proteins that regulate the flow of ions across the plasma membrane and the membranes of intracellular organelles of both excitable and non-excitable cells. Ion channels are vital to a wide variety of biological processes and are prominent components of the nervous system and cardiovascular system, as well as controlling many metabolic functions. Furthermore, ion channels are known to be involved in many disease states and as such have become popular therapeutic targets. For many years now manual patch-clamping has been regarded as one of the best approaches for assaying ion channel function, through direct measurement of ion flow across these membrane proteins. Over the last decade there have been many remarkable breakthroughs in the development of technologies enabling the study of ion channels. One of these breakthroughs is the development of automated planar patch-clamp technology. Automated platforms have demonstrated the ability to generate high-quality data with high throughput capabilities, at great efficiency and reliability. Additional features such as simultaneous intracellular and extracellular perfusion of the cell membrane, current clamp operation, fast compound application, an increasing rate of parallelization, and more recently temperature control have been introduced. Furthermore, in addition to the well-established studies of over-expressed ion channel proteins in cell lines, new generations of planar patch-clamp systems have enabled successful studies of native and primary mammalian cells. This technology is becoming increasingly popular and extensively used both within areas of drug discovery as well as academic research. Many platforms have been developed including NPC-16 Patchliner(®) and SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich), CytoPatch™ (Cytocentrics AG, Rostock), PatchXpress(®) 7000A, IonWorks(®) Quattro and IonWorks Barracuda™, (Molecular Devices, LLC); Dynaflow(®) HT (Cellectricon

  6. Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels

    PubMed Central

    Chowdhury, Sandipan; Haehnel, Benjamin M.

    2014-01-01

    Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4–S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3–5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate. PMID:25311635

  7. Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels.

    PubMed

    Chowdhury, Sandipan; Haehnel, Benjamin M; Chanda, Baron

    2014-11-01

    Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4-S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3-5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate.

  8. Note: High-efficiency energy harvester using double-clamped piezoelectric beams

    SciTech Connect

    Zheng, Yingmei; Wu, Xuan; Parmar, Mitesh; Lee, Dong-weon

    2014-02-15

    In this study, an improvement in energy conversion efficiency has been reported, which is realized by using a double-clamped piezoelectric beam, based on uniaxial stretching strain. The buckling mechanism is applied to maximize axial stress in the double-clamped beam. The voltage generated by using the double-clamped piezoelectric beam is higher than that generated by using other conventional structures, such as bending cantilevers coated/sandwiched with piezoelectric film, which is proven both theoretically and experimentally. The power generation efficiency is enhanced by further optimizing the double-clamped structure. The optimized high-efficiency energy harvester utilizing double-clamped piezoelectric beams generates a peak output power of 80 μW, under an acceleration of 0.1g.

  9. Note: high-efficiency energy harvester using double-clamped piezoelectric beams.

    PubMed

    Zheng, Yingmei; Wu, Xuan; Parmar, Mitesh; Lee, Dong-weon

    2014-02-01

    In this study, an improvement in energy conversion efficiency has been reported, which is realized by using a double-clamped piezoelectric beam, based on uniaxial stretching strain. The buckling mechanism is applied to maximize axial stress in the double-clamped beam. The voltage generated by using the double-clamped piezoelectric beam is higher than that generated by using other conventional structures, such as bending cantilevers coated/sandwiched with piezoelectric film, which is proven both theoretically and experimentally. The power generation efficiency is enhanced by further optimizing the double-clamped structure. The optimized high-efficiency energy harvester utilizing double-clamped piezoelectric beams generates a peak output power of 80 μW, under an acceleration of 0.1g. PMID:24593401

  10. Note: high-efficiency energy harvester using double-clamped piezoelectric beams.

    PubMed

    Zheng, Yingmei; Wu, Xuan; Parmar, Mitesh; Lee, Dong-weon

    2014-02-01

    In this study, an improvement in energy conversion efficiency has been reported, which is realized by using a double-clamped piezoelectric beam, based on uniaxial stretching strain. The buckling mechanism is applied to maximize axial stress in the double-clamped beam. The voltage generated by using the double-clamped piezoelectric beam is higher than that generated by using other conventional structures, such as bending cantilevers coated/sandwiched with piezoelectric film, which is proven both theoretically and experimentally. The power generation efficiency is enhanced by further optimizing the double-clamped structure. The optimized high-efficiency energy harvester utilizing double-clamped piezoelectric beams generates a peak output power of 80 μW, under an acceleration of 0.1g.

  11. Upregulation of voltage-gated Na+ channels by long-term activation of the ghrelin-growth hormone secretagogue receptor in clonal GC somatotropes.

    PubMed

    Dominguez, Belisario; Felix, Ricardo; Monjaraz, Eduardo

    2009-05-01

    A central question in adenohypophyseal cell physiology concerns the role of transmembrane ionic fluxes in the initiation of the hormone secretion process. In the current report, we investigated the effects of the growth hormone (GH) secretagogues ghrelin and GH-releasing peptide-6 (GHRP-6) on the regulation of the functional expression of voltage-gated Na(+) channels using the tumoral somatotrope GC cell line as a model. Cells were cultured under control conditions or in presence of the GH secretagogues (GHS) for 96 h, and Na(+) currents (I(Na)) were characterized in whole cell patch-clamp experiments. GHS treatment significantly increased I(Na) density in a dose-dependent manner. The effects of GHRP-6 were accompanied by an augment in conductance without changes in the kinetics and the voltage dependence of the currents, suggesting an increase in the number of channels in the cell membrane. Sustained inhibition of L-type Ca(2+) channel activity decreased I(Na) density and prevented the effects of the GHS, whereas long-term exposure to an L-channel agonist increased I(Na) density and enhanced the actions of GHRP-6, indicating that Ca(2+) entry through these channels plays a role in the regulation of Na(+) channel expression. Likewise, GHRP-6 failed to enhance Na(+) channel expression in the presence of membrane-permeable inhibitors of protein kinases A and C, as well as the Ca(2+)/calmodulin-dependent kinase II. Conversely, treatment with a cAMP analog or a protein kinase C activator enhanced both basal and GHS-induced secretion of GH measured by enzyme-linked immunoassay, suggesting that GHRP-6 acting through the ghrelin receptor and different signaling pathways enhances Na(+) channel membrane expression, which favors hormone release from GC somatotropes.

  12. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    PubMed

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation. PMID:25825713

  13. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels

    PubMed Central

    Castillo, Karen; Contreras, Gustavo F.; Pupo, Amaury; Torres, Yolima P.; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-01-01

    Being activated by depolarizing voltages and increases in cytoplasmic Ca2+, voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation. PMID:25825713

  14. Whole-Cell Patch-Clamp Recording of Mouse and Rat Inner Hair Cells in the Intact Organ of Corti.

    PubMed

    Goutman, Juan D; Pyott, Sonja J

    2016-01-01

    Whole-cell patch clamping is a widely applied method to record currents across the entire membrane of a cell. This protocol describes application of this method to record currents from the sensory inner hair cells in the intact auditory sensory epithelium, the organ of Corti, isolated from rats or mice. This protocol particularly outlines the basic equipment required, provides instructions for the preparation of solutions and small equipment items, and methodology for recording voltage-activated and evoked synaptic currents from the inner hair cells.

  15. A clamped rectangular plate containing a crack

    NASA Technical Reports Server (NTRS)

    Tang, R.; Erdogan, F.

    1985-01-01

    The general problem of a rectangular plate clamped along two parallel sides and containing a crack parallel to the clamps is considered. The problem is formulated in terms of a system of singular integral equations and the asymptotic behavior of the stress state near the corners is investigated. Numerical examples are considered for a clamped plate without a crack and with a centrally located crack, and the stress intensity factors and the stresses along the clamps are calculated.

  16. 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.

  17. 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.

  18. 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. PMID:27661929

  19. Voltage Drop Compensation Method for Active Matrix Organic Light Emitting Diode Displays

    NASA Astrophysics Data System (ADS)

    Choi, Sang-moo; Ryu, Do-hyung; Kim, Keum-nam; Choi, Jae-beom; Kim, Byung-hee; Berkeley, Brian

    2011-03-01

    In this paper, the conventional voltage drop compensation methods are reviewed and the novel design and driving scheme, the advanced power de-coupled (aPDC) driving method, is proposed to effectively compensate the voltage IR drop of active matrix light emitting diode (AMOLED) displays. The advanced PDC driving scheme can be applied to general AMOLED pixel circuits that have been developed with only minor modification or without requiring modification in pixel circuit. A 14-in. AMOLED panel with the aPDC driving scheme was fabricated. Long range uniformity (LRU) of the 14-in. AMOLED panel was improved from 43% without the aPDC driving scheme, to over 87% at the same brightness by using the scheme and the layout complexity of the panel with new design scheme is less than that of the panel with the conventional design scheme.

  20. New index of pain triggered by spinal activation of voltage-dependent sodium channels.

    PubMed

    Enomoto, Ryugo; Tsukamoto, Mina; Shimoshige, Yukinori; Aoki, Toshiaki; Matsuoka, Nobuya

    2013-12-01

    Voltage-dependent sodium channels (VDSCs) are crucial for pain generation. Here, to develop a new behavioral index of pain induced by spinal VDSC activation, we examined whether intrathecal veratridine injection produced nociceptive behavior. Intrathecal injection of the VDSC opener veratridine in mice dose-dependently induced nociceptive responses, with response times subsequently reduced by administration of morphine or pregabalin. Systemic administration of lidocaine and mexiletine, but not amitriptyline, also decreased this response time. Taken together, these results demonstrated that response time of nociceptive behavior induced by intrathecal veratridine injection is a quantitative index of pain triggered by spinal VDSC activation. PMID:23760511

  1. Voltage-Activated Calcium Channels as Functional Markers of Mature Neurons in Human Olfactory Neuroepithelial Cells: Implications for the Study of Neurodevelopment in Neuropsychiatric Disorders

    PubMed Central

    Solís-Chagoyán, Héctor; Flores-Soto, Edgar; Reyes-García, Jorge; Valdés-Tovar, Marcela; Calixto, Eduardo; Montaño, Luis M.; Benítez-King, Gloria

    2016-01-01

    In adulthood, differentiation of precursor cells into neurons continues in several brain structures as well as in the olfactory neuroepithelium. Isolated precursors allow the study of the neurodevelopmental process in vitro. The aim of this work was to determine whether the expression of functional Voltage-Activated Ca2+ Channels (VACC) is dependent on the neurodevelopmental stage in neuronal cells obtained from the human olfactory epithelium of a single healthy donor. The presence of channel-forming proteins in Olfactory Sensory Neurons (OSN) was demonstrated by immunofluorescent labeling, and VACC functioning was assessed by microfluorometry and the patch-clamp technique. VACC were immunodetected only in OSN. Mature neurons responded to forskolin with a five-fold increase in Ca2+. By contrast, in precursor cells, a subtle response was observed. The involvement of VACC in the precursors’ response was discarded for the absence of transmembrane inward Ca2+ movement evoked by step depolarizations. Data suggest differential expression of VACC in neuronal cells depending on their developmental stage and also that the expression of these channels is acquired by OSN during maturation, to enable specialized functions such as ion movement triggered by membrane depolarization. The results support that VACC in OSN could be considered as a functional marker to study neurodevelopment. PMID:27314332

  2. Free-energy landscape of ion-channel voltage-sensor-domain activation.

    PubMed

    Delemotte, Lucie; Kasimova, Marina A; Klein, Michael L; Tarek, Mounir; Carnevale, Vincenzo

    2015-01-01

    Voltage sensor domains (VSDs) are membrane-bound protein modules that confer voltage sensitivity to membrane proteins. VSDs sense changes in the transmembrane voltage and convert the electrical signal into a conformational change called activation. Activation involves a reorganization of the membrane protein charges that is detected experimentally as transient currents. These so-called gating currents have been investigated extensively within the theoretical framework of so-called discrete-state Markov models (DMMs), whereby activation is conceptualized as a series of transitions across a discrete set of states. Historically, the interpretation of DMM transition rates in terms of transition state theory has been instrumental in shaping our view of the activation process, whose free-energy profile is currently envisioned as composed of a few local minima separated by steep barriers. Here we use atomistic level modeling and well-tempered metadynamics to calculate the configurational free energy along a single transition from first principles. We show that this transition is intrinsically multidimensional and described by a rough free-energy landscape. Remarkably, a coarse-grained description of the system, based on the use of the gating charge as reaction coordinate, reveals a smooth profile with a single barrier, consistent with phenomenological models. Our results bridge the gap between microscopic and macroscopic descriptions of activation dynamics and show that choosing the gating charge as reaction coordinate masks the topological complexity of the network of microstates participating in the transition. Importantly, full characterization of the latter is a prerequisite to rationalize modulation of this process by lipids, toxins, drugs, and genetic mutations.

  3. Free-energy landscape of ion-channel voltage-sensor–domain activation

    PubMed Central

    Delemotte, Lucie; Kasimova, Marina A.; Klein, Michael L.; Tarek, Mounir; Carnevale, Vincenzo

    2015-01-01

    Voltage sensor domains (VSDs) are membrane-bound protein modules that confer voltage sensitivity to membrane proteins. VSDs sense changes in the transmembrane voltage and convert the electrical signal into a conformational change called activation. Activation involves a reorganization of the membrane protein charges that is detected experimentally as transient currents. These so-called gating currents have been investigated extensively within the theoretical framework of so-called discrete-state Markov models (DMMs), whereby activation is conceptualized as a series of transitions across a discrete set of states. Historically, the interpretation of DMM transition rates in terms of transition state theory has been instrumental in shaping our view of the activation process, whose free-energy profile is currently envisioned as composed of a few local minima separated by steep barriers. Here we use atomistic level modeling and well-tempered metadynamics to calculate the configurational free energy along a single transition from first principles. We show that this transition is intrinsically multidimensional and described by a rough free-energy landscape. Remarkably, a coarse-grained description of the system, based on the use of the gating charge as reaction coordinate, reveals a smooth profile with a single barrier, consistent with phenomenological models. Our results bridge the gap between microscopic and macroscopic descriptions of activation dynamics and show that choosing the gating charge as reaction coordinate masks the topological complexity of the network of microstates participating in the transition. Importantly, full characterization of the latter is a prerequisite to rationalize modulation of this process by lipids, toxins, drugs, and genetic mutations. PMID:25535341

  4. Noradrenaline activates a calcium-activated chloride conductance and increases the voltage-dependent calcium current in cultured single cells of rat portal vein.

    PubMed

    Pacaud, P; Loirand, G; Mironneau, C; Mironneau, J

    1989-05-01

    1. Membrane responses were recorded by a patch pipette technique in cultured cells isolated from rat portal vein. Using the whole-cell mode, pressure ejections of noradrenaline evoked depolarization (current clamp) and inward current (voltage clamp) at membrane potentials of -60 to -70 mV. The noradrenaline-induced response was reversibly blocked by prazosin indicating that the response was mediated by alpha 1-adrenoceptors. 2. The ionic mechanism of the noradrenaline-induced inward current was investigated in potassium-free caesium-containing solutions. Alteration of the chloride equilibrium potential produced similar changes in the reversal potential of the noradrenaline-induced current, indicating that noradrenaline opened chloride-selective channels. There was no evidence implicating sodium or calcium as the charge-carrying ion. 3. Caffeine applied in the bathing solution also induced a transient increase in chloride conductance but the noradrenaline-induced response was lost after application of caffeine. This is interpreted to mean that the increase in chloride conductance induced by noradrenaline and caffeine can occur as a consequence of a rise in intracellular calcium concentration depending on release of calcium from the same intracellular stores. 4. In the presence of caffeine, noradrenaline increased both the voltage-dependent calcium and chloride membrane conductances during application of repetitive depolarizing pulses. It is concluded that in isolated cells of the rat portal vein the depolarization in response to noradrenaline is mediated by an increase in chloride conductance depending on both the calcium release from intracellular stores and the increase of the voltage-dependent calcium current. PMID:2470458

  5. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons

    PubMed Central

    Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E.; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

    2016-01-01

    The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels. PMID:27164140

  6. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons.

    PubMed

    Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

    2016-01-01

    The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels. PMID:27164140

  7. A High Frequency Active Voltage Doubler in Standard CMOS Using Offset-Controlled Comparators for Inductive Power Transmission

    PubMed Central

    Lee, Hyung-Min; Ghovanloo, Maysam

    2014-01-01

    In this paper, we present a fully integrated active voltage doubler in CMOS technology using offset-controlled high speed comparators for extending the range of inductive power transmission to implantable microelectronic devices (IMD) and radio-frequency identification (RFID) tags. This active voltage doubler provides considerably higher power conversion efficiency (PCE) and lower dropout voltage compared to its passive counterpart and requires lower input voltage than active rectifiers, leading to reliable and efficient operation with weakly coupled inductive links. The offset-controlled functions in the comparators compensate for turn-on and turn-off delays to not only maximize the forward charging current to the load but also minimize the back current, optimizing PCE in the high frequency (HF) band. We fabricated the active voltage doubler in a 0.5-μm 3M2P std. CMOS process, occupying 0.144 mm2 of chip area. With 1.46 V peak AC input at 13.56 MHz, the active voltage doubler provides 2.4 V DC output across a 1 kΩ load, achieving the highest PCE = 79% ever reported at this frequency. In addition, the built-in start-up circuit ensures a reliable operation at lower voltages. PMID:23853321

  8. From Squid to Mammals with the HH Model through the Nav Channels' Half-Activation-Voltage Parameter.

    PubMed

    Krouchev, Nedialko I; Rattay, Frank; Sawan, Mohamad; Vinet, Alain

    2015-01-01

    The model family analyzed in this work stems from the classical Hodgkin-Huxley model (HHM). for a single-compartment (space-clamp) and continuous variation of the voltage-gated sodium channels (Nav) half-activation-voltage parameter ΔV1/2, which controls the window of sodium-influx currents. Unlike the baseline HHM, its parametric extension exhibits a richer multitude of dynamic regimes, such as multiple fixed points (FP's), bi- and multi-stability (coexistence of FP's and/or periodic orbits). Such diversity correlates with a number of functional properties of excitable neural tissue, such as the capacity or not to evoke an action potential (AP) from the resting state, by applying a minimal absolute rheobase current amplitude. The utility of the HHM rooted in the giant squid for the descriptions of the mammalian nervous system is of topical interest. We conclude that the model's fundamental principles are still valid (up to using appropriate parameter values) for warmer-blooded species, without a pressing need for a substantial revision of the mathematical formulation. We demonstrate clearly that the continuous variation of the ΔV1/2 parameter comes close to being equivalent with recent HHM 'optimizations'. The neural dynamics phenomena described here are nontrivial. The model family analyzed in this work contains the classical HHM as a special case. The validity and applicability of the HHM to mammalian neurons can be achieved by picking the appropriate ΔV1/2 parameter in a significantly broad range of values. For such large variations, in contrast to the classical HHM, the h and n gates' dynamics may be uncoupled--i.e. the n gates may no longer be considered in mere linear correspondence to the h gates. ΔV1/2 variation leads to a multitude of dynamic regimes--e.g. models with either 1 fixed point (FP) or with 3 FP's. These may also coexist with stable and/or unstable periodic orbits. Hence, depending on the initial conditions, the system may behave as either

  9. From Squid to Mammals with the HH Model through the Nav Channels' Half-Activation-Voltage Parameter.

    PubMed

    Krouchev, Nedialko I; Rattay, Frank; Sawan, Mohamad; Vinet, Alain

    2015-01-01

    The model family analyzed in this work stems from the classical Hodgkin-Huxley model (HHM). for a single-compartment (space-clamp) and continuous variation of the voltage-gated sodium channels (Nav) half-activation-voltage parameter ΔV1/2, which controls the window of sodium-influx currents. Unlike the baseline HHM, its parametric extension exhibits a richer multitude of dynamic regimes, such as multiple fixed points (FP's), bi- and multi-stability (coexistence of FP's and/or periodic orbits). Such diversity correlates with a number of functional properties of excitable neural tissue, such as the capacity or not to evoke an action potential (AP) from the resting state, by applying a minimal absolute rheobase current amplitude. The utility of the HHM rooted in the giant squid for the descriptions of the mammalian nervous system is of topical interest. We conclude that the model's fundamental principles are still valid (up to using appropriate parameter values) for warmer-blooded species, without a pressing need for a substantial revision of the mathematical formulation. We demonstrate clearly that the continuous variation of the ΔV1/2 parameter comes close to being equivalent with recent HHM 'optimizations'. The neural dynamics phenomena described here are nontrivial. The model family analyzed in this work contains the classical HHM as a special case. The validity and applicability of the HHM to mammalian neurons can be achieved by picking the appropriate ΔV1/2 parameter in a significantly broad range of values. For such large variations, in contrast to the classical HHM, the h and n gates' dynamics may be uncoupled--i.e. the n gates may no longer be considered in mere linear correspondence to the h gates. ΔV1/2 variation leads to a multitude of dynamic regimes--e.g. models with either 1 fixed point (FP) or with 3 FP's. These may also coexist with stable and/or unstable periodic orbits. Hence, depending on the initial conditions, the system may behave as either

  10. From Squid to Mammals with the HH Model through the Nav Channels’ Half-Activation-Voltage Parameter

    PubMed Central

    Krouchev, Nedialko I.; Rattay, Frank; Sawan, Mohamad; Vinet, Alain

    2015-01-01

    The model family analyzed in this work stems from the classical Hodgkin-Huxley model (HHM). for a single-compartment (space-clamp) and continuous variation of the voltage-gated sodium channels (Nav) half-activation-voltage parameter ΔV1/2, which controls the window of sodium-influx currents. Unlike the baseline HHM, its parametric extension exhibits a richer multitude of dynamic regimes, such as multiple fixed points (FP’s), bi- and multi-stability (coexistence of FP’s and/or periodic orbits). Such diversity correlates with a number of functional properties of excitable neural tissue, such as the capacity or not to evoke an action potential (AP) from the resting state, by applying a minimal absolute rheobase current amplitude. The utility of the HHM rooted in the giant squid for the descriptions of the mammalian nervous system is of topical interest. We conclude that the model’s fundamental principles are still valid (up to using appropriate parameter values) for warmer-blooded species, without a pressing need for a substantial revision of the mathematical formulation. We demonstrate clearly that the continuous variation of the ΔV1/2 parameter comes close to being equivalent with recent HHM ‘optimizations’. The neural dynamics phenomena described here are nontrivial. The model family analyzed in this work contains the classical HHM as a special case. The validity and applicability of the HHM to mammalian neurons can be achieved by picking the appropriate ΔV1/2 parameter in a significantly broad range of values. For such large variations, in contrast to the classical HHM, the h and n gates’ dynamics may be uncoupled - i.e. the n gates may no longer be considered in mere linear correspondence to the h gates. ΔV1/2 variation leads to a multitude of dynamic regimes—e.g. models with either 1 fixed point (FP) or with 3 FP’s. These may also coexist with stable and/or unstable periodic orbits. Hence, depending on the initial conditions, the system may

  11. Modulation of voltage-dependent and inward rectifier potassium channels by 15-epi-lipoxin-A4 in activated murine macrophages: implications in innate immunity.

    PubMed

    Moreno, Cristina; Prieto, Patricia; Macías, Álvaro; Pimentel-Santillana, María; de la Cruz, Alicia; Través, Paqui G; Boscá, Lisardo; Valenzuela, Carmen

    2013-12-15

    Potassium channels modulate macrophage physiology. Blockade of voltage-dependent potassium channels (Kv) by specific antagonists decreases macrophage cytokine production and inhibits proliferation. In the presence of aspirin, acetylated cyclooxygenase-2 loses the activity required to synthesize PGs but maintains the oxygenase activity to produce 15R-HETE from arachidonate. This intermediate product is transformed via 5-LOX into epimeric lipoxins, termed 15-epi-lipoxins (15-epi-lipoxin A4 [e-LXA4]). Kv have been proposed as anti-inflammatory targets. Therefore, we studied the effects of e-LXA4 on signaling and on Kv and inward rectifier potassium channels (Kir) in mice bone marrow-derived macrophages (BMDM). Electrophysiological recordings were performed in these cells by the whole-cell patch-clamp technique. Treatment of BMDM with e-LXA4 inhibited LPS-dependent activation of NF-κB and IκB kinase β activity, protected against LPS activation-dependent apoptosis, and enhanced the accumulation of the Nrf-2 transcription factor. Moreover, treatment of LPS-stimulated BMDM with e-LXA4 resulted in a rapid decrease of Kv currents, compatible with attenuation of the inflammatory response. Long-term treatment of LPS-stimulated BMDM with e-LXA4 significantly reverted LPS effects on Kv and Kir currents. Under these conditions, e-LXA4 decreased the calcium influx versus that observed in LPS-stimulated BMDM. These effects were partially mediated via the lipoxin receptor (ALX), because they were significantly reverted by a selective ALX receptor antagonist. We provide evidence for a new mechanism by which e-LXA4 contributes to inflammation resolution, consisting of the reversion of LPS effects on Kv and Kir currents in macrophages.

  12. Clamping down on clamps and clamp loaders--the eukaryotic replication factor C.

    PubMed

    Mossi, R; Hübscher, U

    1998-06-01

    DNA transactions such as DNA replication and DNA repair require the concerted action of many enzymes, together with other proteins and non-protein cofactors. Among them three main accessory proteins, replication factor C (RF-C), proliferating-cell nuclear antigen (PCNA) and replication protein A (RP-A), are essential for accurate and processive DNA synthesis by DNA polymerases. RF-C is a complex consisting of five polypeptides with distinct functions. RF-C can bind to a template-primer junction and, in the presence of ATP, load the PCNA clamp onto DNA, thereby recruiting DNA polymerases to the site of DNA synthesis. RF-C not only acts as a clamp loader in DNA replication and DNA repair, but there is some evidence that it could be involved in several other processes such as transcription, S-phase checkpoint regulation, apoptosis, differentiation and telomere-length regulation.

  13. Dynamics of Open DNA Sliding Clamps.

    PubMed

    Oakley, Aaron J

    2016-01-01

    A range of enzymes in DNA replication and repair bind to DNA-clamps: torus-shaped proteins that encircle double-stranded DNA and act as mobile tethers. Clamps from viruses (such as gp45 from the T4 bacteriophage) and eukaryotes (PCNAs) are homotrimers, each protomer containing two repeats of the DNA-clamp motif, while bacterial clamps (pol III β) are homodimers, each protomer containing three DNA-clamp motifs. Clamps need to be flexible enough to allow opening and loading onto primed DNA by clamp loader complexes. Equilibrium and steered molecular dynamics simulations have been used to study DNA-clamp conformation in open and closed forms. The E. coli and PCNA clamps appear to prefer closed, planar conformations. Remarkably, gp45 appears to prefer an open right-handed spiral conformation in solution, in agreement with previously reported biophysical data. The structural preferences of DNA clamps in solution have implications for understanding the duty cycle of clamp-loaders. PMID:27148748

  14. Dynamics of Open DNA Sliding Clamps

    PubMed Central

    Oakley, Aaron J.

    2016-01-01

    A range of enzymes in DNA replication and repair bind to DNA-clamps: torus-shaped proteins that encircle double-stranded DNA and act as mobile tethers. Clamps from viruses (such as gp45 from the T4 bacteriophage) and eukaryotes (PCNAs) are homotrimers, each protomer containing two repeats of the DNA-clamp motif, while bacterial clamps (pol III β) are homodimers, each protomer containing three DNA-clamp motifs. Clamps need to be flexible enough to allow opening and loading onto primed DNA by clamp loader complexes. Equilibrium and steered molecular dynamics simulations have been used to study DNA-clamp conformation in open and closed forms. The E. coli and PCNA clamps appear to prefer closed, planar conformations. Remarkably, gp45 appears to prefer an open right-handed spiral conformation in solution, in agreement with previously reported biophysical data. The structural preferences of DNA clamps in solution have implications for understanding the duty cycle of clamp-loaders. PMID:27148748

  15. Stochastic diffusion model of multistep activation in a voltage-dependent K channel

    NASA Astrophysics Data System (ADS)

    Vaccaro, S. R.

    2010-04-01

    The energy barrier to the activated state for the S4 voltage sensor of a K channel is dependent on the electrostatic force between positively charged S4 residues and negatively charged groups on neighboring segments, the potential difference across the membrane, and the dielectric boundary force on the charged residues near the interface between the solvent and the low dielectric region of the membrane gating pore. The variation of the potential function with transverse displacement and rotation of the S4 sensor across the membrane may be derived from a solution of Poisson's equation for the electrostatic potential. By approximating the energy of an S4 sensor along a path between stationary states by a piecewise linear function of the transverse displacement, the dynamics of slow activation, in the millisecond range, may be described by the lowest frequency component of an analytical solution of interacting diffusion equations of Fokker-Planck type for resting and barrier regions. The solution of the Smoluchowski equations for an S4 sensor in an energy landscape with several barriers is in accord with an empirical master equation for multistep activation in a voltage-dependent K channel.

  16. Voltage-sensitive dye imaging of primary motor cortex activity produced by ventral tegmental area stimulation.

    PubMed

    Kunori, Nobuo; Kajiwara, Riichi; Takashima, Ichiro

    2014-06-25

    The primary motor cortex (M1) receives dopaminergic projections from the ventral tegmental area (VTA) through the mesocortical dopamine pathway. However, few studies have focused on changes in M1 neuronal activity caused by VTA activation. To address this issue, we used voltage-sensitive dye imaging (VSD) to reveal the spatiotemporal dynamics of M1 activity induced by single-pulse stimulation of VTA in anesthetized rats. VSD imaging showed that brief electrical stimulation of unilateral VTA elicited a short-latency excitatory-inhibitory sequence of neuronal activity not only in the ipsilateral but also in the contralateral M1. The contralateral M1 response was not affected by pharmacological blockade of ipsilateral M1 activity, but it was completely abolished by corpus callosum transection. Although the VTA-evoked neuronal activity extended throughout the entire M1, we found the most prominent activity in the forelimb area of M1. The 6-OHDA-lesioned VTA failed to evoke M1 activity. Furthermore, both excitatory and inhibitory intact VTA-induced activity was entirely extinguished by blocking glutamate receptors in the target M1. When intracortical microstimulation of M1 was paired with VTA stimulation, the evoked forelimb muscle activity was facilitated or inhibited, depending on the interval between the two stimuli. These findings suggest that VTA neurons directly modulate the excitability of M1 neurons via fast glutamate signaling and, consequently, may control the last cortical stage of motor command processing. PMID:24966388

  17. [Delayed cord clamping in the interest of the newborn child].

    PubMed

    Scherjon, S A; Smit, Y

    2008-06-21

    The importance of delayed cord clamping, both for the preterm and for the term newborn, for the prevention ofneonatal anaemia (during the neonatal period and/or at the age of3 months) and furthermore to reduce the need of blood transfusions, has recently been demonstrated in controlled clinical studies and meta-analyses. Physiological and pathophysiological factors also provide a rationale for delayed cord clamping: neonatal blood volume may increase by 32% if cord clamping is delayed until the umbilical cord has completely stopped pulsating. A slow transition, involving closure of the ductus arteriosus and the foramen ovale cordis and gradual filling of the neonatal systemic circulation, contributes to the opening of the alveoli due to perfusion of the alveolar capillaries. No disadvantages, such as polycythaemia or hyperbilirubinaemia, have been described with regard to preterm neonates, whereas the incidence of intracranial haemorrhages is reduced. Also for the mother, no disadvantages of late clamping have been determined. As a standard procedure, the baby's umbilical cord should not be clamped until at least 3 minutes have passed. One should wait at least 30 seconds during the birth of children for whom a more active approach is necessary. Of all people, these children will benefit from a good Hb level.

  18. High-speed pressure clamp.

    PubMed

    Besch, Stephen R; Suchyna, Thomas; Sachs, Frederick

    2002-10-01

    We built a high-speed, pneumatic pressure clamp to stimulate patch-clamped membranes mechanically. The key control element is a newly designed differential valve that uses a single, nickel-plated piezoelectric bending element to control both pressure and vacuum. To minimize response time, the valve body was designed with minimum dead volume. The result is improved response time and stability with a threefold decrease in actuation latency. Tight valve clearances minimize the steady-state air flow, permitting us to use small resonant-piston pumps to supply pressure and vacuum. To protect the valve from water contamination in the event of a broken pipette, an optical sensor detects water entering the valve and increases pressure rapidly to clear the system. The open-loop time constant for pressure is 2.5 ms for a 100-mmHg step, and the closed-loop settling time is 500-600 micros. Valve actuation latency is 120 micros. The system performance is illustrated for mechanically induced changes in patch capacitance.

  19. Electronics drivers for high voltage dielectric electro active polymer (DEAP) applications

    NASA Astrophysics Data System (ADS)

    Zhang, Zhe; Andersen, Michael A. E.

    2015-04-01

    Dielectric electro active polymer (DEAP) can be used in actuation, sensing and energy harvesting applications, but driving the DEAP based actuators and generators has three main challenges from a power electronics standpoint, i.e. high voltage (around 2.5 kV), nonlinearity, and capacitive behavior. In this paper, electronics divers for heating valves, loud speakers, incremental motors, and energy harvesting are reviewed, studied and developed in accordance with their corresponding specifications. Due to the simplicity and low power capacity (below 10W), the reversible Fly-back converters with both magnetic and piezoelectric transformers are employed for the heating valve and incremental motor application, where only ON/OFF regulation is adopted for energy saving; as for DEAP based energy harvesting, the noisolated Buck/Boost converter is used, due to the system high power capacity (above 100W), but the voltage balancing across the series-connected high voltage IGBTs is a critical issue and accordingly a novel gate driver circuitry is proposed and equipped; due to the requirements of the audio products, such as low distortion and noise, the multi-level Buck converter based Class-D amplifier, because of its high control linearity, is implemented for the loud speaker applications. A synthesis among those converter topologies and control techniques is given; therefore, for those DEAP based applications, their diversity and similarity of electronics drivers, as well as the key technologies employed are analyzed. Therefore a whole picture of how to choose the proper topologies can be revealed. Finally, the design guidelines in order to achieve high efficiency and reliability are discussed.

  20. Patch clamp recordings of retinal bipolar cells in response to extracellular electrical stimulation in wholemount mouse retina.

    PubMed

    Walston, Steven T; Chow, Robert H; Weiland, James D

    2015-01-01

    Retinitis pigmentosa is a family of inherited retinal diseases identified by the degeneration of photoreceptors, which leads to blindness. In efforts to restore vision lost to retinitis pigmentosa, retinal prostheses have been developed to generate visual percepts by electrically stimulating the surviving retinal bipolar and ganglion cells. The response of retinal ganglion cells to electrical stimulation has been characterized through direct measurement. However, the response of bipolar cells has only been inferred by measuring retinal ganglion cell activity. This investigation reports on a novel tissue preparation technique facilitating bipolar cell patch clamp recordings in wholemount retina. We find that bipolar cells respond to extracellular electrical stimuli with time-locked voltage spike depolarizations, which are likely mediated by voltage-gated calcium channels. PMID:26737013

  1. Shunt hybrid active power filter under nonideal voltage based on fuzzy logic controller

    NASA Astrophysics Data System (ADS)

    Dey, Papan; Mekhilef, Saad

    2016-09-01

    In this paper, a synchronous reference frame (SRF) method based on a modified phase lock loop (PLL) circuit is developed for a three-phase four-wire shunt hybrid active power filter (APF). Its performance is analysed under unbalanced grid conditions. The dominant lower order harmonics as well as reactive power can be compensated by the passive elements, whereas the active part mitigates the remaining distortions and improves the power quality. As different control methods show contradictory performance, fuzzy logic controller is considered here for DC-link voltage regulation of the inverter. Extensive simulations of the proposed technique are carried out in a MATLAB-SIMULINK environment. A laboratory prototype has been built on dSPACE1104 platform to verify the feasibility of the suggested SHAPF controller. The simulation and experimental results validate the effectiveness of the proposed technique.

  2. Thermally activated escape from the zero-voltage state in long Josephson junctions

    SciTech Connect

    Castellano, M.G.; Torrioli, G.; Cosmelli, C.; Costantini, A.; Chiarello, F.; Carelli, P.; Rotoli, G.; Cirillo, M.; Kautz, R.L.

    1996-12-01

    We have measured the rate of thermally induced escape from the zero-voltage state in long Josephson junctions of both overlap and in-line geometry as a function of applied magnetic field. The statistical distribution of switching currents is used to evaluate the escape rate and derive an activation energy {Delta}{ital U} for the process. Because long junctions correspond to the continuum limit of multidimensional systems, {Delta}{ital U} is in principle the difference in energy between stationary states in an infinite-dimensional potential. We obtain good agreement between calculated and measured activation energies for junctions with lengths a few times the Josephson penetration depth {lambda}{sub {ital J}}. {copyright} {ital 1996 The American Physical Society.}

  3. Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells.

    PubMed

    Ypey, D L; Weidema, A F; Höld, K M; Van der Laarse, A; Ravesloot, J H; Van Der Plas, A; Nijweide, P J

    1992-12-01

    Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization-activated channels cause cell-inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi channels, OCL may express two other types of voltage-activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell-outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including Cl- and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch-activated channels in chick osteoclasts; other investigators have found stretch-activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage-activated calcium conductances (GCa) in any of the chick bone

  4. 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

  5. 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. PMID:26878552

  6. Advanced motor driven clamped borehole seismic receiver

    DOEpatents

    Engler, Bruce P.; Sleefe, Gerard E.; Striker, Richard P.

    1993-01-01

    A borehole seismic tool including a borehole clamp which only moves perpendicular to the borehole. The clamp is driven by an electric motor, via a right angle drive. When used as a seismic receiver, the tool has a three part housing, two of which are hermetically sealed. Accelerometers or geophones are mounted in one hermetically sealed part, the electric meter in the other hermetically sealed part, and the clamp and right angle drive in the third part. Preferably the tool includes cable connectors at both ends. Optionally a shear plate can be added to the clamp to extend the range of the tool.

  7. Advanced motor driven clamped borehole seismic receiver

    DOEpatents

    Engler, B.P.; Sleefe, G.E.; Striker, R.P.

    1993-02-23

    A borehole seismic tool is described including a borehole clamp which only moves perpendicular to the borehole. The clamp is driven by an electric motor, via a right angle drive. When used as a seismic receiver, the tool has a three part housing, two of which are hermetically sealed. Accelerometers or geophones are mounted in one hermetically sealed part, the electric motor in the other hermetically sealed part, and the clamp and right angle drive in the third part. Preferably the tool includes cable connectors at both ends. Optionally a shear plate can be added to the clamp to extend the range of the tool.

  8. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    PubMed Central

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-01-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes. PMID:26096612

  9. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    NASA Astrophysics Data System (ADS)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  10. Optical electrophysiology for probing function and pharmacology of voltage-gated ion channels

    PubMed Central

    Zhang, Hongkang; Reichert, Elaine; Cohen, Adam E

    2016-01-01

    Voltage-gated ion channels mediate electrical dynamics in excitable tissues and are an important class of drug targets. Channels can gate in sub-millisecond timescales, show complex manifolds of conformational states, and often show state-dependent pharmacology. Mechanistic studies of ion channels typically involve sophisticated voltage-clamp protocols applied through manual or automated electrophysiology. Here, we develop all-optical electrophysiology techniques to study activity-dependent modulation of ion channels, in a format compatible with high-throughput screening. Using optical electrophysiology, we recapitulate many voltage-clamp protocols and apply to Nav1.7, a channel implicated in pain. Optical measurements reveal that a sustained depolarization strongly potentiates the inhibitory effect of PF-04856264, a Nav1.7-specific blocker. In a pilot screen, we stratify a library of 320 FDA-approved compounds by binding mechanism and kinetics, and find close concordance with patch clamp measurements. Optical electrophysiology provides a favorable tradeoff between throughput and information content for studies of NaV channels, and possibly other voltage-gated channels. DOI: http://dx.doi.org/10.7554/eLife.15202.001 PMID:27215841

  11. The activated state of a sodium channel voltage sensor in a membrane environment

    PubMed Central

    Chakrapani, Sudha; Sompornpisut, Pornthep; Intharathep, Pathumwadee; Roux, Benoît; Perozo, Eduardo

    2010-01-01

    Direct structural insights on the fundamental mechanisms of permeation, selectivity, and gating remain unavailable for the Na+ and Ca2+ channel families. Here, we report the spectroscopic structural characterization of the isolated Voltage-Sensor Domain (VSD) of the prokaryotic Na+ channel NaChBac in a lipid bilayer. Site-directed spin-labeling and EPR spectroscopy were carried out for 118 mutants covering all of the VSD. EPR environmental data were used to unambiguously assign the secondary structure elements, define membrane insertion limits, and evaluate the activated conformation of the isolated-VSD in the membrane using restrain-driven molecular dynamics simulations. The overall three-dimensional fold of the NaChBac-VSD closely mirrors those seen in KvAP, Kv1.2, Kv1.2-2.1 chimera, and MlotiK1. However, in comparison to the membrane-embedded KvAP-VSD, the structural dynamics of the NaChBac-VSD reveals a much tighter helix packing, with subtle differences in the local environment of the gating charges and their interaction with the rest of the protein. Using cell complementation assays we show that the NaChBac-VSD can provide a conduit to the transport of ions in the resting or “down” conformation, a feature consistent with our EPR water accessibility measurements in the activated or “up” conformation. These results suggest that the overall architecture of VSD’s is remarkably conserved among K+ and Na+ channels and that pathways for gating-pore currents may be intrinsic to most voltage-sensors. Cell complementation assays also provide information about the putative location of the gating charges in the “down/resting” state and hence a glimpse of the extent of conformational changes during activation. PMID:20207950

  12. A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices

    PubMed Central

    Abdelfattah, Ahmed S.; Farhi, Samouil L.; Zhao, Yongxin; Brinks, Daan; Zou, Peng; Ruangkittisakul, Araya; Platisa, Jelena; Pieribone, Vincent A.; Ballanyi, Klaus; Cohen, Adam E.

    2016-01-01

    Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation. SIGNIFICANCE STATEMENT Fluorescent-protein-based voltage indicators enable imaging of the electrical activity of many genetically targeted neurons with high spatial and temporal resolution. Here, we describe the engineering of a bright red fluorescent protein-based voltage indicator designated as FlicR1 (fluorescent indicator for voltage imaging red). FlicR1 has sufficient speed and sensitivity to report single action potentials and voltage fluctuations at frequencies up to 100 Hz in single-trial recordings with wide-field microscopy. Because it is excitable with yellow light, FlicR1 can be used in conjunction with blue-light-activated

  13. Mechanism of ATP-driven PCNA clamp loading by S. cerevisiae RFC

    PubMed Central

    Chen, Siying; Levin, Mikhail K.; Sakato, Miho; Zhou, Yayan; Hingorani, Manju M.

    2009-01-01

    Circular clamps tether polymerases to DNA, serving as essential processivity factors in genome replication, and function in other critical cellular processes as well. Clamp loaders catalyze clamp assembly onto DNA, and the question of how these proteins construct a topological link between a clamp and DNA remains open, especially the mechanism by which ATP is utilized for the task. Here we describe pre-steady state analysis of ATP hydrolysis, PCNA clamp opening and DNA binding by S. cerevisiae RFC, and present the first kinetic model of a eukaryotic clamp loading reaction validated by global data analysis. ATP binding to multiple RFC subunits initiates a slow conformational change in the clamp loader, enabling it to bind and open PCNA, and bind DNA as well. PCNA opening locks RFC into an active state, and the resulting RFC•ATP•PCNA(open) intermediate is ready for entry of DNA into the clamp. DNA binding commits RFC to ATP hydrolysis, which is followed by PCNA closure and PCNA•DNA release. This model enables quantitative understanding of the multi-step mechanism of a eukaryotic clamp loader, and furthermore facilitates comparative analysis of loaders from diverse organisms. PMID:19285992

  14. Low-Voltage, Low-Power, Organic Light-Emitting Transistors for Active Matrix Displays

    NASA Astrophysics Data System (ADS)

    McCarthy, M. A.; Liu, B.; Donoghue, E. P.; Kravchenko, I.; Kim, D. Y.; So, F.; Rinzler, A. G.

    2011-04-01

    Intrinsic nonuniformity in the polycrystalline-silicon backplane transistors of active matrix organic light-emitting diode displays severely limits display size. Organic semiconductors might provide an alternative, but their mobility remains too low to be useful in the conventional thin-film transistor design. Here we demonstrate an organic channel light-emitting transistor operating at low voltage, with low power dissipation, and high aperture ratio, in the three primary colors. The high level of performance is enabled by a single-wall carbon nanotube network source electrode that permits integration of the drive transistor and the light emitter into an efficient single stacked device. The performance demonstrated is comparable to that of polycrystalline-silicon backplane transistor-driven display pixels.

  15. Diverless pipeline repair clamp: Phase 2

    SciTech Connect

    Miller, J.E.; Lane, B. )

    1992-04-01

    The objective of this project sponsored by the Pipeline Research Committee of the American Gas Association, is to develop a system suitable for repairing small leaks on deepwater pipelines. Phase I of the project, completed in 1990 by Stress Engineering Services, Inc. investigated the types of problems that would have to be overcome to effect a diverless clamp-type repair. Several repair systems were investigated and ten mechanisms were proposed that could be used to secure two clamp halves together. This current Phase 11 effort, is to take two most promising clamp concepts from Phase 1, further evaluate hardware and installation issues, develop conceptual designs, and determine which concept should be carried forward to detailed design. The two concepts evaluated were (1) a bolted split-sleeve clamp suited for ROV installation, and (2) a hydraulically self-actuating clamp requiring only placement on the pipe and actuation by ROV hydraulic hot stabs. Both concepts were evaluated for a 12-inch (324 mm) nominal pipe diameter with an ANSI 900 (15.3 mPa) pressure rating, presuming either system could be adapted to a wider range of pipe sizes and design pressures. Based on the results of this investigation a modified bolted split-sleeve clamp was recommended over the hydraulically self-actuating clamp. The main reasons are (1) the bolted split-sleeve clamp can be adapted to installation by a ROV, (2) sealing and clamping mechanisms borrow from available proven technology, (3) it would require less development effort than the hydraulically self-actuating clamp, and (4) the bolted split-sleeve clamp would probably result in a simpler, less costly design.

  16. Forced-exercise delays neuropathic pain in experimental diabetes: effects on voltage-activated calcium channels.

    PubMed

    Shankarappa, Sahadev A; Piedras-Rentería, Erika S; Stubbs, Evan B

    2011-07-01

    Physical exercise produces a variety of psychophysical effects, including altered pain perception. Elevated levels of centrally produced endorphins or endocannabinoids are implicated as mediators of exercise-induced analgesia. The effect of exercise on the development and persistence of disease-associated acute/chronic pain remains unclear. In this study, we quantified the physiological consequence of forced-exercise on the development of diabetes-associated neuropathic pain. Euglycemic control or streptozotocin (STZ)-induced diabetic adult male rats were subdivided into sedentary or forced-exercised (2-10 weeks, treadmill) subgroups and assessed for changes in tactile responsiveness. Two weeks following STZ-treatment, sedentary rats developed a marked and sustained hypersensitivity to von Frey tactile stimulation. By comparison, STZ-treated diabetic rats undergoing forced-exercise exhibited a 4-week delay in the onset of tactile hypersensitivity that was independent of glucose control. Exercise-facilitated analgesia in diabetic rats was reversed, in a dose-dependent manner, by naloxone. Small-diameter (< 30 μm) DRG neurons harvested from STZ-treated tactile hypersensitive diabetic rats exhibited an enhanced (2.5-fold) rightward (depolarizing) shift in peak high-voltage activated (HVA) Ca(2+) current density with a concomitant appearance of a low-voltage activated (LVA) Ca(2+) current component. LVA Ca(2+) currents present in DRG neurons from hypersensitive diabetic rats exhibited a marked depolarizing shift in steady-state inactivation. Forced-exercise attenuated diabetes-associated changes in HVA Ca(2+) current density while preventing the depolarizing shift in steady-state inactivation of LVA Ca(2+) currents. Forced-exercise markedly delays the onset of diabetes-associated neuropathic pain, in part, by attenuating associated changes in HVA and LVA Ca(2+) channel function within small-diameter DRG neurons possibly by altering opioidergic tone. PMID:21554321

  17. 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.

  18. Effects of pinaverium on voltage-activated calcium channel currents of single smooth muscle cells isolated from the longitudinal muscle of the rabbit jejunum.

    PubMed Central

    Beech, D. J.; MacKenzie, I.; Bolton, T. B.; Christen, M. O.

    1990-01-01

    1. Smooth muscle cells of the longitudinal muscle of the rabbit jejunum were dispersed by enzyme treatment and recordings of membrane current were made in the whole-cell mode by patch clamp technique. The action of pinaverium bromide on the voltage-dependent inward current of single isolated smooth muscle cells was studied in solutions containing normal concentrations of calcium or high concentrations of barium at room temperature. 2. Pinaverium reduced the voltage-dependent inward current with an IC50 of 1.5 microM. This IC50 is similar to those of verapamil, diltiazem and flunarizine on these cells as described by others. Occasionally evidence of a potentiating action of pinaverium on the inward current was seen. 3. Repetitive stimulation of the cells did not increase blockade of inward current by pinaverium unlike the use-dependent blockade seen with verapamil, methoxyverapamil, and diltiazem in these and in other smooth muscle cells. 4. The inactivation of inward current was studied by holding at various potentials for 2 or 10 s before evoking inward current. The voltage at which current was 50% available was changed very little by pinaverium although other calcium entry blockers, for example the dihydropyridines, have been reported to produce appreciable negative shifts which indicate considerable voltage-dependence of their blockade. This may indicate that pinaverium has similar affinities for the closed available and inactivated calcium channel states so that blockade is not appreciably voltage-dependent. PMID:1691676

  19. Effects of pinaverium on voltage-activated calcium channel currents of single smooth muscle cells isolated from the longitudinal muscle of the rabbit jejunum.

    PubMed

    Beech, D J; MacKenzie, I; Bolton, T B; Christen, M O

    1990-02-01

    1. Smooth muscle cells of the longitudinal muscle of the rabbit jejunum were dispersed by enzyme treatment and recordings of membrane current were made in the whole-cell mode by patch clamp technique. The action of pinaverium bromide on the voltage-dependent inward current of single isolated smooth muscle cells was studied in solutions containing normal concentrations of calcium or high concentrations of barium at room temperature. 2. Pinaverium reduced the voltage-dependent inward current with an IC50 of 1.5 microM. This IC50 is similar to those of verapamil, diltiazem and flunarizine on these cells as described by others. Occasionally evidence of a potentiating action of pinaverium on the inward current was seen. 3. Repetitive stimulation of the cells did not increase blockade of inward current by pinaverium unlike the use-dependent blockade seen with verapamil, methoxyverapamil, and diltiazem in these and in other smooth muscle cells. 4. The inactivation of inward current was studied by holding at various potentials for 2 or 10 s before evoking inward current. The voltage at which current was 50% available was changed very little by pinaverium although other calcium entry blockers, for example the dihydropyridines, have been reported to produce appreciable negative shifts which indicate considerable voltage-dependence of their blockade. This may indicate that pinaverium has similar affinities for the closed available and inactivated calcium channel states so that blockade is not appreciably voltage-dependent. PMID:1691676

  20. Time to implement delayed cord clamping.

    PubMed

    McAdams, Ryan M

    2014-03-01

    Immediate umbilical cord clamping after delivery is routine in the United States despite little evidence to support this practice. Numerous trials in both term and preterm neonates have demonstrated the safety and benefit of delayed cord clamping. In premature neonates, delayed cord clamping has been shown to stabilize transitional circulation, lessening needs for inotropic medications and reducing blood transfusions, necrotizing enterocolitis, and intraventricular hemorrhage. In term neonates, delayed cord clamping has been associated with decreased iron-deficient anemia and increased iron stores with potential valuable effects that extend beyond the newborn period, including improvements in long-term neurodevelopment. The failure to more broadly implement delayed cord clamping in neonates ignores published benefits of increased placental blood transfusion at birth and may represent an unnecessary harm for vulnerable neonates.

  1. BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis

    PubMed Central

    Béguin, Pascal; Nagashima, Kazuaki; Mahalakshmi, Ramasubbu N.; Vigot, Réjan; Matsunaga, Atsuko; Miki, Takafumi; Ng, Mei Yong; Ng, Yu Jin Alvin; Lim, Chiaw Hwee; Tay, Hock Soon; Hwang, Le-Ann; Firsov, Dmitri; Tang, Bor Luen; Inagaki, Nobuya; Mori, Yasuo; Seino, Susumu

    2014-01-01

    Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca2+-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca2+ overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain–binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca2+ channel activity at the plasma membrane, resulting in the inhibition of Ca2+-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity. PMID:24751537

  2. BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

    PubMed

    Béguin, Pascal; Nagashima, Kazuaki; Mahalakshmi, Ramasubbu N; Vigot, Réjan; Matsunaga, Atsuko; Miki, Takafumi; Ng, Mei Yong; Ng, Yu Jin Alvin; Lim, Chiaw Hwee; Tay, Hock Soon; Hwang, Le-Ann; Firsov, Dmitri; Tang, Bor Luen; Inagaki, Nobuya; Mori, Yasuo; Seino, Susumu; Launey, Thomas; Hunziker, Walter

    2014-04-28

    Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

  3. Graded boosting of synaptic signals by low-threshold voltage-activated calcium conductance

    PubMed Central

    Carbó Tano, Martín; Vilarchao, María Eugenia

    2015-01-01

    Low-threshold voltage-activated calcium conductances (LT-VACCs) play a substantial role in shaping the electrophysiological attributes of neurites. We have investigated how these conductances affect synaptic integration in a premotor nonspiking (NS) neuron of the leech nervous system. These cells exhibit an extensive neuritic tree, do not fire Na+-dependent spikes, but express an LT-VACC that was sensitive to 250 μM Ni2+ and 100 μM NNC 55-0396 (NNC). NS neurons responded to excitation of mechanosensory pressure neurons with depolarizing responses for which amplitude was a linear function of the presynaptic firing frequency. NNC decreased these synaptic responses and abolished the concomitant widespread Ca2+ signals. Coherent with the interpretation that the LT-VACC amplified signals at the postsynaptic level, this conductance also amplified the responses of NS neurons to direct injection of sinusoidal current. Synaptic amplification thus is achieved via a positive feedback in which depolarizing signals activate an LT-VACC that, in turn, boosts these signals. The wide distribution of LT-VACC could support the active propagation of depolarizing signals, turning the complex NS neuritic tree into a relatively compact electrical compartment. PMID:25972583

  4. A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices.

    PubMed

    Abdelfattah, Ahmed S; Farhi, Samouil L; Zhao, Yongxin; Brinks, Daan; Zou, Peng; Ruangkittisakul, Araya; Platisa, Jelena; Pieribone, Vincent A; Ballanyi, Klaus; Cohen, Adam E; Campbell, Robert E

    2016-02-24

    Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation. PMID:26911693

  5. Voltage-Sensitive Dyes And Imaging Techniques Reveal New Patterns Of Electrical Activity In Heart Cortex

    NASA Astrophysics Data System (ADS)

    Salama, Guy

    1988-04-01

    Voltage-sensitive dyes bind to the plasms membrane of excitable cells (ie., muscle or nerve cells) and exhibit fluorescence and/or absorption changes that vary linearly with changes in transmembrane electrical potential. These potentiometric optical probes can be used to measure local changes in transmembrane potential by monitoring optical signals from dye molecules bound to the surface membrane. Consequently, when excitable cells are stained with such a dye and are stimulated to fire an electrical impulse (ie., an action potential (AP)), the changes in dye fluorescence have the characteristic shape and time course of APs recorded with an intracellular micro-electrode. Potentiometric dyes in conjuction with imaging techniques can now be used to visualize complex patterns and propagation of electrical activity. With photodiode arrays on video imaging techniques, patterns of biological electrical activity can be obtained with high temporal and spatial resolution which could not be obtained by conventional micro-electrodes. These methods reveal new details and offer powerful approaches to study fundamental problem in cardiac electrophysiology, communication in nerve networks, and the organization of cortical neurons.

  6. The monogenean which lost its clamps.

    PubMed

    Justine, Jean-Lou; Rahmouni, Chahrazed; Gey, Delphine; Schoelinck, Charlotte; Hoberg, Eric P

    2013-01-01

    Ectoparasites face a daily challenge: to remain attached to their hosts. Polyopisthocotylean monogeneans usually attach to the surface of fish gills using highly specialized structures, the sclerotized clamps. In the original description of the protomicrocotylid species Lethacotyle fijiensis, described 60 years ago, the clamps were considered to be absent but few specimens were available and this observation was later questioned. In addition, genera within the family Protomicrocotylidae have either clamps of the "gastrocotylid" or the "microcotylid" types; this puzzled systematists because these clamp types are characteristic of distinct, major groups. Discovery of another, new, species of the genus Lethacotyle, has allowed us to explore the nature of the attachment structures in protomicrocotylids. Lethacotyle vera n. sp. is described from the gills of the carangid Caranx papuensis off New Caledonia. It is distinguished from Lethacotyle fijiensis, the only other species of the genus, by the length of the male copulatory spines. Sequences of 28S rDNA were used to build a tree, in which Lethacotyle vera grouped with other protomicrocotylids. The identity of the host fish was confirmed with COI barcodes. We observed that protomicrocotylids have specialized structures associated with their attachment organ, such as lateral flaps and transverse striations, which are not known in other monogeneans. We thus hypothesized that the clamps in protomicrocotylids were sequentially lost during evolution, coinciding with the development of other attachment structures. To test the hypothesis, we calculated the surfaces of clamps and body in 120 species of gastrocotylinean monogeneans, based on published descriptions. The ratio of clamp surface: body surface was the lowest in protomicrocotylids. We conclude that clamps in protomicrocotylids are vestigial organs, and that occurrence of "gastrocotylid" and simpler "microcotylid" clamps within the same family are steps in an

  7. The Monogenean Which Lost Its Clamps

    PubMed Central

    Justine, Jean-Lou; Rahmouni, Chahrazed; Gey, Delphine; Schoelinck, Charlotte; Hoberg, Eric P.

    2013-01-01

    Ectoparasites face a daily challenge: to remain attached to their hosts. Polyopisthocotylean monogeneans usually attach to the surface of fish gills using highly specialized structures, the sclerotized clamps. In the original description of the protomicrocotylid species Lethacotyle fijiensis, described 60 years ago, the clamps were considered to be absent but few specimens were available and this observation was later questioned. In addition, genera within the family Protomicrocotylidae have either clamps of the “gastrocotylid” or the “microcotylid” types; this puzzled systematists because these clamp types are characteristic of distinct, major groups. Discovery of another, new, species of the genus Lethacotyle, has allowed us to explore the nature of the attachment structures in protomicrocotylids. Lethacotyle vera n. sp. is described from the gills of the carangid Caranx papuensis off New Caledonia. It is distinguished from Lethacotyle fijiensis, the only other species of the genus, by the length of the male copulatory spines. Sequences of 28S rDNA were used to build a tree, in which Lethacotyle vera grouped with other protomicrocotylids. The identity of the host fish was confirmed with COI barcodes. We observed that protomicrocotylids have specialized structures associated with their attachment organ, such as lateral flaps and transverse striations, which are not known in other monogeneans. We thus hypothesized that the clamps in protomicrocotylids were sequentially lost during evolution, coinciding with the development of other attachment structures. To test the hypothesis, we calculated the surfaces of clamps and body in 120 species of gastrocotylinean monogeneans, based on published descriptions. The ratio of clamp surface: body surface was the lowest in protomicrocotylids. We conclude that clamps in protomicrocotylids are vestigial organs, and that occurrence of “gastrocotylid” and simpler “microcotylid” clamps within the same family are

  8. Cardioprotective Effect of Histamine H3-Receptor Activation: Pivotal Role of Gβγ-Dependent Inhibition of Voltage-Operated Ca2+ Channels

    PubMed Central

    Morrey, Christopher; Estephan, Rima; Abbott, Geoffrey W.; Levi, Roberto

    2008-01-01

    We previously showed that activation of Gi/o-coupled histamine H3-receptors (H3R) is cardioprotective since it attenuates excessive norepinephrine release from cardiac sympathetic nerves. This action is characterized by a marked decrease in intraneuronal Ca2+ ([Ca2+]i), as Gαi impairs the adenylyl cyclase-cAMP-PKA pathway, and this decreases Ca2+ influx via voltage-operated Ca2+ channels (VOCC). Yet, the Gi/o-derived βγ dimer could directly inhibit VOCC, and the subsequent reduction in Ca2+ influx would be responsible for the H3R-mediated attenuation of transmitter exocytosis. Here, we tested this hypothesis in nerve-growth factor-differentiated rat pheochromocytoma cells (PC12) stably transfected with H3R (PC12-H3) and with the Gβγ scavenger β-ARK1-(495−689)-polypeptide (PC12-H3/β-ARK1). Thus, we evaluated the effects of H3R activation directly on: 1) Ca2+ current (ICa) using the whole-cell patch-clamp technique, and 2) K+-induced exocytosis of endogenous dopamine. H3R activation attenuated both peak ICa and dopamine exocytosis in PC12-H3, but not in PC12-H3/β-ARK1 cells. Moreover, a membrane permeable phosducin-like Gβγ scavenger also prevented the anti-exocytotic effect of H3R activation. In contrast, the H3R-induced attenuation of cAMP accumulation and dopamine exocytosis in response to forskolin were the same in both PC12-H3 and PC12-H3/β-ARK1 cells. Our findings reveal that while Gαi participates in the H3-mediated anti-exocytotic effect when the adenylyl cyclase-cAMP-PKA pathway is stimulated, a direct Gβγ-induced inhibition of VOCC, resulting in an attenuation of ICa plays a pivotal role in the H3R-mediated decrease in [Ca2+]i and associated cardioprotective anti-exocytotic effects. The discovery of this H3R signaling step may offer new therapeutic approaches to cardiovascular diseases characterized by hyperadrenergic activity. PMID:18523159

  9. A comparison of the performance and application differences between manual and automated patch-clamp techniques.

    PubMed

    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

  10. Using atomic force microscopy to investigate patch-clamped nuclear membrane.

    PubMed

    Danker, T; Mazzanti, M; Tonini, R; Rakowska, A; Oberleithner, H

    1997-11-01

    Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure-function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still nuclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3 nm and 0.1 nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments. PMID:9768473

  11. 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

  12. Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels.

    PubMed

    Corzo, Gerardo; Sabo, Jennifer K; Bosmans, Frank; Billen, Bert; Villegas, Elba; Tytgat, Jan; Norton, Raymond S

    2007-02-16

    Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion beta-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded beta-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion beta-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

  13. Active Power Rescheduling for Avoiding Voltage Collapse Using Modified Bare Bones Particle Swarm Optimization

    NASA Astrophysics Data System (ADS)

    Arya, Rajesh; Purey, Pradeep

    2016-06-01

    MW-generation rescheduling is being considered for voltage stability improvement under stressed operating condition. At times it can avoid voltage collapse. This paper describes an algorithm for determination of optimum MW-generation participation pattern for static voltage stability margin enhancement. The optimum search direction has been obtained by employing modified bare born particle swarm optimization technique. Optimum search direction is based on maximization of distance to point of collapse in generation space. Developed algorithm has been implemented on a standard 25 bus test system. Results obtained have been compared with those obtained using standard particle swarm optimization.

  14. An ultra-low-voltage electronic implementation of inertial neuron model with nonmonotonous Liao's activation function.

    PubMed

    Kant, Nasir Ali; Dar, Mohamad Rafiq; Khanday, Farooq Ahmad

    2015-01-01

    The output of every neuron in neural network is specified by the employed activation function (AF) and therefore forms the heart of neural networks. As far as the design of artificial neural networks (ANNs) is concerned, hardware approach is preferred over software one because it promises the full utilization of the application potential of ANNs. Therefore, besides some arithmetic blocks, designing AF in hardware is the most important for designing ANN. While attempting to design the AF in hardware, the designs should be compatible with the modern Very Large Scale Integration (VLSI) design techniques. In this regard, the implemented designs should: only be in Metal Oxide Semiconductor (MOS) technology in order to be compatible with the digital designs, provide electronic tunability feature, and be able to operate at ultra-low voltage. Companding is one of the promising circuit design techniques for achieving these goals. In this paper, 0.5 V design of Liao's AF using sinh-domain technique is introduced. Furthermore, the function is tested by implementing inertial neuron model. The performance of the AF and inertial neuron model have been evaluated through simulation results, using the PSPICE software with the MOS transistor models provided by the 0.18-μm Taiwan Semiconductor Manufacturer Complementary Metal Oxide Semiconductor (TSM CMOS) process. PMID:27030660

  15. Voltage- and calcium-dependent motility of saccular hair bundles

    NASA Astrophysics Data System (ADS)

    Quiñones, Patricia M.; Meenderink, Sebastiaan W. F.; Bozovic, Dolores

    2015-12-01

    Active bundle motility, which is hypothesized to supply feedback for mechanical amplification of signals, is thought to enhance sensitivity and sharpen tuning in vestibular and auditory organs. To study active hair bundle motility, we combined high-speed camera recordings of bullfrog sacculi, which were mounted in a two-compartment chamber, and voltage-clamp of the hair cell membrane potential. Using this paradigm, we measured three types of bundle motions: 1) spontaneous oscillations which can be analyzed to measure the physiological operating range of the transduction channel; 2) a sustained quasi-static movement of the bundle that depends on membrane potential; and 3) a fast, transient and asymmetric movement that resets the bundle position and depends on changes in the membrane potential. These data support a role for both calcium and voltage in the transduction-channel function.

  16. Vibration control of a flexible clamped-clamped plate based on an improved FULMS algorithm and laser displacement measurement

    NASA Astrophysics Data System (ADS)

    Xie, Lingbo; Qiu, Zhi-cheng; Zhang, Xian-min

    2016-06-01

    This paper presents a novel active resonant vibration control experiment of a flexible clamped-clamped plate using an improved filtered-U least mean square (FULMS) algorithm and laser displacement measurement. Different from the widely used PZT sensors or acceleration transducers, the vibration of the flexible clamped-clamped plate is measured by a non-contact laser displacement measurement sensor with higher measurement accuracy and without additional load to the plate. The conventional FULMS algorithm often uses fixed step size and needs reference signal related to the external disturbance signal. However, the fixed step size method cannot obtain a fast convergence speed and it will result in a low residual error. Thus, a variable step size method is investigated. In addition, it is difficult to extract reference signal related to the vibration source directly in the practical application. Therefore, it is practically useful that a reference signal is constructed by both the controller parameters and the vibration residual signal. The experimental results demonstrate that the improved FULMS algorithm has better vibration control effect than the proportional derivative (PD) feedback control algorithm and the fixed step-size control algorithm.

  17. Active voltage contrast imaging of cross-sectional surface of multilayer ceramic capacitor using helium ion microscopy

    NASA Astrophysics Data System (ADS)

    Sakai, C.; Ishida, N.; Masuda, H.; Nagano, S.; Kitahara, M.; Ogata, Y.; Fujita, D.

    2016-08-01

    We studied active voltage contrast (AVC) imaging using helium ion microscopy (HIM). We observed secondary electron (SE) images of the cross-sectional surface of multilayer ceramic capacitors (MLCCs) with and without a voltage applied to the internal electrodes. When no voltage was applied, we obtained an image reflecting the material contrast between the Ni internal electrode region and the BaTiO3 dielectric region of the cross-sectional surface of the MLCC. When a voltage was applied, the electrical potential difference between the grounded and the positively biased internal electrodes affected the contrast (voltage contrast). Moreover, attenuation of the SE intensity from the grounded to the positively biased internal electrodes was observed in the dielectric region. Kelvin probe force microscopy (KPFM) measurements of the contact potential difference (CPD) were performed on the same sample. By using the AVC image from the HIM observation and the CPD image from the KPFM measurement, we could quantitatively evaluate the electrical potential. We think that the results of this study will lead to an expansion in the number of applications of HIM.

  18. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  19. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    NASA Astrophysics Data System (ADS)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  20. Organ protection during aortic cross-clamping.

    PubMed

    Yeung, Kak Khee; Groeneveld, Menno; Lu, Joyce Ja-Ning; van Diemen, Pepijn; Jongkind, Vincent; Wisselink, Willem

    2016-09-01

    Open surgical repair of an aortic aneurysm requires aortic cross-clamping, resulting in temporary ischemia of all organs and tissues supplied by the aorta distal to the clamp. Major complications of open aneurysm repair due to aortic cross-clamping include renal ischemia-reperfusion injury and postoperative colonic ischemia in case of supra- and infrarenal aortic aneurysm repair. Ischemia-reperfusion injury results in excessive production of reactive oxygen species and in oxidative stress, which can lead to multiple organ failure. Several perioperative protective strategies have been suggested to preserve renal function during aortic cross-clamping, such as pharmacotherapy and therapeutic hypothermia of the kidneys. In this chapter, we will briefly discuss the pathophysiology of ischemia-reperfusion injury and the preventative measures that can be taken to avoid abdominal organ injury. Finally, techniques to minimize the risk of complications during and after open aneurysm repair will be presented. PMID:27650341

  1. Activation of conventional kinesin motors in clusters by Shaw voltage-gated K+ channels

    PubMed Central

    Barry, Joshua; Xu, Mingxuan; Gu, Yuanzheng; Dangel, Andrew W.; Jukkola, Peter; Shrestha, Chandra; Gu, Chen

    2013-01-01

    Summary The conventional kinesin motor transports many different cargos to specific locations in neurons. How cargos regulate motor function remains unclear. Here we focus on KIF5, the heavy chain of conventional kinesin, and report that the Kv3 (Shaw) voltage-gated K+ channel, the only known tetrameric KIF5-binding protein, clusters and activates KIF5 motors during axonal transport. Endogenous KIF5 often forms clusters along axons, suggesting a potential role of KIF5-binding proteins. Our biochemical assays reveal that the high-affinity multimeric binding between the Kv3.1 T1 domain and KIF5B requires three basic residues in the KIF5B tail. Kv3.1 T1 competes with the motor domain and microtubules, but not with kinesin light chain 1 (KLC1), for binding to the KIF5B tail. Live-cell imaging assays show that four KIF5-binding proteins, Kv3.1, KLC1 and two synaptic proteins SNAP25 and VAMP2, differ in how they regulate KIF5B distribution. Only Kv3.1 markedly increases the frequency and number of KIF5B-YFP anterograde puncta. Deletion of Kv3.1 channels reduces KIF5 clusters in mouse cerebellar neurons. Therefore, clustering and activation of KIF5 motors by Kv3 regulate the motor number in carrier vesicles containing the channel proteins, contributing not only to the specificity of Kv3 channel transport, but also to the cargo-mediated regulation of motor function. PMID:23487040

  2. Structural analysis of a eukaryotic sliding DNA clamp-clamp loadercomplex.

    SciTech Connect

    Bowman, Gregory D.; O'Donnell, Mike; Kuriyan, John

    2006-06-17

    Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-?-S by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.

  3. Mir environmental effects payload handrail clamp/pointing device

    NASA Technical Reports Server (NTRS)

    Hughes, Stephen J.

    1996-01-01

    The Mir Environmental Effects Payload (MEEP) consists of four International Space Station Alpha (ISSA) Risk mitigation experiments to be transported and deployed in a common carrier. This carrier is to be transported to the Mir Space Station aboard the Space Shuttle and deployed during a US Extravehicular Activity (EVA) on the handrails of the Mir Docking Module (DM). This paper describes the design of the handrail clamp/ pointing device used by the astronauts to attach the carrier to the station.

  4. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  5. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  6. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  7. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures....

  8. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Cable clamps and grips. 18.40 Section 18.40... Requirements § 18.40 Cable clamps and grips. Insulated clamps shall be provided for all portable (trailing) cables to prevent strain on the cable terminals of a machine. Also insulated clamps shall be provided...

  9. 33 CFR 183.532 - Clips, straps, and hose clamps.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Clips, straps, and hose clamps..., straps, and hose clamps. (a) Each clip, strap, and hose clamp must: (1) Be made from a corrosion... under § 183.590, a hose clamp installed on a fuel line system requiring metallic fuel lines or...

  10. 33 CFR 183.532 - Clips, straps, and hose clamps.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Clips, straps, and hose clamps..., straps, and hose clamps. (a) Each clip, strap, and hose clamp must: (1) Be made from a corrosion... under § 183.590, a hose clamp installed on a fuel line system requiring metallic fuel lines or...

  11. 33 CFR 183.532 - Clips, straps, and hose clamps.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Clips, straps, and hose clamps..., straps, and hose clamps. (a) Each clip, strap, and hose clamp must: (1) Be made from a corrosion... under § 183.590, a hose clamp installed on a fuel line system requiring metallic fuel lines or...

  12. 33 CFR 183.532 - Clips, straps, and hose clamps.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Clips, straps, and hose clamps..., straps, and hose clamps. (a) Each clip, strap, and hose clamp must: (1) Be made from a corrosion... under § 183.590, a hose clamp installed on a fuel line system requiring metallic fuel lines or...

  13. 33 CFR 183.532 - Clips, straps, and hose clamps.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Clips, straps, and hose clamps..., straps, and hose clamps. (a) Each clip, strap, and hose clamp must: (1) Be made from a corrosion... under § 183.590, a hose clamp installed on a fuel line system requiring metallic fuel lines or...

  14. Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions.

    PubMed Central

    Church, J; Fletcher, E J; Baxter, K; MacDonald, J F

    1994-01-01

    1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7834201

  15. Novel description of ionic currents recorded with the action potential clamp technique: application to excitatory currents in suprachiasmatic nucleus neurons.

    PubMed

    Clay, John R

    2015-07-01

    The traditional method of recording ionic currents in neurons has been with voltage-clamp steps. Other waveforms such as action potentials (APs) can be used. The AP clamp method reveals contributions of ionic currents that underlie excitability during an AP (Bean BP. Nat Rev Neurosci 8: 451-465, 2007). A novel usage of the method is described in this report. An experimental recording of an AP from the literature is digitized and applied computationally to models of ionic currents. These results are compared with experimental AP-clamp recordings for model verification or, if need be, alterations to the model. The method is applied to the tetrodotoxin-sensitive sodium ion current, INa, and the calcium ion current, ICa, from suprachiasmatic nucleus (SCN) neurons (Jackson AC, Yao GL, Bean BP. J Neurosci 24: 7985-7998, 2004). The latter group reported voltage-step and AP-clamp results for both components. A model of INa is constructed from their voltage-step results. The AP clamp computational methodology applied to that model compares favorably with experiment, other than a modest discrepancy close to the peak of the AP that has not yet been resolved. A model of ICa was constructed from both voltage-step and AP-clamp results of this component. The model employs the Goldman-Hodgkin-Katz equation for the current-voltage relation rather than the traditional linear dependence of this aspect of the model on the Ca(2+) driving force. The long-term goal of this work is a mathematical model of the SCN AP. The method is general. It can be applied to any excitable cell.

  16. An Ultrasonic Clamp for Bloodless Partial Nephrectomy

    NASA Astrophysics Data System (ADS)

    Lafon, Cyril; Bouchoux, Guillaume; Murat, François Joseph; Birer, Alain; Theillère, Yves; Chapelon, Jean Yves; Cathignol, Dominique

    2007-05-01

    Maximum conservation of the kidney is preferable through partial nephrectomy for patients at risk of disease recurrence of renal cancers. Haemostatic tools are needed in order to achieve bloodless surgery and reduce post surgery morbidity. Two piezo-ceramic transducers operating at a frequency of 4 MHz were mounted on each arm of a clamp. When used for coagulation purposes, two transducers situated on opposite arms of the clamp were driven simultaneously. Heat delivery was optimized as each transducers mirrored back to targeted tissues the wave generated by the opposite transducer. Real-time treatment monitoring with an echo-based technique was also envisaged with this clamp. Therapy was periodically interrupted so one transducer could generate a pulse. The echo returning from the opposite transducer was treated. Coagulation necroses were obtained in vitro on substantial thicknesses (23-38mm) of pig liver over exposure durations ranging from 30s to 130s, and with acoustic intensities of less than 15W/cm2 per transducer. Both kidneys of two pigs were treated in vivo with the clamp (14.5W/cm2 for 90s), and the partial nephrectomies performed proved to be bloodless. In vitro and in vivo, wide transfixing lesions corresponded to an echo energy decrease superior to -10dB and parabolic form of the time of flight versus treatment time. In conclusion, this ultrasound clamp has proven to be an excellent mean for achieving monitored haemostasis in kidney.

  17. Force-clamp laser trapping of rapidly interacting molecules

    NASA Astrophysics Data System (ADS)

    Capitanio, Marco; Monico, Carina; Vanzi, Francesco; Pavone, Francesco S.

    2013-06-01

    Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay of only ˜10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  18. E-beam high voltage switching power supply

    DOEpatents

    Shimer, Daniel W.; Lange, Arnold C.

    1997-01-01

    A high power, solid state power supply is described for producing a controllable, constant high voltage output under varying and arcing loads suitable for powering an electron beam gun or other ion source. The present power supply is most useful for outputs in a range of about 100-400 kW or more. The power supply is comprised of a plurality of discrete switching type dc-dc converter modules, each comprising 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, and an output rectifier for producing a dc voltage at the output of each module. The inputs to the converter modules are fed from a common dc rectifier/filter and are linked together in parallel through decoupling networks to suppress high frequency input interactions. The outputs of the converter modules are linked together in series and connected to the input of the transmission line to the load through a decoupling and line matching network. The dc-dc converter modules are phase activated such that for n modules, each module is activated equally 360.degree./n out of phase with respect to a successive module. The phased activation of the converter modules, combined with the square current waveforms out of the step up transformers, allows the power supply to operate with greatly reduced output capacitance values which minimizes the stored energy available for discharge into an electron beam gun or the like during arcing. The present power supply also provides dynamic response to varying loads by controlling the voltage regulator duty cycle using simulated voltage feedback signals and voltage feedback loops. Circuitry is also provided for sensing incipient arc currents reflected at the output of the power supply and for simultaneously decoupling the power supply circuitry from the arcing load.

  19. E-beam high voltage switching power supply

    DOEpatents

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

    1997-03-11

    A high power, solid state power supply is described for producing a controllable, constant high voltage output under varying and arcing loads suitable for powering an electron beam gun or other ion source. The present power supply is most useful for outputs in a range of about 100-400 kW or more. The power supply is comprised of a plurality of discrete switching type dc-dc converter modules, each comprising 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, and an output rectifier for producing a dc voltage at the output of each module. The inputs to the converter modules are fed from a common dc rectifier/filter and are linked together in parallel through decoupling networks to suppress high frequency input interactions. The outputs of the converter modules are linked together in series and connected to the input of the transmission line to the load through a decoupling and line matching network. The dc-dc converter modules are phase activated such that for n modules, each module is activated equally 360{degree}/n out of phase with respect to a successive module. The phased activation of the converter modules, combined with the square current waveforms out of the step up transformers, allows the power supply to operate with greatly reduced output capacitance values which minimizes the stored energy available for discharge into an electron beam gun or the like during arcing. The present power supply also provides dynamic response to varying loads by controlling the voltage regulator duty cycle using simulated voltage feedback signals and voltage feedback loops. Circuitry is also provided for sensing incipient arc currents reflected at the output of the power supply and for simultaneously decoupling the power supply circuitry from the arcing load. 7 figs.

  20. Voltage-sensitive potassium channels in Limulus ventral photoreceptors

    PubMed Central

    1978-01-01

    The steady-state slope conductance of Limulus ventral photoreceptors increases markedly when the membrane is depolarized from rest. The ionic basis of this rectification has been examined with a voltage- clamp technique. Tail currents that occur when membrane potential is repolarized after having been depolarized have been identified. The tail currents reverse direction at a voltage that becomes more positive when Ko is increased. Rectification is reduced by extracellular 4- aminopyridine and by intracellular injection of tetra-ethyl-ammonium (TEA). These results indicate that the membrane rectification around resting potential is due primarily to voltage-sensitive K+ channels. The increase in gK caused by depolarization is not mediated by a voltage-dependent rise in in Cai++, since intracellular injection of Ca++ causes a decrease rather than an increase in slope conductance. TEA can be used to examine the functional role of the K+ channels because it blocks them without substantially affecting the light- activated Na+ conductance. The effect of TEA on response-intensity curves shows that the K+ channels serve to compress the voltage range of receptor potentials. PMID:621492

  1. Clamping instability and van der Waals forces in carbon nanotube mechanical resonators.

    PubMed

    Aykol, Mehmet; Hou, Bingya; Dhall, Rohan; Chang, Shun-Wen; Branham, William; Qiu, Jing; Cronin, Stephen B

    2014-05-14

    We investigate the role of weak clamping forces, typically assumed to be infinite, in carbon nanotube mechanical resonators. Due to these forces, we observe a hysteretic clamping and unclamping of the nanotube device that results in a discrete drop in the mechanical resonance frequency on the order of 5-20 MHz, when the temperature is cycled between 340 and 375 K. This instability in the resonant frequency results from the nanotube unpinning from the electrode/trench sidewall where it is bound weakly by van der Waals forces. Interestingly, this unpinning does not affect the Q-factor of the resonance, since the clamping is still governed by van der Waals forces above and below the unpinning. For a 1 μm device, the drop observed in resonance frequency corresponds to a change in nanotube length of approximately 50-65 nm. On the basis of these findings, we introduce a new model, which includes a finite tension around zero gate voltage due to van der Waals forces and shows better agreement with the experimental data than the perfect clamping model. From the gate dependence of the mechanical resonance frequency, we extract the van der Waals clamping force to be 1.8 pN. The mechanical resonance frequency exhibits a striking temperature dependence below 200 K attributed to a temperature-dependent slack arising from the competition between the van der Waals force and the thermal fluctuations in the suspended nanotube. PMID:24758201

  2. Planar silicon patch-clamp electrodes integrated with polydimethylsiloxane microfluidics

    NASA Astrophysics Data System (ADS)

    Nagarah, John Michael

    The patch-clamp technique allows one to probe single ion channels and macroscopic ion channel activity in their native environment and resolve their activity as their physical and chemical surroundings are varied. The traditional method of patch-clamping cells involves bringing a clean, flame-polished glass pipette tip with a 1-2 mum diameter pore into contact with a cell membrane to form a high electrical resistance seal. This technique is the gold standard for cellular electrophysiology investigations because it allows the observation of single ion channel protein dynamics as well as activity from an ensemble ion channels from a single cell. Furthermore, any drug approved by federal drug agencies must be screened against particular ion channels with the patch-clamp technique. However, this technique by its nature is serial, time consuming, difficult when exchanging pipette solutions, and difficult to integrate with other technologies. These reasons have prompted several investigators to explore alternative approaches to traditional pipette patch-clamping to increase the throughput of measurements. Herein, I describe the development of a silicon-wafer based device platform that enables the measurement of ion channel activities. The electrical nature of the cell/wafer seal is characterized for several pore design variations. The majority of gigaohm seals obtained falls in the range of 10-20GO. The cell-attached and whole cell configurations are demonstrated. Whole cell ion channel activity originating from various cell fines is consistent with the more traditional micropipette patch-clamp recordings. The silicon fabrication methods developed, although novel, utilize established semiconductor technologies, making them amenable to batch fabrication techniques. I integrate these silicon devices with PDMS microfluidics with monolithic valves, allowing ultra-fast solution exchange as low as tens of milliseconds for the extracellular solution. Furthermore, I developed a

  3. Fiber optic accelerometer based on clamped beam

    NASA Astrophysics Data System (ADS)

    Zhang, Wentao; Li, Fang

    2013-01-01

    In this paper a fiber optic accelerometer (FOA) based on camped beam is proposed. The clamped beam is used as the elastic element and a mass installed on the clamped beam is used as the inertial element. The accelerometer is based on a fiber optic Michelson interferometer and has a sensing arm and a reference arm. The optical fiber of the sensing arm is wrapped on the clamped beam and the mass, which are both cylinder shaped. The sensitivity of the FOA is analyzed based on the theory of elasticity; the frequency response is analyzed based on the theory of vibration. Experiment is carried out to test the performance of the fiber optic accelerometer. The experiment results show a high sensitivity and a flat frequency response within the low frequency range of 5-250 Hz, which agrees well with the theoretical result.

  4. Gating Kinetics of the Cyclic-GMP-Activated Channel of Retinal Rods: Flash Photolysis and Voltage-Jump Studies

    NASA Astrophysics Data System (ADS)

    Karpen, Jeffrey W.; Zimmerman, Anita L.; Stryer, Lubert; Baylor, Denis A.

    1988-02-01

    The gating kinetics of the cGMP-activated cation channel of salamander retinal rods have been studied in excised membrane patches. Relaxations in patch current were observed after two kinds of perturbation: (i) fast jumps of cGMP concentration, generated by laser flash photolysis of a cGMP ester (``caged'' cGMP), and (ii) membrane voltage jumps, which perturb activation of the channel by cGMP. In both methods the speed of activation increased with the final cGMP concentration. The results are explained by a simple kinetic model in which activation involves three sequential cGMP binding steps with bimolecular rate constants close to the diffusion-controlled limit; fully liganded channels undergo rapid open-closed transitions. Voltage perturbs activation by changing the rate constant for channel closing, which increases with hyperpolarization. Intramolecular transitions of the fully liganded channel limit the kinetics of activation at high cGMP concentrations (>50 μ M), whereas at physiological cGMP concentrations (<5 μ M), the kinetics of activation are limited by the third cGMP binding step. The channel appears to be optimized for rapid responses to changes in cytoplasmic cGMP concentration.

  5. Threshold-Voltage-Shift Compensation and Suppression Method Using Hydrogenated Amorphous Silicon Thin-Film Transistors for Large Active Matrix Organic Light-Emitting Diode Displays

    NASA Astrophysics Data System (ADS)

    Oh, Kyonghwan; Kwon, Oh-Kyong

    2012-03-01

    A threshold-voltage-shift compensation and suppression method for active matrix organic light-emitting diode (AMOLED) displays fabricated using a hydrogenated amorphous silicon thin-film transistor (TFT) backplane is proposed. The proposed method compensates for the threshold voltage variation of TFTs due to different threshold voltage shifts during emission time and extends the lifetime of the AMOLED panel. Measurement results show that the error range of emission current is from -1.1 to +1.7% when the threshold voltage of TFTs varies from 1.2 to 3.0 V.

  6. Modulation of Exciton Generation in Organic Active Planar pn Heterojunction: Toward Low Driving Voltage and High-Efficiency OLEDs Employing Conventional and Thermally Activated Delayed Fluorescent Emitters.

    PubMed

    Chen, Dongcheng; Liu, Kunkun; Gan, Lin; Liu, Ming; Gao, Kuo; Xie, Gaozhan; Ma, Yuguang; Cao, Yong; Su, Shi-Jian

    2016-08-01

    Organic light-emitting diodes (OLEDs) combining low driving voltage and high efficiency are designed by employing conventional and thermally activated delayed fluorescence emitters through modulation of excitons generated at the planar p-n heterojunction region. To date, this approach enables the highest power efficiency for yellow-green emitting fluorescent OLEDs with a simplified structure.

  7. Perspectives on implementing delayed cord clamping.

    PubMed

    Leslie, Mayri Sagady

    2015-01-01

    Expanding evidence supports delayed cord clamping (DCC) for both term and preterm infants. This article explores issues that may be keeping early cord clamping (ECC) in place as usual practice. Professional organizations almost universally recommend DCC for preterm infants, but some reserve recommending it for term infants only in resource-poor settings. Concerns about polycythemia and jaundice persist in the literature, while years of published randomized controlled trials do not support the assumptions behind the concerns. New data suggest that DCC may improve resuscitative efforts in compromised infants. Multiple perspectives are offered for consideration when thinking about incorporating DCC into practice.

  8. Ion channels activated by light in Limulus ventral photoreceptors

    PubMed Central

    1986-01-01

    The light-activated conductance of Limulus ventral photoreceptors was studied using the patch-clamp technique. Channels (40 pS) were observed whose probability of opening was greatly increased by light. In some cells the latency of channel activation was nearly the same as that of the macroscopic response, while in other cells the channel latency was much greater. Like the macroscopic conductance, channel activity was reduced by light adaptation but enhanced by the intracellular injection of the calcium chelator EGTA. The latter observation indicates that channel activation was not a secondary result of the light-induced rise in intracellular calcium. A two-microelectrode voltage-clamp method was used to measure the voltage dependence of the light-activated macroscopic conductance. It was found that this conductance is constant over a wide voltage range more negative than zero, but it increases markedly at positive voltages. The single channel currents measured over this same voltage range show that the single channel conductance is independent of voltage, but that channel gating properties are dependent on voltage. Both the mean channel open time and the opening rate increase at positive voltages. These properties change in a manner consistent with the voltage dependence of the macroscopic conductance. The broad range of similarities between the macroscopic and single channel currents supports the conclusion that the 40-pS channel that we have observed is the principal channel underlying the response to light in these photoreceptors. PMID:2419481

  9. Active medical implants and occupational safety--measurement and numerical calculation of interference voltage.

    PubMed

    Gustrau, F; Bahr, A; Goltz, S; Eggert, S

    2002-01-01

    Low frequency electric and magnetic fields may interfere with implanted cardiac pacemakers causing a life-threatening malfunction of the device. In order to assess the safety of workers in the vicinity of industrial electrical devices the interference voltage at the input port of a pacemaker is an important measure. In order to investigate the coupling of fields emanating from electrical devices a numerical method for the calculation of interference voltages is presented and applied to the investigation of homogeneous electric and magnetic fields in the frequency range from 50 Hz to 1 MHz. Implantation of the pacemaker in the right pectoral, left pectoral and abdominal area using a realistic model of the human body as well as different grounding conditions are considered. The numerical method is successfully validated by measurements and shows good agreement with results in the literature.

  10. Multiphosphine-Oxide Hosts for Ultralow-Voltage-Driven True-Blue Thermally Activated Delayed Fluorescence Diodes with External Quantum Efficiency beyond 20.

    PubMed

    Zhang, Jing; Ding, Dongxue; Wei, Ying; Han, Fuquan; Xu, Hui; Huang, Wei

    2016-01-20

    Highly efficient low-voltage-driven -true-blue thermally activated -delayed fluorescence diodes are realized through employing a tri-phosphine oxide host (2,2',4-tris(di(phenyl) -phosphoryl)-diphenylether (DPETPO)) with a record external quantum efficiency of 23.0% and the lowest onset voltage of 2.8 V to date.

  11. Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

    PubMed Central

    Marzahn, Melissa R.; Hayner, Jaclyn N.; Meyer, Jennifer A.; Bloom, Linda B.

    2014-01-01

    DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+ family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the S. cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the β-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway. PMID:25450506

  12. Application of low-voltage field-emission SEM to the study of internal pore structures of activated carbon

    SciTech Connect

    Liu, J.; Ornberg, R.L.

    1996-12-31

    Activated carbon has interesting and useful properties for industrial applications. It has been used extensively in purification, separation, chemical recovery and catalysis. To achieve a predictable performance of activated carbon materials, it is necessary to develop a comprehensive understanding of the pore structure including pore size, pore shape, and pore surface chemistry. Macropores (> 50 nm), mesopores (2-50 nm) and micropores (< 2 nm) generally coexist in activated carbon. It is thus desirable to synthesize activated carbon with controlled pore structures to optimize its performance. We previously reported the characterization of the surface pore structure of activated carbon by field emission SEM (FESEM) and the examination of the internal pore structure by HAADF/HRTEM techniques. However, both HAADF and HRTEM techniques give only limited information about the carbon pore structure. We report here some preliminary observation of the internal pore structure of activated carbon by high resolution low voltage FESEM technique.

  13. The sliding clamp tethers the endonuclease domain of MutL to DNA

    PubMed Central

    Pillon, Monica C.; Babu, Vignesh M. P.; Randall, Justin R.; Cai, Jiudou; Simmons, Lyle A.; Sutton, Mark D.; Guarné, Alba

    2015-01-01

    The sliding clamp enhances polymerase processivity and coordinates DNA replication with other critical DNA processing events including translesion synthesis, Okazaki fragment maturation and DNA repair. The relative binding affinity of the sliding clamp for its partners determines how these processes are orchestrated and is essential to ensure the correct processing of newly replicated DNA. However, while stable clamp interactions have been extensively studied; dynamic interactions mediated by the sliding clamp remain poorly understood. Here, we characterize the interaction between the bacterial sliding clamp (β-clamp) and one of its weak-binding partners, the DNA mismatch repair protein MutL. Disruption of this interaction causes a mild mutator phenotype in Escherichia coli, but completely abrogates mismatch repair activity in Bacillus subtilis. We stabilize the MutL-β interaction by engineering two cysteine residues at variable positions of the interface. Using disulfide bridge crosslinking, we have stabilized the E. coli and B. subtilis MutL-β complexes and have characterized their structures using small angle X-ray scattering. We find that the MutL-β interaction greatly stimulates the endonuclease activity of B. subtilis MutL and supports this activity even in the absence of the N-terminal region of the protein. PMID:26384423

  14. A monitor and control system for high voltage, gating, and triggering of a scintillating fiber active target

    SciTech Connect

    Baumbaugh, B.; Bishop, J.; Gardner, R.W.; Mountain, R.J.; Ruchti, R.; Baumbaugh, A.; Knickerbocker, K.

    1987-10-01

    A monitor and control system has been designed, constructed and tested at Notre Dame for the purpose of controlling all aspects of a Scintillating Fiber Active Target system used in High Energy Physics Experimentation. The SFT Active Target system requires control of high voltages, gating, trigger counters, and monitoring. In addition, it resides in a radioactive area with very limited access. The control system uses a Leading Edge microcomputer, two specialized Z80-based processors, associated DACs, ADCs, discrete semiconductors, linear ICs, and TTL and MECL logic. All of the hardware and software is custom-built; its design and performance is discussed. 5 refs., 4 figs.

  15. Functional heterogeneity of the four voltage sensors of a human L-type calcium channel

    PubMed Central

    Pantazis, Antonios; Savalli, Nicoletta; Sigg, Daniel; Neely, Alan; Olcese, Riccardo

    2014-01-01

    Excitation-evoked Ca2+ influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca2+ entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I–IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening. PMID:25489110

  16. Structure of a Small-Molecule Inhibitor of a DNA Polymerase Sliding Clamp

    SciTech Connect

    Georgescu, R.; Yurieva, O; Kim, S; Kuriyan, J; Kong, X; O'Donnell, M

    2008-01-01

    DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli ?-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with ? and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to ?. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with ?, and that the ?-clamp may represent an antibiotic target.

  17. Self-tuning behavior of a clamped-clamped beam with sliding proof mass for broadband energy harvesting

    NASA Astrophysics Data System (ADS)

    Pillatsch, P.; Miller, L. M.; Halvorsen, E.; Wright, P. K.; Yeatman, E. M.; Holmes, A. S.

    2013-12-01

    Real world systems rarely vibrate at a single resonance frequency and the frequencies drift over time. Tunable devices exist, but generally need additional energy to achieve frequency adaptation. This means that the benefits in power output from this tuning need to be large enough to power the mechanism itself. Passively self-tuning systems go into resonance without requiring active control. This paper focuses on a passively self-tuning system with a proof mass that can slide freely along a clamped-clamped beam. Under external vibration, the slider moves along the beam until the system goes into resonance. A proof-of-concept design is introduced using either a copper or a steel beam and a 3D-printed ABS thermoplastic proof mass. Successful self-tuning is demonstrated in both cases. The frequencies range from 80 - 140 Hz at accelerations as low as 0.007 g rms. Results show the resonance of the beam and the position of the slider along the beam with time. Furthermore, the dynamic magnification and the proof mass position at resonance are discussed, together with the inherent non-linearities of double-clamped beam resonators. The findings support the hypothesis that the effect of the ratio between proof mass and beam mass outweighs the Duffing spring stiffening effects.

  18. Patch-clamp amplifiers on a chip.

    PubMed

    Weerakoon, Pujitha; Culurciello, Eugenio; Yang, Youshan; Santos-Sacchi, Joseph; Kindlmann, Peter J; Sigworth, Fred J

    2010-10-15

    We present the first, fully integrated, two-channel implementation of a patch-clamp measurement system. With this "PatchChip" two simultaneous whole-cell recordings can be obtained with rms noise of 8pA in a 10kHz bandwidth. The capacitance and series-resistance of the electrode can be compensated up to 10pF and 100MΩ respectively under computer control. Recordings of hERG and Na(v) 1.7 currents demonstrate the system's capabilities, which are on par with large, commercial patch-clamp instrumentation. By reducing patch-clamp amplifiers to a millimeter size micro-chip, this work paves the way to the realization of massively parallel, high-throughput patch-clamp systems for drug screening and ion-channel research. The PatchChip is implemented in a 0.5μm silicon-on-sapphire process; its size is 3×3mm(2) and the power consumption is 5mW per channel with a 3.3V power supply.

  19. [Spinal cord ischemia following subrenal aortic clamping].

    PubMed

    Battisti, G; Marigliani, M; Stio, F; Iavarone, C

    1990-01-01

    The paraplegia caused by an aortic clamping just below the Renal artery is a rare but very complication in aortic surgery. Such a complication is even rarer if we consider the few cases reported in literature following a reconstructive surgery for occlusive chronic diseases of aortiliac axes. The authors have studied the case of a patient bearing the syndrome of Leriche; this one had an aortic clamping below the kidney and soon after developed an acute ischaemic syndrome below the spinal medulla with flaccid paraparesis, anal and vesical sphincteric diseases and persistence of deep tactile sensibility. After a reconstruction of vascular anatomy of the medulla they emphasize the importance, in such a disease, of the "arteria radicularis magna" of Adamkievicz and its place of origin. After they discuss the severe physioopathologic moments that are connected: with the direct ischaemia following aortic clamping in the cases where the arteria radicularis magna rises at a level lower than the clamping itself; with the embolism or thrombosis caused by surgical manipulation peroperatively (it might be the cause of paraplegia more frequent in aneurysmectomia surgery); with the severe hypotension per- and post operatively for the existence of arteriosclerotic disease of the lumbar arteries. Finally they analyses preoperatively diagnostic possibilities and per operatively methods used in preventing this sort of complication.

  20. Patch-clamp amplifiers on a chip

    PubMed Central

    Weerakoon, Pujitha; Culurciello, Eugenio; Yang, Youshan; Santos-Sacchi, Joseph; Kindlmann, Peter J.; Sigworth, Fred J.

    2010-01-01

    We present the first, fully-integrated, two-channel implementation of a patch-clamp measurement system. With this “PatchChip” two simultaneous whole-cell recordings can be obtained with rms noise of 8 pA in a 10 kHz bandwidth. The capacitance and series-resistance of the electrode can be compensated up to 10 pF and 100 MΩ respectively under computer control. Recordings of hERG and Nav 1.7 currents demonstrate the system's capabilities, which are on par with large, commercial patch-clamp instrumentation. By reducing patch-clamp amplifiers to a millimeter size micro-chip, this work paves the way to the realization of massively-parallel, high-throughput patch-clamp systems for drug screening and ion-channel research. The PatchChip is implemented in a 0.5 μm silicon-on-sapphire process; its size is 3 × 3 mm2 and the power consumption is 5 mW per channel with a 3.3 V power supply. PMID:20637803

  1. Π-Clamp-mediated cysteine conjugation.

    PubMed

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J; Santos, Michael S; Van Voorhis, Troy; Pentelute, Bradley L

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the 'π-clamp', that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  2. Analytical chemistry: Clamping down on cancer detection

    NASA Astrophysics Data System (ADS)

    Gorodetskaya, Irina A.; Gorodetsky, Alon A.

    2015-07-01

    An electrochemical clamp assay that enables the rapid and sensitive detection of nucleic acids containing single base mutations has now been developed. It has been shown to differentiate between cancer patient samples featuring a specific mutation, and controls from healthy donors or other cancer patients, all directly in unprocessed serum.

  3. Π-Clamp-mediated cysteine conjugation.

    PubMed

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J; Santos, Michael S; Van Voorhis, Troy; Pentelute, Bradley L

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the 'π-clamp', that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics. PMID:26791894

  4. Clamp and Gas Nozzle for TIG Welding

    NASA Technical Reports Server (NTRS)

    Gue, G. B.; Goller, H. L.

    1982-01-01

    Tool that combines clamp with gas nozzle is aid to tungsten/inert-gas (TIG) welding in hard-to-reach spots. Tool holds work to be welded while directing a stream of argon gas at weld joint, providing an oxygen-free environment for tungsten-arc welding.

  5. Development of voltage-activated potassium currents in cultured cerebellar granule neurons under different growth conditions.

    PubMed

    Gorter, J A; Aronica, E; Hack, N J; Balázs, R; Wadman, W J

    1995-07-01

    1. The functional expression of two potassium currents in cultured cerebellar granule cells was investigated with the whole cell patch-clamp technique in relation to development and growth condition. Cells were grown in medium containing different concentrations of potassium: 25 mM (K25) and 40 mM (K40), together referred to as "high K+"; 10 mM (K10) or "low K+"; and K10 with 100 microM N-methyl-D-aspartate (KNMDA). All conditions are known to influence maturation and survival of granule cells in culture. 2. At 2 days in vitro (DIV) the membrane capacitance, taken as index of membrane surface area, was the same for cells grown in each growth condition. At 7-9 DIV it had increased in each condition, but to a substantially larger extent in cells grown in KNMDA, K25, and K40 than in cells grown in K10. During development the input resistance only decreased in cells grown in KNMDA and high K+. 3. A delayed potassium current (IK) and a fast transient potassium current (IA) could both be recorded at 2 DIV in each growth condition, although a few neurons only expressed the IK. The IK was partially suppressed by tetraethylammonium (5 mM), whereas IA was predominantly sensitive to 4-aminopyridine (5 mM). 4. Normalized for cell capacitance, the specific IA conductance hardly changed during development in cells grown in high K+ and KNMDA. Cells in K10, however, displayed an IA with totally different properties in 23 of 24 cells; the specific IA conductance in these cells was considerably smaller at 7-9 DIV, suggesting a deletion of these channels during development.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. The TCF C-clamp DNA binding domain expands the Wnt transcriptome via alternative target recognition

    PubMed Central

    Hoverter, Nate P.; Zeller, Michael D.; McQuade, Miriam M.; Garibaldi, Angela; Busch, Anke; Selwan, Elizabeth M.; Hertel, Klemens J.; Baldi, Pierre; Waterman, Marian L.

    2014-01-01

    LEF/TCFs direct the final step in Wnt/β-catenin signalling by recruiting β-catenin to genes for activation of transcription. Ancient, non-vertebrate TCFs contain two DNA binding domains, a High Mobility Group box for recognition of the Wnt Response Element (WRE; 5′-CTTTGWWS-3′) and the C-clamp domain for recognition of the GC-rich Helper motif (5′-RCCGCC-3′). Two vertebrate TCFs (TCF-1/TCF7 and TCF-4/TCF7L2) use the C-clamp as an alternatively spliced domain to regulate cell-cycle progression, but how the C-clamp influences TCF binding and activity genome-wide is not known. Here, we used a doxycycline inducible system with ChIP-seq to assess how the C-clamp influences human TCF1 binding genome-wide. Metabolic pulse-labeling of nascent RNA with 4′Thiouridine was used with RNA-seq to connect binding to the Wnt transcriptome. We find that the C-clamp enables targeting to a greater number of gene loci for stronger occupancy and transcription regulation. The C-clamp uses Helper sites concurrently with WREs for gene targeting, but it also targets TCF1 to sites that do not have readily identifiable canonical WREs. The coupled ChIP-seq/4′Thiouridine-seq analysis identified new Wnt target genes, including additional regulators of cell proliferation. Thus, C-clamp containing isoforms of TCFs are potent transcriptional regulators with an expanded transcriptome directed by C-clamp-Helper site interactions. PMID:25414359

  7. A novel biophysical model on calcium and voltage dual dependent gating of calcium-activated chloride channel.

    PubMed

    Zhang, Suhua; Chen, Yafei; An, Hailong; Liu, Hui; Li, Junwei; Pang, Chunli; Ji, Qing; Zhan, Yong

    2014-08-21

    Ca(2+)-activated Cl(-) channels (CaCCs) are anion-selective channels and involved in physiological processes such as electrolyte/fluid secretion, smooth muscle excitability, and olfactory perception which critically depend on the Ca(2+) and voltage dual-dependent gating of channels. However, how the Ca(2+) and voltage regulate the gating of CaCCs still unclear. In this work, the authors constructed a biophysical model to illustrate the dual-dependent gating of CaCCs. For validation, we applied our model on both native CaCCs and exogenous TMEM16A which is thought to be the molecular basis of CaCCs. Our data show that the native CaCCs may share universal gating mechanism. We confirmed the assumption that by binding with the channel, Ca(2+) decreases the energy-barrier to open the channel, but not changes the voltage-sensitivity. For TMEM16A, our model indicates that the exogenous channels show different Ca(2+) dependent gating mechanism from the native ones. These results advance the understanding of intracellular Ca(2+) and membrane potential regulation in CaCCs, and shed new light on its function in aspect of physiology and pharmacology.

  8. β1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

    PubMed

    Castillo, Juan P; Sánchez-Rodríguez, Jorge E; Hyde, H Clark; Zaelzer, Cristian A; Aguayo, Daniel; Sepúlveda, Romina V; Luk, Louis Y P; Kent, Stephen B H; Gonzalez-Nilo, Fernando D; Bezanilla, Francisco; Latorre, Ramón

    2016-06-01

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

  9. Menthol pain relief through cumulative inactivation of voltage-gated sodium channels.

    PubMed

    Gaudioso, Christelle; Hao, Jizhe; Martin-Eauclaire, Marie-France; Gabriac, Mélanie; Delmas, Patrick

    2012-02-01

    Menthol is a natural compound of plant origin known to produce cool sensation via the activation of the TRPM8 channel. It is also frequently part of topical analgesic drugs available in a pharmacy, although its mechanism of action is still unknown. Compelling evidence indicates that voltage-gated Na(+) channels are critical for experiencing pain sensation. We tested the hypothesis that menthol may block voltage-gated Na(+) channels in dorsal root ganglion (DRG) neurons. By use of a patch clamp, we evaluated the effects of menthol application on tetrodotoxin (TTX)-resistant Nav1.8 and Nav1.9 channel subtypes in DRG neurons, and on TTX-sensitive Na(+) channels in immortalized DRG neuron-derived F11 cells. The results indicate that menthol inhibits Na(+) channels in a concentration-, voltage-, and frequency-dependent manner. Menthol promoted fast and slow inactivation states, causing use-dependent depression of Na(+) channel activity. In current clamp recordings, menthol inhibited firing at high-frequency stimulation with minimal effects on normal neuronal activity. We found that low concentrations of menthol cause analgesia in mice, relieving pain produced by a Na(+) channel-targeting toxin. We conclude that menthol is a state-selective blocker of Nav1.8, Nav1.9, and TTX-sensitive Na(+) channels, indicating a role for Na(+) channel blockade in the efficacy of menthol as topical analgesic compound. PMID:22172548

  10. Menthol pain relief through cumulative inactivation of voltage-gated sodium channels.

    PubMed

    Gaudioso, Christelle; Hao, Jizhe; Martin-Eauclaire, Marie-France; Gabriac, Mélanie; Delmas, Patrick

    2012-02-01

    Menthol is a natural compound of plant origin known to produce cool sensation via the activation of the TRPM8 channel. It is also frequently part of topical analgesic drugs available in a pharmacy, although its mechanism of action is still unknown. Compelling evidence indicates that voltage-gated Na(+) channels are critical for experiencing pain sensation. We tested the hypothesis that menthol may block voltage-gated Na(+) channels in dorsal root ganglion (DRG) neurons. By use of a patch clamp, we evaluated the effects of menthol application on tetrodotoxin (TTX)-resistant Nav1.8 and Nav1.9 channel subtypes in DRG neurons, and on TTX-sensitive Na(+) channels in immortalized DRG neuron-derived F11 cells. The results indicate that menthol inhibits Na(+) channels in a concentration-, voltage-, and frequency-dependent manner. Menthol promoted fast and slow inactivation states, causing use-dependent depression of Na(+) channel activity. In current clamp recordings, menthol inhibited firing at high-frequency stimulation with minimal effects on normal neuronal activity. We found that low concentrations of menthol cause analgesia in mice, relieving pain produced by a Na(+) channel-targeting toxin. We conclude that menthol is a state-selective blocker of Nav1.8, Nav1.9, and TTX-sensitive Na(+) channels, indicating a role for Na(+) channel blockade in the efficacy of menthol as topical analgesic compound.

  11. Dynamics and Stability of Pinned-Clamped and Clamped-Pinned Cylindrical Shells Conveying Fluid

    NASA Astrophysics Data System (ADS)

    Misra, A. K.; Wong, S. S. T.; Païdoussis, M. P.

    2001-11-01

    The paper examines the dynamics and stability of fluid-conveying cylindrical shells having pinned-clamped or clamped-pinned boundary conditions, where ``pinned'' is an abbreviation for ``simply supported''. Flügge's equations are used to describe the shell motion, while the fluid-dynamic perturbation pressure is obtained utilizing the linearized potential flow theory. The solution is obtained using two methods - the travelling wave method and the Fourier-transform approach. The results obtained by both methods suggest that the negative damping of the clamped-pinned systems and positive damping of the pinned-clamped systems, observed by previous investigators for any arbitrarily small flow velocity, are simply numerical artefacts; this is reinforced by energy considerations, in which the work done by the fluid on the shell is shown to be zero. Hence, it is concluded that both systems are conservative.

  12. Benzonatate inhibition of voltage-gated sodium currents.

    PubMed

    Evans, M Steven; Maglinger, G Benton; Fletcher, Anita M; Johnson, Stephen R

    2016-02-01

    Benzonatate was FDA-approved in 1958 as an antitussive. Its mechanism of action is thought to be anesthesia of vagal sensory nerve fibers that mediate cough. Vagal sensory neurons highly express the Nav1.7 subtype of voltage-gated sodium channels, and inhibition of this channel inhibits the cough reflex. Local anesthetics inhibit voltage-gated sodium channels, but there are no reports of whether benzonatate affects these channels. Our hypothesis is that benzonatate inhibits Nav1.7 voltage-gated sodium channels. We used whole cell voltage clamp recording to test the effects of benzonatate on voltage-gated sodium (Na(+)) currents in two murine cell lines, catecholamine A differentiated (CAD) cells, which express primarily Nav1.7, and N1E-115, which express primarily Nav1.3. We found that, like local anesthetics, benzonatate strongly and reversibly inhibits voltage-gated Na(+) channels. Benzonatate causes both tonic and phasic inhibition. It has greater effects on channel inactivation than on activation, and its potency is much greater at depolarized potentials, indicating inactivated-state-specific effects. Na(+) currents in CAD cells and N1E-115 cells are similarly affected, indicating that benzonatate is not Na(+) channel subtype-specific. Benzonatate is a mixture of polyethoxy esters of 4-(butylamino) benzoic acid having varying degrees of hydrophobicity. We found that Na(+) currents are inhibited most potently by a benzonatate fraction containing the 9-ethoxy component. Detectable effects of benzonatate occur at concentrations as low as 0.3 μM, which has been reported in humans. We conclude that benzonatate has local anesthetic-like effects on voltage-gated sodium channels, including Nav1.7, which is a possible mechanism for cough suppression by the drug.

  13. Benzonatate inhibition of voltage-gated sodium currents.

    PubMed

    Evans, M Steven; Maglinger, G Benton; Fletcher, Anita M; Johnson, Stephen R

    2016-02-01

    Benzonatate was FDA-approved in 1958 as an antitussive. Its mechanism of action is thought to be anesthesia of vagal sensory nerve fibers that mediate cough. Vagal sensory neurons highly express the Nav1.7 subtype of voltage-gated sodium channels, and inhibition of this channel inhibits the cough reflex. Local anesthetics inhibit voltage-gated sodium channels, but there are no reports of whether benzonatate affects these channels. Our hypothesis is that benzonatate inhibits Nav1.7 voltage-gated sodium channels. We used whole cell voltage clamp recording to test the effects of benzonatate on voltage-gated sodium (Na(+)) currents in two murine cell lines, catecholamine A differentiated (CAD) cells, which express primarily Nav1.7, and N1E-115, which express primarily Nav1.3. We found that, like local anesthetics, benzonatate strongly and reversibly inhibits voltage-gated Na(+) channels. Benzonatate causes both tonic and phasic inhibition. It has greater effects on channel inactivation than on activation, and its potency is much greater at depolarized potentials, indicating inactivated-state-specific effects. Na(+) currents in CAD cells and N1E-115 cells are similarly affected, indicating that benzonatate is not Na(+) channel subtype-specific. Benzonatate is a mixture of polyethoxy esters of 4-(butylamino) benzoic acid having varying degrees of hydrophobicity. We found that Na(+) currents are inhibited most potently by a benzonatate fraction containing the 9-ethoxy component. Detectable effects of benzonatate occur at concentrations as low as 0.3 μM, which has been reported in humans. We conclude that benzonatate has local anesthetic-like effects on voltage-gated sodium channels, including Nav1.7, which is a possible mechanism for cough suppression by the drug. PMID:26386152

  14. Firing up the Amplifier:. Temperature, Pressure and Voltage Jump Studies on Ohc Motor Capacitance

    NASA Astrophysics Data System (ADS)

    Santos-Sacchi, Joseph; Song, Lei; Li, Xiantao

    2009-02-01

    The outer hair cell (OHC) possesses molecular motors that drive electromotility and cochlear amplification. Here we look at the effects of fast perturbations of biophysical forces that affect the OHC, including voltage, temperature and pressure, on the electrical signature of motor activity, namely nonlinear capacitance (NLC). Under whole cell voltage clamp, we measure changes in NLC at fixed holding voltages following steps in voltage, jumps in temperature induced by ir laser, or jumps in intracellular turgor pressure. In each case we find time dependent changes in NLC resulting from induced shifts of the NLC function across the voltage axis. The time course of these shifts depends on the stimulus, with voltage jumps inducing shifts with fast exponential components less than a millisecond. Those induced by temperature and pressure are within the tens of millisecond ranges and may be limited by cellular and experimental constraints that voltage is able to overcome. The overall observation of time dependent changes to the electromotility function upon stimulation indicates a more complex mechanism than provided by a simple two-state Boltzmann model.

  15. 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

  16. 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.

  17. A monitor and control system for high voltage, gating, and triggering of a scintillating fiber active target

    SciTech Connect

    Baumbaugh, B.; Bishop, J.; Gardner, R.W.; Mountain, R.J.; Ruchti, R.; Baumbaugh, A.; Knickerbocker, K.

    1988-02-01

    A monitor and control system has been designed, constructed and tested at Notre Dame for the purpose of controlling all aspects of a Scintillating Fiber Acxtive Target system used in High Energy Physics Experimentation. The SFT Active Target system requires control of high voltages, gating, trigger counters, and monitoring. In addition, it resides in a radioactive area with very limited access. The control system uses a Leading Edge microcomputer, two specialized Z80-based processors, associated DACs, ADCs, discrete semiconductors, linear ICs and TTL and MECL logic. All of the hardware and software is custom-built; its design and performance is discussed.

  18. Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes.

    PubMed

    Das, Debasis; Krantz, Bryan A

    2016-08-23

    Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins-protective antigen (PA), lethal factor (LF), and edema factor-translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LF-derived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/helix-to-coil cooperativity. PMID:27506790

  19. A new technique for multiple re-use of planar patch clamp chips.

    PubMed

    Kao, Liyo; Abuladze, Natalia; Shao, Xuesi M; McKeegan, Kevin; Kurtz, Ira

    2012-07-15

    The patch clamp technique is widely used for recording the activity of ion channels in single cells and lipid bilayers. Most platforms utilize borosilicate glass configured as a pipette, however more recently planar patch clamp chips have been developed that require less technical expertise. Planar patch clamp chips in systems like the Nanion Port-a-Patch are useful in that they allow more rapid throughput in drug screening studies. This technique also has the ability to perform rapid solution changes from the intracellular side. A current drawback with the planar patch clamp chips is the need to utilize a separate chip for each experiment. This increases the cost of each experiment and is due to the fact that the ∼1μm aperture used for cell attachment is thought to retain cellular debris thereby preventing subsequent cell attachment and formation of GΩ seals. In the present study we have for the first time solved the technical problem of developing a simple protocol for re-use of Nanion planar patch clamp chips. The re-use methodology is demonstrated in whole cell patch clamp studies of HEK-293 cells expressing the electrogenic sodium bicarbonate cotransporter NBCe1-A in protocols involving external and internal solution changes, and CHO-K1 cells with incorporated gramicidin channels. PMID:22609774

  20. Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes

    PubMed Central

    Das, Debasis; Krantz, Bryan A.

    2016-01-01

    Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins—protective antigen (PA), lethal factor (LF), and edema factor—translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LF-derived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/helix-to-coil cooperativity. PMID:27506790

  1. Voltage-Independent Calcium Release in Heart Muscle

    NASA Astrophysics Data System (ADS)

    Niggli, Ernst; Lederer, W. Jonathan

    1990-10-01

    The Ca2+ that activates contraction in heart muscle is regulated as in skeletal muscle by processes that depend on voltage and intracellular Ca2+ and involve a positive feedback system. How the initial electrical signal is amplified in heart muscle has remained controversial, however. Analogous protein structures from skeletal muscle and heart muscle have been identified physiologically and sequenced; these include the Ca2+ channel of the sarcolemma and the Ca2+ release channel of the sarcoplasmic reticulum. Although the parallels found in cardiac and skeletal muscles have provoked valuable experiments in both tissues, separation of the effects of voltage and intracellular Ca2+ on sarcoplasmic reticulum Ca2+ release in heart muscle has been imperfect. With the use of caged Ca2+ and flash photolysis in voltage-clamped heart myocytes, effects of membrane potential in heart muscle cells on Ca2+ release from intracellular stores have been studied. Unlike the response in skeletal muscle, voltage across the sarcolemma of heart muscle does not affect the release of Ca2+ from the sarcoplasmic reticulum, suggesting that other regulatory processes are needed to control Ca2+-induced Ca2+ release.

  2. A low-voltage high-speed terahertz spatial light modulator using active metamaterial

    NASA Astrophysics Data System (ADS)

    Rout, Saroj; Sonkusale, Sameer R.

    2016-11-01

    An all solid-state metamaterial based terahertz (THz) spatial light modulator (SLM) is presented which uses high mobility 2DEG to manipulate the metamaterial resonant frequency (0.45 THz) leading to terahertz wave modulation. The 2DEG is created by embedding pseudomorphic high-electron mobility transistors in the capacitive gap of each electrical-LC resonator, allowing the charge density to be controlled with very low voltage (1 V) and modulating speeds up to 10 MHz while consuming sub-milliwatt power. We have demonstrated our SLM as a 2 × 2 pixel array operating around 0.45 THz by raster scanning a 6 × 6 image of an occluded metal object behind a thick polystyrene screen using a single-pixel THz imaging setup.

  3. VOLTAGE REGULATOR

    DOEpatents

    Von Eschen, R.L.; Scheele, P.F.

    1962-04-24

    A transistorized voltage regulator which provides very close voitage regulation up to about 180 deg F is described. A diode in the positive line provides a constant voltage drop from the input to a regulating transistor emitter. An amplifier is coupled to the positive line through a resistor and is connected between a difference circuit and the regulating transistor base which is negative due to the difference in voltage drop across thc diode and the resistor so that a change in the regulator output causes the amplifier to increase or decrease the base voltage and current and incrcase or decrease the transistor impedance to return the regulator output to normal. (AEC)

  4. Single molecule study of a processivity clamp sliding on DNA

    SciTech Connect

    Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A; Barsky, D

    2007-07-05

    Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.

  5. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Carotid artery clamp. 882.5175 Section 882.5175...) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Therapeutic Devices § 882.5175 Carotid artery clamp. (a) Identification. A carotid artery clamp is a device that is surgically placed around a patient's carotid...

  6. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Carotid artery clamp. 882.5175 Section 882.5175...) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Therapeutic Devices § 882.5175 Carotid artery clamp. (a) Identification. A carotid artery clamp is a device that is surgically placed around a patient's carotid...

  7. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Carotid artery clamp. 882.5175 Section 882.5175...) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Therapeutic Devices § 882.5175 Carotid artery clamp. (a) Identification. A carotid artery clamp is a device that is surgically placed around a patient's carotid...

  8. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Carotid artery clamp. 882.5175 Section 882.5175...) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Therapeutic Devices § 882.5175 Carotid artery clamp. (a) Identification. A carotid artery clamp is a device that is surgically placed around a patient's carotid...

  9. 21 CFR 882.5175 - Carotid artery clamp.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carotid artery clamp. 882.5175 Section 882.5175...) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Therapeutic Devices § 882.5175 Carotid artery clamp. (a) Identification. A carotid artery clamp is a device that is surgically placed around a patient's carotid...

  10. 21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Neurosurgical head holder (skull clamp). 882.4460... (CONTINUED) MEDICAL DEVICES NEUROLOGICAL DEVICES Neurological Surgical Devices § 882.4460 Neurosurgical head holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used...

  11. 30 CFR 18.40 - Cable clamps and grips.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... prevent strain on both ends of each cable or cord leading from a machine to a detached or separately... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Cable clamps and grips. 18.40 Section 18.40... Requirements § 18.40 Cable clamps and grips. Insulated clamps shall be provided for all portable...

  12. 33 CFR 183.560 - Hose clamps: Installation.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Hose clamps: Installation. 183... Hose clamps: Installation. Each hose clamp on a hose from the fuel tank to the fuel inlet connection on the engine, a hose between the fuel pump and the carburetor, or a vent line must: (a) Be used...

  13. 33 CFR 183.560 - Hose clamps: Installation.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Hose clamps: Installation. 183... Hose clamps: Installation. Each hose clamp on a hose from the fuel tank to the fuel inlet connection on the engine, a hose between the fuel pump and the carburetor, or a vent line must: (a) Be used...

  14. 33 CFR 183.560 - Hose clamps: Installation.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Hose clamps: Installation. 183... Hose clamps: Installation. Each hose clamp on a hose from the fuel tank to the fuel inlet connection on the engine, a hose between the fuel pump and the carburetor, or a vent line must: (a) Be used...

  15. 33 CFR 183.560 - Hose clamps: Installation.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Hose clamps: Installation. 183... Hose clamps: Installation. Each hose clamp on a hose from the fuel tank to the fuel inlet connection on the engine, a hose between the fuel pump and the carburetor, or a vent line must: (a) Be used...

  16. 33 CFR 183.560 - Hose clamps: Installation.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Hose clamps: Installation. 183... Hose clamps: Installation. Each hose clamp on a hose from the fuel tank to the fuel inlet connection on the engine, a hose between the fuel pump and the carburetor, or a vent line must: (a) Be used...

  17. High-field actively detuneable transverse electromagnetic (TEM) coil with low-bias voltage for high-power RF transmission.

    PubMed

    Avdievich, Nikolai I; Bradshaw, Ken; Kuznetsov, Andrey M; Hetherington, Hoby P

    2007-06-01

    The design and construction of a 4T (170 MHz) transverse electromagnetic (TEM) actively detuneable quadrature head coil is described. Conventional schemes for active detuning require high negative bias voltages (>300 V) to prevent leakage of RF pulses with amplitudes of 1-2 kW. To extend the power handling capacity and avoid the use of high DC bias voltages, we developed an alternate method of detuning the volume coil. In this method the PIN diodes in the detuning circuits are shorted when the RF volume coil is tuned, and negatively biased with -12 V when the coil is detuned. To preserve the high Q(U)/Q(L) ratio of the TEM coil, we modified the method of Nabetani and Watkins (Proceedings of the 13th Annual Meeting of ISMRM, Kyoto, Japan, 2004, abstract 1574) by utilizing a high-impedance (approximately 200 Omega), lumped-element, quarter-wavelength transformer. A Q(U) of 500 was achieved for the detuneable TEM, such that incorporation of the detuning network had minimal effect (<1 dB) on the performance of the coil in vivo. PMID:17534919

  18. Combining optical tweezers and patch clamp for studies of cell membrane electromechanics

    NASA Astrophysics Data System (ADS)

    Qian, Feng; Ermilov, Sergey; Murdock, David; Brownell, William E.; Anvari, Bahman

    2004-09-01

    We have designed and implemented a novel experimental setup which combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. In this system, optical tweezers provide measurement of forces at piconewton scale, and the patch-clamp technique allows control of the cell transmembrane potential. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement from the trapping center is monitored by a quadrant photodetector for dynamic measurements of tether force. Fluorescent beads and the corresponding fluorescence imaging optics are used to eliminate the shadow of the cell projected on the quadrant photodetector. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. A unique feature of this setup is that the patch-clamp headstage and the manipulator for the recording pipette are mounted on a piezoelectric stage, preventing relative movements between the cell and the patch pipette during the process of tether pulling. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from mammalian cochlear outer hair cells and human embryonic kidney cells are presented.

  19. Intermittent selective clamping improves rat liver regeneration by attenuating oxidative and endoplasmic reticulum stress.

    PubMed

    Ben Mosbah, I; Duval, H; Mbatchi, S-F; Ribault, C; Grandadam, S; Pajaud, J; Morel, F; Boudjema, K; Compagnon, P; Corlu, A

    2014-03-06

    Intermittent clamping of the portal trial is an effective method to avoid excessive blood loss during hepatic resection, but this procedure may cause ischemic damage to liver. Intermittent selective clamping of the lobes to be resected may represent a good alternative as it exposes the remnant liver only to the reperfusion stress. We compared the effect of intermittent total or selective clamping on hepatocellular injury and liver regeneration. Entire hepatic lobes or only lobes to be resected were subjected twice to 10 min of ischemia followed by 5 min of reperfusion before hepatectomy. We provided evidence that the effect of intermittent clamping can be damaging or beneficial depending to its mode of application. Although transaminase levels were similar in all groups, intermittent total clamping impaired liver regeneration and increased apoptosis. In contrast, intermittent selective clamping improved liver protein secretion and hepatocyte proliferation when compared with standard hepatectomy. This beneficial effect was linked to better adenosine-5'-triphosphate (ATP) recovery, nitric oxide production, antioxidant activities and endoplasmic reticulum adaptation leading to limit mitochondrial damage and apoptosis. Interestingly, transient and early chaperone inductions resulted in a controlled activation of the unfolded protein response concomitantly to endothelial nitric oxide synthase, extracellular signal-regulated kinase-1/2 (ERK1/2) and p38 MAPK activation that favors liver regeneration. Endoplasmic reticulum stress is a central target through which intermittent selective clamping exerts its cytoprotective effect and improves liver regeneration. This procedure could be applied as a powerful protective modality in the field of living donor liver transplantation and liver surgery.

  20. Intermittent selective clamping improves rat liver regeneration by attenuating oxidative and endoplasmic reticulum stress.

    PubMed

    Ben Mosbah, I; Duval, H; Mbatchi, S-F; Ribault, C; Grandadam, S; Pajaud, J; Morel, F; Boudjema, K; Compagnon, P; Corlu, A

    2014-01-01

    Intermittent clamping of the portal trial is an effective method to avoid excessive blood loss during hepatic resection, but this procedure may cause ischemic damage to liver. Intermittent selective clamping of the lobes to be resected may represent a good alternative as it exposes the remnant liver only to the reperfusion stress. We compared the effect of intermittent total or selective clamping on hepatocellular injury and liver regeneration. Entire hepatic lobes or only lobes to be resected were subjected twice to 10 min of ischemia followed by 5 min of reperfusion before hepatectomy. We provided evidence that the effect of intermittent clamping can be damaging or beneficial depending to its mode of application. Although transaminase levels were similar in all groups, intermittent total clamping impaired liver regeneration and increased apoptosis. In contrast, intermittent selective clamping improved liver protein secretion and hepatocyte proliferation when compared with standard hepatectomy. This beneficial effect was linked to better adenosine-5'-triphosphate (ATP) recovery, nitric oxide production, antioxidant activities and endoplasmic reticulum adaptation leading to limit mitochondrial damage and apoptosis. Interestingly, transient and early chaperone inductions resulted in a controlled activation of the unfolded protein response concomitantly to endothelial nitric oxide synthase, extracellular signal-regulated kinase-1/2 (ERK1/2) and p38 MAPK activation that favors liver regeneration. Endoplasmic reticulum stress is a central target through which intermittent selective clamping exerts its cytoprotective effect and improves liver regeneration. This procedure could be applied as a powerful protective modality in the field of living donor liver transplantation and liver surgery. PMID:24603335

  1. π-Clamp-mediated cysteine conjugation

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  2. Carbon nanotube-clamped metal atomic chain

    PubMed Central

    Tang, Dai-Ming; Yin, Li-Chang; Li, Feng; Liu, Chang; Yu, Wan-Jing; Hou, Peng-Xiang; Wu, Bo; Lee, Young-Hee; Ma, Xiu-Liang; Cheng, Hui-Ming

    2010-01-01

    Metal atomic chain (MAC) is an ultimate one-dimensional structure with unique physical properties, such as quantized conductance, colossal magnetic anisotropy, and quantized magnetoresistance. Therefore, MACs show great potential as possible components of nanoscale electronic and spintronic devices. However, MACs are usually suspended between two macroscale metallic electrodes; hence obvious technical barriers exist in the interconnection and integration of MACs. Here we report a carbon nanotube (CNT)-clamped MAC, where CNTs play the roles of both nanoconnector and electrodes. This nanostructure is prepared by in situ machining a metal-filled CNT, including peeling off carbon shells by spatially and elementally selective electron beam irradiation and further elongating the exposed metal nanorod. The microstructure and formation process of this CNT-clamped MAC are explored by both transmission electron microscopy observations and theoretical simulations. First-principles calculations indicate that strong covalent bonds are formed between the CNT and MAC. The electrical transport property of the CNT-clamped MAC was experimentally measured, and quantized conductance was observed. PMID:20427743

  3. π-Clamp Mediated Cysteine Conjugation

    PubMed Central

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; Van Voorhis, Troy; Pentelute, Bradley L.

    2016-01-01

    Site-selective functionalization of complex molecules is a grand challenge in chemistry. Protecting groups or catalysts must be used to selectively modify one site among many that are similarly reactive. General strategies are rare such the local chemical environment around the target site is tuned for selective transformation. Here we show a four amino acid sequence (Phe-Cys-Pro-Phe), which we call the “π-clamp”, tunes the reactivity of its cysteine thiol for the site-selective conjugation with perfluoroaromatic reagents. We used the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues (e.g. antibodies and cysteine-based enzymes), which was impossible with prior cysteine modification methods. The modified π-clamp antibodies retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates (ADCs) for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach for site-selective chemistry and provides opportunities to modify biomolecules for research and therapeutics. PMID:26791894

  4. A Highly Active Low Voltage Redox Mediator for Enhanced Rechargeability of Lithium-Oxygen Batteries.

    PubMed

    Kundu, Dipan; Black, Robert; Adams, Brian; Nazar, Linda F

    2015-12-23

    Owing to its high theoretical specific energy, the Li-oxygen battery is one of the fundamentally most promising energy storage systems, but also one of the most challenging. Poor rechargeability, involving the oxidation of insoluble and insulating lithium peroxide (Li2O2), has remained the "Achilles' heel" of this electrochemical energy storage system. We report here on a new redox mediator tris[4-(diethylamino)phenyl]amine (TDPA), that-at 3.1 V-exhibits the lowest and closest potential redox couple compared to the equilibrium voltage of the Li-oxygen cell of those reported to date, with a second couple also at a low potential of 3.5 V. We show it is a soluble "catalyst" capable of lowering the Li2O2 charging potential by >0.8 V without requiring direct electrical contact of the peroxide and that it also facilitates high discharge capacities. Its chemical and electrochemical stability, fast diffusion kinetics, and two dynamic redox potentials represent a significant advance in oxygen-evolution catalysis. It enables Li-O2 cells that can be recharged more than 100 cycles with average round-trip efficiencies >80%, opening a new avenue for practical Li-oxygen batteries.

  5. A Highly Active Low Voltage Redox Mediator for Enhanced Rechargeability of Lithium–Oxygen Batteries

    PubMed Central

    2015-01-01

    Owing to its high theoretical specific energy, the Li-oxygen battery is one of the fundamentally most promising energy storage systems, but also one of the most challenging. Poor rechargeability, involving the oxidation of insoluble and insulating lithium peroxide (Li2O2), has remained the “Achilles’ heel” of this electrochemical energy storage system. We report here on a new redox mediator tris[4-(diethylamino)phenyl]amine (TDPA), that—at 3.1 V—exhibits the lowest and closest potential redox couple compared to the equilibrium voltage of the Li-oxygen cell of those reported to date, with a second couple also at a low potential of 3.5 V. We show it is a soluble “catalyst” capable of lowering the Li2O2 charging potential by >0.8 V without requiring direct electrical contact of the peroxide and that it also facilitates high discharge capacities. Its chemical and electrochemical stability, fast diffusion kinetics, and two dynamic redox potentials represent a significant advance in oxygen-evolution catalysis. It enables Li–O2 cells that can be recharged more than 100 cycles with average round-trip efficiencies >80%, opening a new avenue for practical Li-oxygen batteries. PMID:27163015

  6. Toxins targeting voltage-activated Ca2+ channels and their potential biomedical applications.

    PubMed

    Gandini, María A; Sandoval, Alejandro; Felix, Ricardo

    2015-01-01

    Voltage-gated Ca(2+) (CaV) channels are transmembrane proteins primarily formed by an ion-conducting α 1 subunit that can associate with auxiliary β and α2δ subunits. Ca(2+) entering the cell through these channels serves as a versatile second messenger of electrical signaling, initiating numerous different cellular processes ranging from gene expression to cell fertilization, neuronal transmission and cell death. CaV channels, as other ion channels, are targets for numerous ligands including naturally occurring peptide toxins. Some of these peptide toxins are invaluable tools for studying their structure and function and have potential therapeutic applications. Here, we present an overview of the current knowledge regarding the structure and function of CaV channels as well as their role in human disease, and highlight some of the growing applications of peptide toxins targeting CaV channels. Analysis and understanding of the molecular strategy used by these peptide toxins might allow the design of novel classes of therapeutic agents acting on specific targets with high selectivity and efficacy.

  7. Asymmetric synthesis of crambescin A-C carboxylic acids and their inhibitory activity on voltage-gated sodium channels.

    PubMed

    Nakazaki, Atsuo; Nakane, Yoshiki; Ishikawa, Yuki; Yotsu-Yamashita, Mari; Nishikawa, Toshio

    2016-06-21

    Synthesis of both enantiomers of crambescin B carboxylic acid is described. A cis-enyne starting material was epoxidized under the conditions of Katsuki asymmetric epoxidation to give 95% ee of the epoxide, which was transformed to crambescin B carboxylic acid via bromocation-triggered cascade cyclization as the key step. Enantiomerically pure crambescin A and C carboxylic acids were also synthesized from the product of the cascade reaction. Structure-activity relationship (SAR) studies against voltage-gated sodium channel (VGSC) inhibition using those synthetic compounds revealed that the natural enantiomer of crambescin B carboxylic acid was most active and comparable to tetrodotoxin, and the unalkylated cyclic guanidinium structure is indispensible, while the carboxylate moiety is not important. The absolute stereochemistry of crambescin A was determined by a comparison of the methyl ester derived from natural crambescin A with that derived from the stereochemically defined crambescin A carboxylic acid synthesized in this study.

  8. Peripheral benzodiazepine receptor regulates vascular endothelial activations via suppression of the voltage-dependent anion channel-1.

    PubMed

    Joo, Hee Kyoung; Lee, Yu Ran; Lim, Sun Young; Lee, Eun Ji; Choi, Sunga; Cho, Eun Jung; Park, Myoung Soo; Ryoo, Sungwoo; Park, Jin Bong; Jeon, Byeong Hwa

    2012-05-01

    Peripheral benzodiazepine receptor (PBR) is a multifunctional protein mainly found on the outer mitochondrial membrane. PBR expression is increased by tumor necrosis factor-α (TNF-α) in endothelial cells. Adenoviral overexpression of PBR inhibits monocyte adhesion, VCAM-1, and ICAM-1 expression in TNF-α-activated endothelial cells. Rotenone, cyclosporine A, and bongkrekic acid suppress TNF-α-induced VCAM-1 expression. Overexpression of PBR inhibits voltage-dependent anion channel-1 (VDAC-1) expression and the silencing of PBR increases VDAC-1 expression in endothelial cells. Moreover, TNF-α-induced VCAM-1 expression is suppressed by VDAC-1 gene silencing. PBR overexpression significantly decreases TNF-α-induced mitochondrial reactive oxygen species and MnSOD expression. These results suggest that PBR can inhibit endothelial activation and this action is related to the inhibition of mitochondrial ROS and/or VDAC-1 expression in endothelial cells.

  9. Direct Effect of Remifentanil and Glycine Contained in Ultiva® on Nociceptive Transmission in the Spinal Cord: In Vivo and Slice Patch Clamp Analyses

    PubMed Central

    Sumie, Makoto; Shiokawa, Hiroaki; Yamaura, Ken; Karashima, Yuji; Hoka, Sumio; Yoshimura, Megumu

    2016-01-01

    Background Ultiva® is commonly administered intravenously for analgesia during general anaesthesia and its main constituent remifentanil is an ultra-short-acting μ-opioid receptor agonist. Ultiva® is not approved for epidural or intrathecal use in clinical practice. Previous studies have reported that Ultiva® provokes opioid-induced hyperalgesia by interacting with spinal dorsal horn neurons. Ultiva® contains glycine, an inhibitory neurotransmitter but also an N-methyl-D-aspartate receptor co-activator. The presence of glycine in the formulation of Ultiva® potentially complicates its effects. We examined how Ultiva® directly affects nociceptive transmission in the spinal cord. Methods We made patch-clamp recordings from substantia gelatinosa (SG) neurons in the adult rat spinal dorsal horn in vivo and in spinal cord slices. We perfused Ultiva® onto the SG neurons and analysed its effects on the membrane potentials and synaptic responses activated by noxious mechanical stimuli. Results Bath application of Ultiva® hyperpolarized membrane potentials under current-clamp conditions and produced an outward current under voltage-clamp conditions. A barrage of excitatory postsynaptic currents (EPSCs) evoked by the stimuli was suppressed by Ultiva®. Miniature EPSCs (mEPSCs) were depressed in frequency but not amplitude. Ultiva®-induced outward currents and suppression of mEPSCs were not inhibited by the μ-opioid receptor antagonist naloxone, but were inhibited by the glycine receptor antagonist strychnine. The Ultiva®-induced currents demonstrated a specific equilibrium potential similar to glycine. Conclusions We found that intrathecal administration of Ultiva® to SG neurons hyperpolarized membrane potentials and depressed presynaptic glutamate release predominantly through the activation of glycine receptors. No Ultiva®-induced excitatory effects were observed in SG neurons. Our results suggest different analgesic mechanisms of Ultiva® between intrathecal

  10. 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

  11. Functional segregation of voltage-activated calcium channels in motoneurons of the dorsal motor nucleus of the vagus

    PubMed Central

    Cooper, Garry; Lasser-Katz, Efrat; Simchovitz, Alon; Sharon, Ronit; Soreq, Hermona; Surmeier, D. James

    2015-01-01

    Calcium influx elevates mitochondrial oxidant stress (mOS) in dorsal motor nucleus of the vagus (DMV) neurons that are prone to Lewy body pathologies in presymptomatic Parkinson's disease (PD) patients. In experimental PD models, treatment with isradipine, the dihydropyridine with the highest affinity to Cav1.3 channels, prevents subthreshold calcium influx via Cav1.3 channels into midbrain dopamine neurons and protects them from mOS. In DMV neurons, isradipine is also effective in reducing mOS despite overwhelming evidence that subthreshold calcium influx is negligible compared with spike-triggered influx. To solve this conundrum we combined slice electrophysiology, two-photon laser scanning microscopy, mRNA profiling, and computational modeling. We find that the unusually depolarized subthreshold voltage trajectory of DMV neurons is positioned between the relatively hyperpolarized activation curve of Cav1.3 channels and that of other high-voltage activated (HVA) calcium channels, thus creating a functional segregation between Cav1.3 and HVA calcium channels. The HVA channels flux the bulk of calcium during spikes but can only influence pacemaking through their coupling to calcium-activated potassium currents. In contrast, Cav1.3 currents, which we show to be more than an order-of-magnitude smaller than the HVA calcium currents, are able to introduce sufficient inward current to speed up firing. However, Kv4 channels that are constitutively open in the subthreshold range guarantee slow pacemaking, despite the depolarizing action of Cav1.3 and other pacemaking currents. We propose that the efficacy of isradipine in preventing mOS in DMV neurons arises from its mixed effect on Cav1.3 channels and on HVA Cav1.2 channels. PMID:26156385

  12. Current advances in invertebrate vision: insights from patch-clamp studies of photoreceptors in apposition eyes.

    PubMed

    Frolov, Roman V

    2016-08-01

    Traditional electrophysiological research on invertebrate photoreceptors has been conducted in vivo, using intracellular recordings from intact compound eyes. The only exception used to be Drosophila melanogaster, which was exhaustively studied by both intracellular recording and patch-clamp methods. Recently, several patch-clamp studies have provided new information on the biophysical properties of photoreceptors of diverse insect species, having both apposition and neural superposition eyes, in the contexts of visual ecology, behavior, and ontogenesis. Here, I discuss these and other relevant results, emphasizing differences between fruit flies and other species, between photoreceptors of diurnal and nocturnal insects, properties of distinct functional types of photoreceptors, postembryonic developmental changes, and relationships between voltage-gated potassium channels and visual ecology. PMID:27250910

  13. Unpinning the Open-Circuit Voltage in Organic Solar Cells through Tuning Ternary Blend Active Layer Morphology

    NASA Astrophysics Data System (ADS)

    Khlyabich, Petr; Thompson, Barry; Loo, Yueh-Lin

    2015-03-01

    The use of ternary, as opposed to binary, blends having complementary absorption in active layers of organic bulk heterojunction solar cells is a simple approach to increase overall light absorption. While the open-circuit voltage (Voc) of such solar cells have generally been shown to be pinned by the smallest energy level difference between the donor and acceptor constituents, there have been materials systems, that when incorporated into active layers of solar cells, exhibit composition dependent and tunable Voc. Herein, we demonstrate that this Voc tunability in ternary blend solar cells is correlated with the morphology of the active layer. Chemical compatibility between the constituents in the blend, as probed by grazing-incidence X-ray diffraction (GIXD) measurements, affords Voc tuning. The constituents need not ``co-crystallize'' limited miscibility between the constituents in the active layers of solar cells affords Voc tunability. Poor physical interactions between the constituent domains within the active layers, on the other hand, result in devices that exhibit an invariant Voc that is pinned by the smallest energy level difference between the donor(s) and the acceptor(s). Our morphological studies thus support the proposed alloying model that was put forth originally.

  14. Voltage-sensitive dye imaging reveals improved topographic activation of cortex in response to manipulation of thalamic microstimulation parameters

    NASA Astrophysics Data System (ADS)

    Wang, Qi; Millard, Daniel C.; Zheng, He J. V.; Stanley, Garrett B.

    2012-04-01

    Voltage-sensitive dye imaging was used to quantify in vivo, network level spatiotemporal cortical activation in response to electrical microstimulation of the thalamus in the rat vibrissa pathway. Thalamic microstimulation evoked a distinctly different cortical response than natural sensory stimulation, with response to microstimulation spreading over a larger area of cortex and being topographically misaligned with the cortical column to which the stimulated thalamic region projects. Electrical stimulation with cathode-leading asymmetric waveforms reduced this topographic misalignment while simultaneously increasing the spatial specificity of the cortical activation. Systematically increasing the asymmetry of the microstimulation pulses revealed a continuum between symmetric and asymmetric stimulation that gradually reduced the topographic bias. These data strongly support the hypothesis that manipulation of the electrical stimulation waveform can be used to selectively activate specific neural elements. Specifically, our results are consistent with the prediction that cathode-leading asymmetric waveforms preferentially stimulate cell bodies over axons, while symmetric waveforms preferentially activate axons over cell bodies. The findings here provide some initial steps toward the design and optimization of microstimulation of neural circuitry, and open the door to more sophisticated engineering tools, such as nonlinear system identification techniques, to develop technologies for more effective control of activity in the nervous system.

  15. Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles

    PubMed Central

    Aimon, Sophie; Manzi, John; Schmidt, Daniel; Poveda Larrosa, Jose Antonio; Bassereau, Patricia; Toombes, Gilman E. S.

    2011-01-01

    Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability. PMID:21998666

  16. Negligible substrate clamping effect on piezoelectric response in (111)-epitaxial tetragonal Pb(Zr, Ti)O{sub 3} films

    SciTech Connect

    Yamada, Tomoaki; Yasumoto, Jun; Ito, Daisuke; Yoshino, Masahito; Nagasaki, Takanori; Sakata, Osami; Imai, Yasuhiko; Kiguchi, Takanori; Shiraishi, Takahisa; Shimizu, Takao; Funakubo, Hiroshi

    2015-08-21

    The converse piezoelectric responses of (111)- and (001)-epitaxial tetragonal Pb(Zr{sub 0.35}Ti{sub 0.65})O{sub 3} [PZT] films were compared to investigate the orientation dependence of the substrate clamping effect. Synchrotron X-ray diffraction (XRD) and piezoelectric force microscopy revealed that the as-grown (111)-PZT film has a polydomain structure with normal twin boundaries that are changed by the poling process to inclined boundaries, as predicted by Romanov et al. [Phys. Status Solidi A 172, 225 (1999)]. Time-resolved synchrotron XRD under bias voltage showed the negligible impact of substrate clamping on the piezoelectric response in the (111)-PZT film, unlike the case for (001)-PZT film. The origin of the negligible clamping effect in the (111)-PZT film is discussed from the viewpoint of the elastic properties and the compensation of lattice distortion between neighboring domains.

  17. Negligible substrate clamping effect on piezoelectric response in (111)-epitaxial tetragonal Pb(Zr, Ti)O3 films

    NASA Astrophysics Data System (ADS)

    Yamada, Tomoaki; Yasumoto, Jun; Ito, Daisuke; Sakata, Osami; Imai, Yasuhiko; Kiguchi, Takanori; Shiraishi, Takahisa; Shimizu, Takao; Funakubo, Hiroshi; Yoshino, Masahito; Nagasaki, Takanori

    2015-08-01

    The converse piezoelectric responses of (111)- and (001)-epitaxial tetragonal Pb(Zr0.35Ti0.65)O3 [PZT] films were compared to investigate the orientation dependence of the substrate clamping effect. Synchrotron X-ray diffraction (XRD) and piezoelectric force microscopy revealed that the as-grown (111)-PZT film has a polydomain structure with normal twin boundaries that are changed by the poling process to inclined boundaries, as predicted by Romanov et al. [Phys. Status Solidi A 172, 225 (1999)]. Time-resolved synchrotron XRD under bias voltage showed the negligible impact of substrate clamping on the piezoelectric response in the (111)-PZT film, unlike the case for (001)-PZT film. The origin of the negligible clamping effect in the (111)-PZT film is discussed from the viewpoint of the elastic properties and the compensation of lattice distortion between neighboring domains.

  18. Anticoagulation management during cross-clamping and bypass.

    PubMed

    Lander, H; Zammert, M; FitzGerald, D

    2016-09-01

    Anticoagulation is required for successful implementation of cardiopulmonary bypass (CPB), as well as for surgeries requiring temporary aortic occlusion. It is well established that both coagulation and fibrinolysis are activated during CPB (Teufelsbauer et al., 1992) [1]. Appropriate dosing, monitoring, and maintenance of anticoagulation are essential to prevent devastating thrombosis of the CPB circuit or the occluded aorta and to minimize the activation of the hemostatic system. Although numerous novel anticoagulants have been developed over the past decade, unfractionated heparin remains the primary anticoagulant utilized during these types of procedures, with monitoring systems primarily based upon the activated clotting time and/or heparin concentration. This article will review the current state of anticoagulation management during cross-clamp and CPB. PMID:27650345

  19. Biological cell controllable patch-clamp microchip

    NASA Astrophysics Data System (ADS)

    Penmetsa, Siva; Nagrajan, Krithika; Gong, Zhongcheng; Mills, David; Que, Long

    2010-12-01

    A patch-clamp (PC) microchip with cell sorting and positioning functions is reported, which can avoid drawbacks of random cell selection or positioning for a PC microchip. The cell sorting and positioning are enabled by air bubble (AB) actuators. AB actuators are pneumatic actuators, in which air pressure is generated by microheaters within sealed microchambers. The sorting, positioning, and capturing of 3T3 cells by this type of microchip have been demonstrated. Using human breast cancer cells MDA-MB-231 as the model, experiments have been demonstrated by this microchip as a label-free technical platform for real-time monitoring of the cell viability.

  20. Relative motion of transmembrane segments S0 and S4 during voltage sensor activation in the human BK(Ca) channel.

    PubMed

    Pantazis, Antonios; Kohanteb, Azadeh P; Olcese, Riccardo

    2010-12-01

    Large-conductance voltage- and Ca(2+)-activated K(+) (BK(Ca)) channel α subunits possess a unique transmembrane helix referred to as S0 at their N terminus, which is absent in other members of the voltage-gated channel superfamily. Recently, S0 was found to pack close to transmembrane segments S3 and S4, which are important components of the BK(Ca) voltage-sensing apparatus. To assess the role of S0 in voltage sensitivity, we optically tracked protein conformational rearrangements from its extracellular flank by site-specific labeling with an environment-sensitive fluorophore, tetramethylrhodamine maleimide (TMRM). The structural transitions resolved from the S0 region exhibited voltage dependence similar to that of charge-bearing transmembrane domains S2 and S4. The molecular determinant of the fluorescence changes was identified in W203 at the extracellular tip of S4: at hyperpolarized potential, W203 quenches the fluorescence of TMRM labeling positions at the N-terminal flank of S0. We provide evidence that upon depolarization, W203 (in S4) moves away from the extracellular region of S0, lifting its quenching effect on TMRM fluorescence. We suggest that S0 acts as a pivot component against which the voltage-sensitive S4 moves upon depolarization to facilitate channel activation.

  1. Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3'-5' exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp.

    PubMed

    Khanam, Taran; Rai, Niyati; Ramachandran, Ravishankar

    2015-10-01

    The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel 239 QLRFPKK245 motif in the DNA-binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding-groove (PBG) and the C-terminal of β-clamp located on different domains interact with XthA. The β-clamp-XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β-clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence.

  2. The ethylene bis-dithiocarbamate fungicide Mancozeb activates voltage-gated KCNQ2 potassium channel.

    PubMed

    Li, Ping; Zhu, Jin; Kong, Qingya; Jiang, Baifeng; Wan, Xia; Yue, Jinfeng; Li, Min; Jiang, Hualiang; Li, Jian; Gao, Zhaobing

    2013-06-01

    Mancozeb (manganese/zinc ethylene bis-dithiocarbamate) is an organometallic fungicide that has been associated with human neurotoxicity and neurodegeneration. In a high-throughput screen for modulators of KCNQ2 channel, a fundamental player modulating neuronal excitability, Mancozeb, was found to significantly potentiate KCNQ2 activity. Mancozeb was validated electrophysiologically as a KCNQ2 activator with an EC50 value of 0.92±0.23μM. Further examination showed that manganese but not zinc ethylene bis-dithiocarbamate is the active component for the positive modulation effects. In addition, the compounds are effective when the metal ions are substituted by iron but lack potentiation activity when the metal ions are substituted by sodium, signifying the importance of the metal ion. However, the iron (Fe(3+)) alone, organic ligands alone or the mixture of iron with the organic ligand did not show any potentiation effect, suggesting as the active ingredient is a specific complex rather than two separate additive or synergistic components. Our study suggests that potentiation on KCNQ2 potassium channels might be the possible mechanism of Mancozeb toxicity in the nervous system. PMID:23542819

  3. Response of delayed (K+) channels to the time-dependent clamping function in squid giant axon. I. Ascending ramps.

    PubMed

    Starzak, M E; Senft, J P; Starzak, R J

    1977-01-01

    Squid giant axons are voltage-clamped with ascending potential ramps whose slopes range from 0.5 mV/msec to 60 mV/msec and delayed (K+) currents are observed. The parametric current-voltage curves exhibit a delay period of minimal current followed by a rapid increase of current toward a final steady state. Both the initial delay and the slope of the subsequent rising phase increase with increasing ramp slope. When the Hodgkin-Huxley equations are used to generate theoretical current-voltage curves, the sharp difference between the delay and rising phases is muted and the ramp slope must be increased to produce an adequate representation of the data. A muted biphasic response is also observed when the current-voltage curves are generated using modified Hodgkin-Huxley parameters and a correction for K+ accumulation in the periaxonal space. These modified equations provide an accurate fit for step-potential clamp current data. Since the ramp experiments include all relevant clamping potentials, the experiments provide a sensitive test for kinetic models of K+ on flow in the delayed (K+) channels of squid giant axon.

  4. Voltage-dependent conductance changes in the store-operated Ca2+ current ICRAC in rat basophilic leukaemia cells

    PubMed Central

    Bakowski, Daniel; Parekh, Anant B

    2000-01-01

    Tight-seal whole-cell patch-clamp experiments were carried out in order to investigate the effects of different holding potentials on the rate of development and amplitude of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia (RBL-1) cells. ICRAC was monitored at −80 mV from fast voltage ramps, spanning 200 mV in 50 ms. At hyperpolarised potentials, the macroscopic CRAC conductance was lower than that seen at depolarised potentials. The conductance increased almost 5-fold over the voltage range −60 to +40 mV and was seen when the stores were depleted either by the combination of IP3 and thapsigargin in high Ca2+ buffer, or passively with 10 mm EGTA or BAPTA. The voltage-dependent conductance of the CRAC channels could not be fully accounted for by Ca2+-dependent fast inactivation, nor by other slower inhibitory mechanisms. It also did not seem to involve intracellular Mg2+ or the polycations spermine and spermidine. Voltage step relaxation experiments revealed that the voltage-dependent conductance changes developed and reversed slowly, with a time constant of several seconds at −60 mV. In the presence of physiological levels of intracellular Ca2+ buffers, ICRAC was barely detectable when cells were clamped at −60 mV and dialysed with IP3 and thapsigargin, but at 0 mV the current in low Ca2+ buffer was as large as that seen in high Ca2+ buffer. Our results suggest that CRAC channels exhibit slow voltage-dependent conductance changes which can triple the current amplitude over the physiological range of voltages normally encountered by these cells. The role of this conductance change and possible underlying mechanisms are discussed. PMID:11101641

  5. α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor activation protects against phencyclidine-induced caspase-3 activity by activating voltage-gated calcium channels.

    PubMed

    Timpe, Jennifer M; Wang, Cheng Z; Kim, Jisoo; Johnson, Kenneth M

    2014-12-01

    Phencyclidine (PCP) is a noncompetitive, open channel blocker of the N-methyl-D-aspartate (NMDA) receptor-ion channel complex. When administered to immature animals, it is known to cause apoptotic neurodegeneration in several regions, and this is followed by olanzapine-sensitive, schizophrenia-like behaviors in late adolescence and adulthood. Clarification of its mechanism of action could yield data that would help to inform the treatment of schizophrenia. In our initial experiments, we found that α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) inhibited PCP-induced apoptosis in organotypic neonatal rat brain slices in a concentration-dependent and 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive manner. Calcium signaling pathways are widely implicated in apoptosis, and PCP prevents calcium influx through NMDA receptor channels. We therefore hypothesized that AMPA could protect against this effect by activation of voltage-dependent calcium channels (VDCCs). In support of this hypothesis, pretreatment with the calcium channel blocker cadmium chloride eliminated AMPA-mediated protection against PCP. Furthermore, the L-type VDCC inhibitor nifedipine (10 µM) fully abrogated the effects of AMPA, suggesting that L-type VDCCs are required for AMPA-mediated protection against PCP-induced neurotoxicity. Whereas the P/Q-type inhibitor ω-agatoxin TK (200 nM) reduced AMPA protection by 51.7%, the N-type VDCC inhibitor ω-conotoxin (2 µM) had no effect. Decreased AMPA-mediated protection following cotreatment with K252a, a TrkB inhibitor, suggests that brain-derived neurotrophic factor signaling plays an important role. By analogy, these results suggest that activation of L-type, and to a lesser extent P/Q-type, VDCCs might be advantageous in treating conditions associated with diminished NMDAergic activity during early development. PMID:24995437

  6. In Vivo Mesoscopic Voltage-Sensitive Dye Imaging of Brain Activation

    PubMed Central

    Tang, Qinggong; Tsytsarev, Vassiliy; Frank, Aaron; Wu, Yalun; Chen, Chao-wei; Erzurumlu, Reha S.; Chen, Yu

    2016-01-01

    Functional mapping of brain activity is important in elucidating how neural networks operate in the living brain. The whisker sensory system of rodents is an excellent model to study peripherally evoked neural activity in the central nervous system. Each facial whisker is represented by discrete modules of neurons all along the pathway leading to the neocortex. These modules are called “barrels” in layer 4 of the primary somatosensory cortex. Their location (approximately 300–500 μm below cortical surface) allows for convenient imaging of whisker-evoked neural activity in vivo. Fluorescence laminar optical tomography (FLOT) provides depth-resolved fluorescence molecular information with an imaging depth of a few millimeters. Angled illumination and detection configurations can improve both resolution and penetration depth. We applied angled FLOT (aFLOT) to record 3D neural activities evoked in the whisker system of mice by deflection of a single whisker in vivo. A 100 μm capillary and a pair of microelectrodes were inserted to the mouse brain to test the capability of the imaging system. The results show that it is possible to obtain 3D functional maps of the sensory periphery in the brain. This approach can be broadly applicable to functional imaging of other brain structures. PMID:27125318

  7. In Vivo Mesoscopic Voltage-Sensitive Dye Imaging of Brain Activation

    NASA Astrophysics Data System (ADS)

    Tang, Qinggong; Tsytsarev, Vassiliy; Frank, Aaron; Wu, Yalun; Chen, Chao-Wei; Erzurumlu, Reha S.; Chen, Yu

    2016-04-01

    Functional mapping of brain activity is important in elucidating how neural networks operate in the living brain. The whisker sensory system of rodents is an excellent model to study peripherally evoked neural activity in the central nervous system. Each facial whisker is represented by discrete modules of neurons all along the pathway leading to the neocortex. These modules are called “barrels” in layer 4 of the primary somatosensory cortex. Their location (approximately 300–500 μm below cortical surface) allows for convenient imaging of whisker-evoked neural activity in vivo. Fluorescence laminar optical tomography (FLOT) provides depth-resolved fluorescence molecular information with an imaging depth of a few millimeters. Angled illumination and detection configurations can improve both resolution and penetration depth. We applied angled FLOT (aFLOT) to record 3D neural activities evoked in the whisker system of mice by deflection of a single whisker in vivo. A 100 μm capillary and a pair of microelectrodes were inserted to the mouse brain to test the capability of the imaging system. The results show that it is possible to obtain 3D functional maps of the sensory periphery in the brain. This approach can be broadly applicable to functional imaging of other brain structures.

  8. Optimization of a clamped plate silencer.

    PubMed

    Wang, Chunqi; Han, Jun; Huang, Lixi

    2007-02-01

    A previous theoretical study [L. Huang, J. Acoust. Soc. Am. 119, 2628-2638 (2006)] shows that, in a duct, a simply supported plate covering a side-branch rigid cavity can function effectively as a wave reflector over a broad range of low to medium frequencies. In this study, analytical formulation is extended to the boundary condition of clamped plate, which is easier to implement in practice. The theoretical model is tested experimentally using balsawood, which has a very high stiffness to mass ratio. The spectral peaks and shapes of the measured TL are in agreement with those calculated theoretically, attempts are also made to account for the considerable sound absorption in the rig. Further numerical studies based on the validated model show that, for a uniform plate, the optimal stopband is narrower and the lower band limit is worse than that of the simply supported configuration. However, a wave reflector using nonuniform, clamped plates with thinner ends out-performs the simply supported configuration in every aspect. Analyses show that the improvement is attributed to the increased acoustic radiation efficiency over the bulk length of the nonuniform plate, which behaves more like a rigid plate.

  9. Laser-assisted patch clamping: a methodology

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

    Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

  10. Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

    PubMed Central

    Jung, Sangyong; Oshima-Takago, Tomoko; Chakrabarti, Rituparna; Wong, Aaron B.; Jing, Zhizi; Yamanbaeva, Gulnara; Picher, Maria Magdalena; Wojcik, Sonja M.; Göttfert, Fabian; Predoehl, Friederike; Michel, Katrin; Hell, Stefan W.; Schoch, Susanne; Strenzke, Nicola; Wichmann, Carolin; Moser, Tobias

    2015-01-01

    Ca2+ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated CaV1.3 Ca2+ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca2+ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic CaV1.3 Ca2+ channels, resulting in reduced synaptic Ca2+ influx. Using superresolution microscopy, we found that Ca2+ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca2+ current, whereas the apparent Ca2+ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca2+ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca2+ channels at IHC AZs and are required for normal hearing. PMID:26034270

  11. Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells.

    PubMed

    Pappone, P A; Lucero, M T

    1988-06-01

    We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.

  12. Effects of intermittent 60-Hz high voltage electric fields on metabolism, activity, and temperature in mice

    SciTech Connect

    Rosenbergy, R.S; Duffy, P.H.; Sacher, G.A.

    1981-01-01

    Transient effects of 100-kV/m extremely low frequency electric fields were studied in the white footed deermouse, Peromyscus leucopus. Gross motor activity, carbon dioxide production, oxygen consumption, and core body temperature were monitored before, during, and after intermittent field exposures (four hour-long exposures, at one-hour intervals). Thirty-four mice were exposed in cages with plastic floors floating above ground potential, and 21 mice were exposed in cages with grounded metal floor plates. The first field exposure produced an immediate, transient increase of activity and gas measures during the inactive phase of the circadian cycle. All measures returned to baseline levels before the second exposure and were not significantly changed throughout the remainder of the exposures. The rapid habituation of field-induced arousal suggests that significant metabolic changes will not be measured in experiments in which the interval between exposure and measurement is greater than two hours.

  13. Attachment ability of a clamp-bearing fish parasite, Diplozoon paradoxum (Monogenea), on gills of the common bream, Abramis brama.

    PubMed

    Wong, Wey-Lim; Gorb, Stanislav N

    2013-08-15

    Monogeneans, which are mainly fish ectoparasites, use various types of haptoral (posterior) attachment apparatus to secure their attachment onto their hosts. However, it remains unclear how strongly a monogenean can attach onto its host. In the present study, we aimed for the first time to (1) measure pull-off forces required to detach a pair of clamp-bearing monogeneans, Diplozoon paradoxum, from gills of Abramis brama and (2) determine the contribution of muscles to the clamp movements. A mean force of 6.1±2.7 mN (~246 times the animals' weight) was required to dislodge a paired D. paradoxum vertically from the gills. There were significant differences (P<0.05, Tukey test) between the widths of clamp openings in D. paradoxum treated in three different solutions: the widest clamp openings were observed in the monogeneans treated in 100 mmol l(-1) potassium chloride solution (58.26±13.44 μm), followed by those treated in 20 mmol l(-1) magnesium chloride solution (37.91±7.58 μm), and finally those treated in filtered lake water (20.16±8.63 μm). This suggests that the closing of the clamps is probably not due to the continuous contraction of extrinsic muscles but is caused by the elasticity of the clamp material and that muscle activity is required for clamp opening.

  14. 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

  15. Response reliability observed with voltage-sensitive dye imaging of cortical layer 2/3: the probability of activation hypothesis.

    PubMed

    Gollnick, Clare A; Millard, Daniel C; Ortiz, Alexander D; Bellamkonda, Ravi V; Stanley, Garrett B

    2016-06-01

    A central assertion in the study of neural processing is that our perception of the environment directly reflects the activity of our sensory neurons. This assertion reinforces the intuition that the strength of a sensory input directly modulates the amount of neural activity observed in response to that sensory feature: an increase in the strength of the input yields a graded increase in the amount of neural activity. However, cortical activity across a range of sensory pathways can be sparse, with individual neurons having remarkably low firing rates, often exhibiting suprathreshold activity on only a fraction of experimental trials. To compensate for this observed apparent unreliability, it is assumed that instead the local population of neurons, although not explicitly measured, does reliably represent the strength of the sensory input. This assumption, however, is largely untested. In this study, using wide-field voltage-sensitive dye (VSD) imaging of the somatosensory cortex in the anesthetized rat, we show that whisker deflection velocity, or stimulus strength, is not encoded by the magnitude of the population response at the level of cortex. Instead, modulation of whisker deflection velocity affects the likelihood of the cortical response, impacting the magnitude, rate of change, and spatial extent of the cortical response. An ideal observer analysis of the cortical response points to a probabilistic code based on repeated sampling across cortical columns and/or time, which we refer to as the probability of activation hypothesis. This hypothesis motivates a range of testable predictions for both future electrophysiological and future behavioral studies. PMID:26864758

  16. Bothriurus bonariensis scorpion venom activates voltage-dependent sodium channels in insect and mammalian nervous systems.

    PubMed

    Dos Santos, Douglas Silva; Carvalho, Evelise Leis; de Lima, Jeferson Camargo; Breda, Ricardo Vaz; Oliveira, Raquel Soares; de Freitas, Thiago Carrazoni; Salamoni, Simone Denise; Domingues, Michelle Flores; Piovesan, Angela Regina; Boldo, Juliano Tomazzoni; de Assis, Dênis Reis; da Costa, Jaderson Costa; Dal Belo, Cháriston André; Pinto, Paulo Marcos

    2016-10-25

    Animal venoms have been widely recognized as a major source of biologically active molecules. Bothriurus bonariensis, popularly known as black scorpion, is the arthropod responsible for the highest number of accidents involving scorpion sting in Southern Brazil. Here we reported the first attempt to investigate the neurobiology of B. bonariensis venom (BBV) in the insect and mammalian nervous system. BBV (32 μg/g) induced a slow neuromuscular blockade in the in vivo cockroach nerve-muscle preparations (70 ± 4%, n = 6, p < 0.001), provoking repetitive twitches and significantly decreasing the frequency of spontaneous leg action potentials (SNCAPs) from 82 ± 3 min(-1) to 36 ± 1.3 min(-1) (n = 6, p < 0.05), without affecting the amplitude. When tested in primary cultures of rat hippocampal cells, BBV induced a massive increase of Ca(2+) influx (250 ± 1% peak increase, n = 3, p < 0.0001). The disturbance of calcium homeostasis induced by BBV on the mammalian central nervous system was not accompanied by cellular death and was prevented by the co-treatment of the hippocampal cells with tetrodotoxin, a selective sodium channel blocker. The results suggest that the biological activity of BBV is mostly related to a modulation of sodium channels function. Our biological activity survey suggests that BBV may have a promising insecticidal and therapeutic potential. PMID:27544632

  17. Voltage clustering in redox-active ligand complexes: mitigating electronic communication through choice of metal ion

    DOE PAGES

    Zarkesh, Ryan A.; Ichimura, Andrew S.; Monson, Todd C.; Tomson, Neil C.; Anstey, Mitchell R.

    2016-02-01

    We used the redox-active bis(imino)acenapthene (BIAN) ligand to synthesize homoleptic aluminum, chromium, and gallium complexes of the general formula (BIAN)3M. The resulting compounds were characterized using X-ray crystallography, NMR, EPR, magnetic susceptibility and cyclic voltammetry measurements and modeled using both DFT and ab initio wavefunction calculations to compare the orbital contributions of main group elements and transition metals in ligand-based redox events. Ultimately, complexes of this type have the potential to improve the energy density and electrolyte stability of grid-scale energy storage technologies, such as redox flow batteries, through thermodynamically-clustered redox events.

  18. Voltage clustering in redox-active ligand complexes: mitigating electronic communication through choice of metal ion.

    PubMed

    Zarkesh, Ryan A; Ichimura, Andrew S; Monson, Todd C; Tomson, Neil C; Anstey, Mitchell R

    2016-06-14

    The redox-active bis(imino)acenapthene (BIAN) ligand was used to synthesize homoleptic aluminum, chromium, and gallium complexes of the general formula (BIAN)3M. The resulting compounds were characterized using X-ray crystallography, NMR, EPR, magnetic susceptibility and cyclic voltammetry measurements and modeled using both DFT and ab initio wavefunction calculations to compare the orbital contributions of main group elements and transition metals in ligand-based redox events. Complexes of this type have the potential to improve the energy density and electrolyte stability of grid-scale energy storage technologies, such as redox flow batteries, through thermodynamically-clustered redox events. PMID:26998892

  19. Active-Matrix Organic Light Emission Diode Pixel Circuit for Suppressing and Compensating for the Threshold Voltage Degradation of Hydrogenated Amorphous Silicon Thin Film Transistors

    NASA Astrophysics Data System (ADS)

    Shin, Hee-Sun; Lee, Won-Kyu; Park, Sang-Guen; Kuk, Seung-Hee; Han, Min-Koo

    2009-03-01

    A new hydrogenated amorphous silicon (a-Si:H) thin film transistor (TFT) pixel circuit for active-matrix organic light emission diodes (AM-OLEDs), which significantly compensates the OLED current degradation by memorizing the threshold voltage of driving TFT and suppresses the threshold voltage shift of a-Si:H TFTs by negative bias annealing, is proposed and fabricated. During the first half of each frame, the driving TFT of the proposed pixel circuit supplies current to the OLED, which is determined by modified data voltage in the compensation scheme. The proposed pixel circuit was able to compensate the threshold voltage shift of the driving TFT as well as the OLED. During the remaining half of each frame, the proposed pixel circuit induces the recovery of the threshold voltage degradation of a-Si:H TFTs owing to the negative bias annealing. The experimental results show that the proposed pixel circuit was able to successfully compensate for the OLED current degradation and suppress the threshold voltage degradation of the driving TFT.

  20. Patch-clamp array with on-chip electronics, optics, flow control and mechanical actuation.

    SciTech Connect

    James, Conrad D.; Okandan, Murat; Draper, Bruce Leroy; Mani, Seethambal S.

    2003-07-01

    Fast and quantitative analysis of cellular activity, signaling and responses to external stimuli is a crucial capability and it has been the goal of several projects focusing on patch clamp measurements. To provide the maximum functionality and measurement options, we have developed a patch clamp array device that incorporates on-chip electronics, mechanical, optical and microfluidic coupling as well as cell localization through fluid flow. The preliminary design, which integrated microfluidics, electrodes and optical access, was fabricated and tested. In addition, new designs which further combine mechanical actuation, on-chip electronics and various electrode materials with the previous designs are currently being fabricated.

  1. Voltage dependence of Hodgkin-Huxley rate functions for a multistage K+ channel voltage sensor within a membrane

    NASA Astrophysics Data System (ADS)

    Vaccaro, S. R.

    2014-11-01

    The activation of a K+channel sensor in two sequential stages during a voltage clamp may be described as the translocation of a Brownian particle in an energy landscape with two large barriers between states. A solution of the Smoluchowski equation for a square-well approximation to the potential function of the S4 voltage sensor satisfies a master equation and has two frequencies that may be determined from the forward and backward rate functions. When the higher-frequency terms have small amplitude, the solution reduces to the relaxation of a rate equation, where the derived two-state rate functions are dependent on the relative magnitude of the forward rates (α and γ ) and the backward rates (β and δ ) for each stage. In particular, the voltage dependence of the Hodgkin-Huxley rate functions for a K+channel may be derived by assuming that the rate functions of the first stage are large relative to those of the second stage—α ≫γ and β ≫δ . For a Shaker IR K+ channel, the first forward and backward transitions are rate limiting (α <γ and δ ≪β ), and for an activation process with either two or three stages, the derived two-state rate functions also have a voltage dependence that is of a similar form to that determined for the squid axon. The potential variation generated by the interaction between a two-stage K+ ion channel and a noninactivating Na+ ion channel is determined by the master equation for K+channel activation and the ionic current equation when the Na+channel activation time is small, and if β ≪δ and α ≪γ , the system may exhibit a small amplitude oscillation between spikes, or mixed-mode oscillation, in which the slow closed state modulates the K+ ion channel conductance in the membrane.

  2. Voltage-gated Sodium Channel Activity Promotes Cysteine Cathepsin-dependent Invasiveness and Colony Growth of Human Cancer Cells.

    PubMed

    Gillet, Ludovic; Roger, Sébastien; Besson, Pierre; Lecaille, Fabien; Gore, Jacques; Bougnoux, Philippe; Lalmanach, Gilles; Le Guennec, Jean-Yves

    2009-03-27

    Voltage-gated sodium channels (Na(V)) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of Na(V)1.5 and associated auxiliary beta-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role ( approximately 65%) in cancer invasiveness. Matrigel invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on Na(V) function. Inhibiting Na(V) has no consequence for cathepsins at the transcription, translation, and secretion levels. However, Na(V) activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Na(v) enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases. PMID:19176528

  3. Spectral infrared hemispherical reflectance measurements for LDEF tray clamps

    NASA Technical Reports Server (NTRS)

    Cromwell, B. K.; Shepherd, S. D.; Pender, C. W.; Wood, B. E.

    1993-01-01

    Infrared hemispherical reflectance measurements that were made on 58 chromic acid anodized tray clamps from LDEF are described. The measurements were made using a hemiellipsoidal mirror reflectometer with interferometer for wavelengths between 2-15 microns. The tray clamps investigated were from locations about the entire spacecraft and provided the opportunity for comparing the effects of atomic oxygen at each location. Results indicate there was essentially no dependence on atomic oxygen fluence for the surfaces studied, but there did appear to be a slight dependence on solar radiation exposure. The reflectances of the front sides of the tray clamps consistently were slightly higher than for the protected rear tray clamp surfaces.

  4. An Optimal Cell Detection Technique for Automated Patch Clamping

    NASA Technical Reports Server (NTRS)

    McDowell, Mark; Gray, Elizabeth

    2004-01-01

    While there are several hardware techniques for the automated patch clamping of cells that describe the equipment apparatus used for patch clamping, very few explain the science behind the actual technique of locating the ideal cell for a patch clamping procedure. We present a machine vision approach to patch clamping cell selection by developing an intelligent algorithm technique that gives the user the ability to determine the good cell to patch clamp in an image within one second. This technique will aid the user in determining the best candidates for patch clamping and will ultimately save time, increase efficiency and reduce cost. The ultimate goal is to combine intelligent 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 amount of human intervention. We present a unique technique that identifies good patch clamping cell candidates based on feature metrics of a cell's (x, y) position, major axis length, minor axis length, area, elongation, roundness, smoothness, angle of orientation, thinness and whether or not the cell is only particularly in the field of view. A patent is pending for this research.

  5. Structure-function analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin signaling.

    PubMed

    Ravindranath, Aditi J; Ravindranath, Aditi; Cadigan, Ken M

    2014-01-01

    The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila. PMID:24465946

  6. Structural Features and Functional Dependency on β-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL.

    PubMed

    Fukui, Kenji; Baba, Seiki; Kumasaka, Takashi; Yano, Takato

    2016-08-12

    In early reactions of DNA mismatch repair, MutS recognizes mismatched bases and activates MutL endonuclease to incise the error-containing strand of the duplex. DNA sliding clamp is responsible for directing the MutL-dependent nicking to the newly synthesized/error-containing strand. In Bacillus subtilis MutL, the β-clamp-interacting motif (β motif) of the C-terminal domain (CTD) is essential for both in vitro direct interaction with β-clamp and in vivo repair activity. A large cluster of negatively charged residues on the B. subtilis MutL CTD prevents nonspecific DNA binding until β clamp interaction neutralizes the negative charge. We found that there are some bacterial phyla whose MutL endonucleases lack the β motif. For example, the region corresponding to the β motif is completely missing in Aquifex aeolicus MutL, and critical amino acid residues in the β motif are not conserved in Thermus thermophilus MutL. We then revealed the 1.35 Å-resolution crystal structure of A. aeolicus MutL CTD, which lacks the β motif but retains the metal-binding site for the endonuclease activity. Importantly, there was no negatively charged cluster on its surface. It was confirmed that CTDs of β motif-lacking MutLs, A. aeolicus MutL and T. thermophilus MutL, efficiently incise DNA even in the absence of β-clamp and that β-clamp shows no detectable enhancing effect on their activity. In contrast, CTD of Streptococcus mutans, a β motif-containing MutL, required β-clamp for the digestion of DNA. We propose that MutL endonucleases are divided into three subfamilies on the basis of their structural features and dependence on β-clamp. PMID:27369079

  7. Structure-Function Analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin Signaling

    PubMed Central

    Ravindranath, Aditi; Cadigan, Ken M.

    2014-01-01

    The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila. PMID:24465946

  8. Structure-function analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin signaling.

    PubMed

    Ravindranath, Aditi J; Ravindranath, Aditi; Cadigan, Ken M

    2014-01-01

    The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila.

  9. Voltage-dependent K channels in protoplasts of trap-lobe cells of Dionaea muscipula.

    PubMed

    Iijima, T; Hagiwara, S

    1987-01-01

    The outward rectification of the K+ current in mesophyll cell protoplasts from trap-lobes of Dionaea muscipula was studied with the patch-clamp technique. The rectification had instantaneous and time-dependent components. Changes in [K+]i strongly affected the conductance voltage relation of the plasma membrane while changes in [K+]o had little effect on the relation. Thus, the outward rectification depends on the membrane voltage and the concentration of intracellular K+. Corresponding single-channel activities were observed both in the intact membrane (cell-attached recording) and in excised patches. The single-channel conductance was about 3.3 pS with symmetrical solutions containing 30 mM K+.

  10. Suppression of voltage-gated Na(+) channels and neuronal excitability by imperatorin.

    PubMed

    Wu, King-Chuen; Chen, Yi-Hung; Cheng, Ka-Shun; Kuo, Yueh-Hsiung; Yang, Chin-Tsang; Wong, Kar-Lok; Tu, Yuan-Kun; Chan, Paul; Leung, Yuk-Man

    2013-12-01

    Imperatorin is a naturally occurring furocoumarin compound isolated from plants such as Angelica archangelica and Cnidium monnieri. It has multiple pharmacological effects including anticonvulsant effects. Here we determined the effects of imperatorin on voltage-gated Na(+) channels (VGSC) using whole-cell patch clamp techniques in differentiated neuronal NG108-15 cells. We showed that imperatorin inhibited VGSC; such inhibition did not show state-dependence. Imperatorin caused a left shift in the steady-state inactivation curve without affecting activation gating. The inhibition of VGSC by imperatorin displayed a mild frequency-dependence. Imperatorin was also shown to inhibit VGSC and action potential amplitude without affecting voltage-gated K(+) channels in rat hippocampal CA1 neurons. In conclusion, our results suggest that imperatorin dampens neuronal excitability by inhibiting VGSC. PMID:24113522

  11. Electrodic voltages in the presence of dissolved sulfide: Implications for monitoring natural microbial activity

    SciTech Connect

    Slater, L.; Ntarlagiannis, D.; Yee, N.; O'Brien, M.; Zhang, C.; Williams, K. H.

    2008-10-01

    There is growing interest in the development of new monitoring strategies for obtaining spatially extensive data diagnostic of microbial processes occurring in the earth. Open-circuit potentials arising from variable redox conditions in the fluid local-to-electrode surfaces (electrodic potentials) were recorded for a pair of silver-silver chloride electrodes in a column experiment, whereby a natural wetland soil containing a known community of sulfate reducers was continuously fed with a sulfate-rich nutrient medium. Measurements were made between five electrodes equally spaced along the column and a reference electrode placed on the column inflow. The presence of a sulfate reducing microbial population, coupled with observations of decreasing sulfate levels, formation of black precipitate (likely iron sulfide),elevated solid phase sulfide, and a characteristic sulfurous smell, suggest microbial-driven sulfate reduction (sulfide generation) in our column. Based on the known sensitivity of a silver electrode to dissolved sulfide concentration, we interpret the electrodic potentials approaching 700 mV recorded in this experiment as an indicator of the bisulfide (HS-) concentration gradients in the column. The measurement of the spatial and temporal variation in these electrodic potentials provides a simple and rapid method for monitoring patterns of relative HS- concentration that are indicative of the activity of sulfate-reducing bacteria. Our measurements have implications both for the autonomous monitoring of anaerobic microbial processes in the subsurface and the performance of self-potential electrodes, where it is critical to isolate, and perhaps quantify, electrochemical interfaces contributing to observed potentials.

  12. Imaging voltage in neurons

    PubMed Central

    Peterka, Darcy S.; Takahashi, Hiroto; Yuste, Rafael

    2011-01-01

    In the last decades, imaging membrane potential has become a fruitful approach to study neural circuits, especially in invertebrate preparations with large, resilient neurons. At the same time, particularly in mammalian preparations, voltage imaging methods suffer from poor signal to noise and secondary side effects, and they fall short of providing single-cell resolution when imaging of the activity of neuronal populations. As an introduction to these techniques, we briefly review different voltage imaging methods (including organic fluorophores, SHG chromophores, genetic indicators, hybrid, nanoparticles and intrinsic approaches), and illustrate some of their applications to neuronal biophysics and mammalian circuit analysis. We discuss their mechanisms of voltage sensitivity, from reorientation, electrochromic or electro-optical phenomena, to interaction among chromophores or membrane scattering, and highlight their advantages and shortcomings, commenting on the outlook for development of novel voltage imaging methods. PMID:21220095

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

    SciTech Connect

    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 on 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.

  14. The α2δ-1 subunit remodels CaV1.2 voltage sensors and allows Ca2+ influx at physiological membrane potentials.

    PubMed

    Savalli, Nicoletta; Pantazis, Antonios; Sigg, Daniel; Weiss, James N; Neely, Alan; Olcese, Riccardo

    2016-08-01

    Excitation-evoked calcium influx across cellular membranes is strictly controlled by voltage-gated calcium channels (CaV), which possess four distinct voltage-sensing domains (VSDs) that direct the opening of a central pore. The energetic interactions between the VSDs and the pore are critical for tuning the channel's voltage dependence. The accessory α2δ-1 subunit is known to facilitate CaV1.2 voltage-dependent activation, but the underlying mechanism is unknown. In this study, using voltage clamp fluorometry, we track the activation of the four individual VSDs in a human L-type CaV1.2 channel consisting of α1C and β3 subunits. We find that, without α2δ-1, the channel complex displays a right-shifted voltage dependence such that currents mainly develop at nonphysiological membrane potentials because of very weak VSD-pore interactions. The presence of α2δ-1 facilitates channel activation by increasing the voltage sensitivity (i.e., the effective charge) of VSDs I-III. Moreover, the α2δ-1 subunit also makes VSDs I-III more efficient at opening the channel by increasing the coupling energy between VSDs II and III and the pore, thus allowing Ca influx within the range of physiological membrane potentials. PMID:27481713

  15. Space Life Sciences Directorate's Position on the Physiological Effects of Exposing the Crewmemeber to Low-Voltage Electrical Hazards During Extravehicular Activity

    NASA Technical Reports Server (NTRS)

    Hamilton, Douglas; Kramer, Leonard; Mikatarian, Ron; Polk, James; Duncan, Michael; Koontz, Steven

    2010-01-01

    The models predict that, for low voltage exposures in the space suit, physiologically active current could be conducted across the crew member causing catastrophic hazards. Future work with Naval Health Research Center Detachment Directed Energy Bio-effects Laboratory is being proposed to analyze additional current paths across the human torso and upper limbs. These models may need to be verified with human studies.

  16. Mild Alkalization Acutely Triggers the Warburg Effect by Enhancing Hexokinase Activity via Voltage-Dependent Anion Channel Binding.

    PubMed

    Quach, Cung Hoa Thien; Jung, Kyung-Ho; Lee, Jin Hee; Park, Jin Won; Moon, Seung Hwan; Cho, Young Seok; Choe, Yearn Seong; Lee, Kyung-Han

    2016-01-01

    To fully understand the glycolytic behavior of cancer cells, it is important to recognize how it is linked to pH dynamics. Here, we evaluated the acute effects of mild acidification and alkalization on cancer cell glucose uptake and glycolytic flux and investigated the role of hexokinase (HK). Cancer cells exposed to buffers with graded pH were measured for 18F-fluorodeoxyglucose (FDG) uptake, lactate production and HK activity. Subcellular localization of HK protein was assessed by western blots and confocal microscopy. The interior of T47D breast cancer cells was mildly alkalized to pH 7.5 by a buffer pH of 7.8, and this was accompanied by rapid increases of FDG uptake and lactate extrusion. This shift toward glycolytic flux led to the prompt recovery of a reversed pH gradient. In contrast, mild acidification rapidly reduced cellular FDG uptake and lactate production. Mild acidification decreased and mild alkalization increased mitochondrial HK translocation and enzyme activity. Cells transfected with specific siRNA against HK-1, HK-2 and voltage-dependent anion channel (VDAC)1 displayed significant attenuation of pH-induced changes in FDG uptake. Confocal microscopy showed increased co-localization of HK-1 and HK-2 with VDAC1 by alkaline treatment. In isolated mitochondria, acidic pH increased and alkaline pH decreased release of free HK-1 and HK-2 from the mitochondrial pellet into the supernatant. Furthermore, experiments using purified proteins showed that alkaline pH promoted co-immunoprecipitation of HK with VDAC protein. These findings demonstrate that mild alkalization is sufficient to acutely trigger cancer cell glycolytic flux through enhanced activity of HK by promoting its mitochondrial translocation and VDAC binding. This process might serve as a mechanism through which cancer cells trigger the Warburg effect to maintain a dysregulated pH. PMID:27479079

  17. Mild Alkalization Acutely Triggers the Warburg Effect by Enhancing Hexokinase Activity via Voltage-Dependent Anion Channel Binding

    PubMed Central

    Lee, Jin Hee; Park, Jin Won; Moon, Seung Hwan; Cho, Young Seok; Choe, Yearn Seong; Lee, Kyung-Han

    2016-01-01

    To fully understand the glycolytic behavior of cancer cells, it is important to recognize how it is linked to pH dynamics. Here, we evaluated the acute effects of mild acidification and alkalization on cancer cell glucose uptake and glycolytic flux and investigated the role of hexokinase (HK). Cancer cells exposed to buffers with graded pH were measured for 18F-fluorodeoxyglucose (FDG) uptake, lactate production and HK activity. Subcellular localization of HK protein was assessed by western blots and confocal microscopy. The interior of T47D breast cancer cells was mildly alkalized to pH 7.5 by a buffer pH of 7.8, and this was accompanied by rapid increases of FDG uptake and lactate extrusion. This shift toward glycolytic flux led to the prompt recovery of a reversed pH gradient. In contrast, mild acidification rapidly reduced cellular FDG uptake and lactate production. Mild acidification decreased and mild alkalization increased mitochondrial HK translocation and enzyme activity. Cells transfected with specific siRNA against HK-1, HK-2 and voltage-dependent anion channel (VDAC)1 displayed significant attenuation of pH-induced changes in FDG uptake. Confocal microscopy showed increased co-localization of HK-1 and HK-2 with VDAC1 by alkaline treatment. In isolated mitochondria, acidic pH increased and alkaline pH decreased release of free HK-1 and HK-2 from the mitochondrial pellet into the supernatant. Furthermore, experiments using purified proteins showed that alkaline pH promoted co-immunoprecipitation of HK with VDAC protein. These findings demonstrate that mild alkalization is sufficient to acutely trigger cancer cell glycolytic flux through enhanced activity of HK by promoting its mitochondrial translocation and VDAC binding. This process might serve as a mechanism through which cancer cells trigger the Warburg effect to maintain a dysregulated pH. PMID:27479079

  18. Spexin Enhances Bowel Movement through Activating L-type Voltage-dependent Calcium Channel via Galanin Receptor 2 in Mice

    PubMed Central

    Lin, Cheng-yuan; Zhang, Man; Huang, Tao; Yang, Li-ling; Fu, Hai-bo; Zhao, Ling; Zhong, Linda LD; Mu, Huai-xue; Shi, Xiao-ke; Leung, Christina FP; Fan, Bao-min; Jiang, Miao; Lu, Ai-ping; Zhu, Li-xin; Bian, Zhao-xiang

    2015-01-01

    A novel neuropeptide spexin was found to be broadly expressed in various endocrine and nervous tissues while little is known about its functions. This study investigated the role of spexin in bowel movement and the underlying mechanisms. In functional constipation (FC) patients, serum spexin levels were significantly decreased. Consistently, in starved mice, the mRNA of spexin was significantly decreased in intestine and colon. Spexin injection increased the velocity of carbon powder propulsion in small intestine and decreased the glass beads expulsion time in distal colon in mice. Further, spexin dose-dependently stimulated the intestinal/colonic smooth muscle contraction. Galanin receptor 2 (GALR2) antagonist M871, but not Galanin receptor 3 (GALR3) antagonist SNAP37899, effectively suppressed the stimulatory effects of spexin on intestinal/colonic smooth muscle contraction, which could be eliminated by extracellular [Ca2+] removal and L-type voltage-dependentCa2+ channel (VDCC) inhibitor nifedipine. Besides, spexin dramatically increased the [Ca2+]i in isolated colonic smooth muscle cells. These data indicate that spexin can act on GALR2 receptor to regulate bowel motility by activating L-type VDCC. Our findings provide evidence for important physiological roles of spexin in GI functions. Selective action on spexin pathway might have therapeutic effects on GI diseases with motility disorders. PMID:26160593

  19. Combination Space Station Handrail Clamp and Pointing Device

    NASA Technical Reports Server (NTRS)

    Hughes, Stephen J. (Inventor)

    1999-01-01

    A device for attaching an experiment carrier to a space station handrail is provided. The device has two major components, a clamping mechanism for attachment to a space station handrail, and a pointing carrier on which an experiment package can be mounted and oriented. The handrail clamp uses an overcenter mechanism and the carrier mechanism uses an adjustable preload ball and socket for carrier positioning. The handrail clamp uses a stack of disk springs to provide a spring loaded button. This configuration provides consistent clamping force over a range of possible handrail thicknesses. Three load points are incorporated in the clamping mechanism thereby spreading the clamping load onto three separate points on the handrail. A four bar linkage is used to provide for a single actuation lever for all three load points. For additional safety, a secondary lock consisting of a capture plate and push lock keeps the clamp attached to the handrail in the event of main clamp failure. For the carrier positioning mechanism, a ball in a spring loaded socket uses friction to provide locking torque; however. the ball and socket are torque limited so that the ball ran slip under kick loads (125 pounds or greater). A lead screw attached to disk spring stacks is used to provide an adjustable spring force on the socket. A locking knob is attached to the lead screw to allow for hand manipulation of the lead screw.

  20. Mitochondria are clamped to vacuoles for lipid transport.

    PubMed

    Klecker, Till; Westermann, Benedikt

    2014-07-14

    In this issue of Developmental Cell, Elbaz-Alon et al. (2014) and Hönscher et al. (2014) describe a contact site between mitochondria and the lysosome-like yeast vacuole named vCLAMP (vacuole and mitochondria patch). They show that vCLAMP plays a role in lipid exchange, thereby linking mitochondria to the endomembrane system.

  1. 21 CFR 876.5160 - Urological clamp for males.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Urological clamp for males. 876.5160 Section 876.5160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for...

  2. 21 CFR 876.5160 - Urological clamp for males.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Urological clamp for males. 876.5160 Section 876.5160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for...

  3. 21 CFR 876.5160 - Urological clamp for males.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Urological clamp for males. 876.5160 Section 876.5160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for...

  4. 21 CFR 876.5160 - Urological clamp for males.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Urological clamp for males. 876.5160 Section 876.5160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for...

  5. 21 CFR 876.5160 - Urological clamp for males.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Urological clamp for males. 876.5160 Section 876.5160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for...

  6. Catch and Patch: A Pipette-Based Approach for Automating Patch Clamp That Enables Cell Selection and Fast Compound Application.

    PubMed

    Danker, Timm; Braun, Franziska; Silbernagl, Nikole; Guenther, Elke

    2016-03-01

    Manual patch clamp, the gold standard of electrophysiology, represents a powerful and versatile toolbox to stimulate, modulate, and record ion channel activity from membrane fragments and whole cells. The electrophysiological readout can be combined with fluorescent or optogenetic methods and allows for ultrafast solution exchanges using specialized microfluidic tools. A hallmark of manual patch clamp is the intentional selection of individual cells for recording, often an essential prerequisite to generate meaningful data. So far, available automation solutions rely on random cell usage in the closed environment of a chip and thus sacrifice much of this versatility by design. To parallelize and automate the traditional patch clamp technique while perpetuating the full versatility of the method, we developed an approach to automation, which is based on active cell handling and targeted electrode placement rather than on random processes. This is achieved through an automated pipette positioning system, which guides the tips of recording pipettes with micrometer precision to a microfluidic cell handling device. Using a patch pipette array mounted on a conventional micromanipulator, our automated patch clamp process mimics the original manual patch clamp as closely as possible, yet achieving a configuration where recordings are obtained from many patch electrodes in parallel. In addition, our implementation is extensible by design to allow the easy integration of specialized equipment such as ultrafast compound application tools. The resulting system offers fully automated patch clamp on purposely selected cells and combines high-quality gigaseal recordings with solution switching in the millisecond timescale. PMID:26991363

  7. Transparent Pixel Circuit with Threshold Voltage Compensation Using ZnO Thin-Film Transistors for Active-Matrix Organic Light Emitting Diode Displays

    NASA Astrophysics Data System (ADS)

    Yang, Ik-Seok; Kwon, Oh-Kyong

    2009-03-01

    A transparent pixel circuit with a threshold voltage compensating scheme using ZnO thin-film transistors (TFTs) for active-matrix organic light emitting diode (AMOLED) displays is proposed. This circuit consists of five n-type ZnO TFTs and two capacitors and can compensate for the threshold voltage variation of ZnO TFTs in real time. From simulation results, the maximum deviation of the emission current of the pixel circuit with a threshold voltage variation of ±1 V is determined to be less than 10 nA. From measurement results, it is verified that the maximum deviation of measured emission currents with measurement position in a glass substrate is less than 15 nA in a higher current range, and the deviation of emission current with time is less than 3%.

  8. Evaluation of clamp effects on LMFBR piping systems

    SciTech Connect

    Jones, G.L.

    1980-01-01

    Loop-type liquid metal breeder reactor plants utilize thin-wall piping to mitigate through-wall thermal gradients due to rapid thermal transients. These piping loops require a support system to carry the combined weight of the pipe, coolant and insulation and to provide attachments for seismic restraints. The support system examined here utilizes an insulated pipe clamp designed to minimize the stresses induced in the piping. To determine the effect of these clamps on the pipe wall a non-linear, two-dimensional, finite element model of the clamp, insulation and pipe wall was used to determine the clamp/pipe interface load distributions which were then applied to a three-dimensional, finite element model of the pipe. The two-dimensional interaction model was also utilized to estimate the combined clamp/pipe stiffness.

  9. Cerebral oxygenation and processed EEG response to clamping and shunting during carotid endarterectomy under general anesthesia.

    PubMed

    Perez, William; Dukatz, Christopher; El-Dalati, Sami; Duncan, James; Abdel-Rasoul, Mahmoud; Springer, Andrew; Go, Michael R; Dzwonczyk, Roger

    2015-12-01

    Clamping and shunting during carotid endarterectomy (CEA) surgery causes changes in cerebral blood flow. The purpose of this study was to assess and compare, side by side, the cerebral oxygenation (rSO2) and processed electroencephalogram (EEG) response bilaterally to carotid artery clamping and shunting in patients undergoing CEA under general anesthesia. With institutional approval and written informed consent, patients undergoing CEA under general anesthesia and routine carotid artery shunting were recorded bilaterally, simultaneously and continuously with an rSO2 and processed EEG monitor. The response of the monitors during carotid artery clamping and shunting were assessed and compared between monitors and bilaterally within each monitor. Sixty-nine patients were included in the study. At clamping the surgical-side and contralateral-side rSO2 dropped significantly below the baseline incision value (-17.6 and -9.4% respectively). After shunting, the contralateral-side rSO2 returned to baseline while the surgical-side rSO2 remained significantly below baseline (-9.0%) until the shunt was removed following surgery. At clamping the surgical-side and contralateral-side processed EEG also dropped below baseline (-19.9 and -20.6% respectively). However, following shunt activation, the processed EEG returned bilaterally to baseline. During the course of this research, we found the rSO2 monitor to be clinically more robust (4.4% failure rate) than the processed EEG monitor (20.0% failure rate). There was no correlation between the rSO2 or processed EEG changes that occurred immediately after clamping and the degree of surgical side stenosis measured pre-operatively. Both rSO2 and processed EEG respond to clamping and shunting during CEA. Cerebral oximetry discriminates between the surgical and contralateral side during surgery. The rSO2 monitor is more reliable in the real-world clinical setting. Future studies should focus on developing algorithms based on these

  10. 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).

  11. Clamping the Mec1/ATR checkpoint kinase into action.

    PubMed

    Majka, Jerzy; Burgers, Peter M J

    2007-05-15

    The yeast checkpoint protein kinase Mec1, the ortholog of human ATR, is the essential upstream regulator of the cell cycle checkpoint in response to DNA damage and to stalling of DNA replication forks. The activity of Mec1/ATR is not directly regulated by the DNA substrates that signal checkpoint activation. Rather the signal appears to be transduced to Mec1 by factors that interact with the signaling DNA substrates. One of these factors, the DNA damage checkpoint clamp Rad17-Mec3-Ddc1 (human 9-1-1) is loaded onto gapped DNA resulting from the partial repair of DNA damage, and the Ddc1 subunit of this complex activates Mec1. In vertebrate cells, the TopBP1 protein (Cut5 in S. pombe and Dpb11 in S. cervisiae) that is also required for establishment of the replication fork, functions during replication fork dysfunction to activate ATR. Both mechanisms of activation generally upregulate the kinase activity towards all downstream targets. PMID:17495536

  12. Parallel input parallel output high voltage bi-directional converters for driving dielectric electro active polymer actuators

    NASA Astrophysics Data System (ADS)

    Thummala, P.; Zhang, Z.; Andersen, M. A. E.; Rahimullah, S.

    2014-03-01

    Dielectric electroactive polymer (DEAP) actuators are capacitive devices which provide mechanical motions when charged electrically. The charging characteristics of a DEAP actuator depends on its size, voltage applied to its electrodes, and its operating frequency. The main idea of this paper is to design and implement driving circuits for the DEAP actuators for their use in various applications. This paper presents implementation of parallel input, parallel output, high voltage (~2.5 kV) bi-directional DC-DC converters for driving the DEAP actuators. The topology is a bidirectional flyback DC-DC converter incorporating commercially available high voltage MOSFETs (4 kV) and high voltage diodes (5 kV). Although the average current of the aforementioned devices is limited to 300 mA and 150 mA, respectively, connecting the outputs of multiple converters in parallel can provide a scalable design. This enables operating the DEAP actuators in various static and dynamic applications e.g. positioning, vibration generation or damping, and pumps. The proposed idea is experimentally verified by connecting three high voltage converters in parallel to operate a single DEAP actuator. The experimental results with both film capacitive load and the DEAP actuator are shown for a maximum charging voltage of 2 kV.

  13. Patch clamp studies of human sperm under physiological ionic conditions reveal three functionally and pharmacologically distinct cation channels.

    PubMed

    Mansell, S A; Publicover, S J; Barratt, C L R; Wilson, S M

    2014-05-01

    Whilst fertilizing capacity depends upon a K(+) conductance (GK) that allows the spermatozoon membrane potential (Vm) to be held at a negative value, the characteristics of this conductance in human sperm are virtually unknown. We therefore studied the biophysical/pharmacological properties of the K(+) conductance in spermatozoa from normal donors held under voltage/current clamp in the whole cell recording configuration. Our standard recording conditions were designed to maintain quasi-physiological, Na(+), K(+) and Cl(-) gradients. Experiments that explored the effects of ionic substitution/ion channel blockers upon membrane current/potential showed that resting Vm was dependent upon a hyperpolarizing K(+) current that flowed via channels that displayed only weak voltage dependence and limited (∼7-fold) K(+) versus Na(+) selectivity. This conductance was blocked by quinidine (0.3 mM), bupivacaine (3 mM) and clofilium (50 µM), NNC55-0396 (2 µM) and mibefradil (30 µM), but not by 4-aminopyridine (2 mM, 4-AP). Progesterone had no effect upon the hyperpolarizing K(+) current. Repolarization after a test depolarization consistently evoked a transient inward 'tail current' (ITail) that flowed via a second population of ion channels with poor (∼3-fold) K(+) versus Na(+) selectivity. The activity of these channels was increased by quinidine, 4-AP and progesterone. Vm in human sperm is therefore dependent upon a hyperpolarizing K(+) current that flows via channels that most closely resemble those encoded by Slo3. Although 0.5 µM progesterone had no effect upon these channels, this hormone did activate the pharmacologically distinct channels that mediate ITail. In conclusion, this study reveals three functionally and pharmacologically distinct cation channels: Ik, ITail, ICatSper.

  14. Whole-cell Patch-clamp Recordings in Brain Slices.

    PubMed

    Segev, Amir; Garcia-Oscos, Francisco; Kourrich, Saïd

    2016-01-01

    Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices, a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular, when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals

  15. Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

    PubMed Central

    Collins, Marcus D.; Gordon, Sharona E.

    2013-01-01

    The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment. Electroformation is an efficient mean to produce giant liposomes >10 μm in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory. We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results. PMID:23851612

  16. Simple circuit to improve electric field homogeneity in contour-clamped homogeneous electric field chambers.

    PubMed

    Herrera, José A; Canino, Carlos A; López-Cánovas, Lilia; Gigato, Regnar; Riverón, Ana Maria

    2003-04-01

    We redesigned contour-clamped homogeneous electric field (CHEF) circuitry to eliminate crossover distortion, to set identical potentials at electrodes of each equipotential pair and to drive pairs with transistors in emitter follower stages. An equipotential pair comprised the two electrodes set at the same potential to provide electric field homogeneity inside of the hexagonal array. The new circuitry consisted of two identical circuits, each having a resistor ladder, diodes and transistors. Both circuits were interconnected by diodes that controlled the current flow to electrodes when the array was energized in the 'A' or 'B' direction of the electric field. The total number of transistors was two-thirds of the total number of electrodes. Average voltage deviation from potentials expected at electrodes to achieve a homogeneous electric field was 0.06 V, whereas 0.44 V was obtained with another circuit that used transistors in push-pull stages. The new voltage clamp unit is cheap, generated homogeneous electric field, and gave reproducible and undistorted DNA band patterns.

  17. Simple circuit to improve electric field homogeneity in contour-clamped homogeneous electric field chambers.

    PubMed

    Herrera, José A; Canino, Carlos A; López-Cánovas, Lilia; Gigato, Regnar; Riverón, Ana Maria

    2003-04-01

    We redesigned contour-clamped homogeneous electric field (CHEF) circuitry to eliminate crossover distortion, to set identical potentials at electrodes of each equipotential pair and to drive pairs with transistors in emitter follower stages. An equipotential pair comprised the two electrodes set at the same potential to provide electric field homogeneity inside of the hexagonal array. The new circuitry consisted of two identical circuits, each having a resistor ladder, diodes and transistors. Both circuits were interconnected by diodes that controlled the current flow to electrodes when the array was energized in the 'A' or 'B' direction of the electric field. The total number of transistors was two-thirds of the total number of electrodes. Average voltage deviation from potentials expected at electrodes to achieve a homogeneous electric field was 0.06 V, whereas 0.44 V was obtained with another circuit that used transistors in push-pull stages. The new voltage clamp unit is cheap, generated homogeneous electric field, and gave reproducible and undistorted DNA band patterns. PMID:12707904

  18. 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

  19. Expression of voltage dependent potassium currents in freshly dissociated rat articular chondrocytes.

    PubMed

    Ponce, Arturo

    2006-01-01

    The electrophysiological properties of voltage dependent potassium channels from freshly dissociated rat articular chondrocytes were studied. The resting membrane potential (-42.7+/-2.0 mV) was significantly depolarized by increasing concentrations of external potassium. No change was observed when external chloride concentration was varied. Addition of TEA, 4AP, alpha-Dendrotoxin and charybdotoxin depolarized resting membrane potential. Whole cell patch clamp studies revealed the presence of outwardly rectifying currents whose kinetic and pharmacological properties suggest the expression of voltage dependent potassium channels. Two kinds of currents were observed under the same experimental conditions. The first one, most frequently observed (80%), starts activating near -50 mV, with V(1/2)=-18 mV, G(max)=0.30 pS/pF. The second kind was observed in only 10% of cases; It activates near -40 mV, with(1/2)=+28.35 mV, G(max)=0.28 pS/pF pA/pF and does not inactivates. Inactivating currents were significantly inhibited by TEA (IC(50)=1.45 mM), 4AP (IC(50)=0.64 mM), CTX (IC(50) = 10 nM), alpha-Dendrotoxin (IC(50) < 100 nM) and Margatoxin (IC(50)=28.5 nM). These results show that rat chondrocytes express voltage dependent potassium currents and suggest a role of voltage-dependent potassium channels in regulating membrane potential of rat chondrocytes.

  20. High-voltage integrated active quenching circuit for single photon count rate up to 80 Mcounts/s.

    PubMed

    Acconcia, Giulia; Rech, Ivan; Gulinatti, Angelo; Ghioni, Massimo

    2016-08-01

    Single photon avalanche diodes (SPADs) have been subject to a fast improvement in recent years. In particular, custom technologies specifically developed to fabricate SPAD devices give the designer the freedom to pursue the best detector performance required by applications. A significant breakthrough in this field is represented by the recent introduction of a red enhanced SPAD (RE-SPAD) technology, capable of attaining a good photon detection efficiency in the near infrared range (e.g. 40% at a wavelength of 800 nm) while maintaining a remarkable timing resolution of about 100ps full width at half maximum. Being planar, the RE-SPAD custom technology opened the way to the development of SPAD arrays particularly suited for demanding applications in the field of life sciences. However, to achieve such excellent performance custom SPAD detectors must be operated with an external active quenching circuit (AQC) designed on purpose. Next steps toward the development of compact and practical multichannel systems will require a new generation of monolithically integrated AQC arrays. In this paper we present a new, fully integrated AQC fabricated in a high-voltage 0.18 µm CMOS technology able to provide quenching pulses up to 50 Volts with fast leading and trailing edges. Although specifically designed for optimal operation of RE-SPAD devices, the new AQC is quite versatile: it can be used with any SPAD detector, regardless its fabrication technology, reaching remarkable count rates up to 80 Mcounts/s and generating a photon detection pulse with a timing jitter as low as 119 ps full width at half maximum. The compact design of our circuit has been specifically laid out to make this IC a suitable building block for monolithically integrated AQC arrays.

  1. High-voltage integrated active quenching circuit for single photon count rate up to 80 Mcounts/s.

    PubMed

    Acconcia, Giulia; Rech, Ivan; Gulinatti, Angelo; Ghioni, Massimo

    2016-08-01

    Single photon avalanche diodes (SPADs) have been subject to a fast improvement in recent years. In particular, custom technologies specifically developed to fabricate SPAD devices give the designer the freedom to pursue the best detector performance required by applications. A significant breakthrough in this field is represented by the recent introduction of a red enhanced SPAD (RE-SPAD) technology, capable of attaining a good photon detection efficiency in the near infrared range (e.g. 40% at a wavelength of 800 nm) while maintaining a remarkable timing resolution of about 100ps full width at half maximum. Being planar, the RE-SPAD custom technology opened the way to the development of SPAD arrays particularly suited for demanding applications in the field of life sciences. However, to achieve such excellent performance custom SPAD detectors must be operated with an external active quenching circuit (AQC) designed on purpose. Next steps toward the development of compact and practical multichannel systems will require a new generation of monolithically integrated AQC arrays. In this paper we present a new, fully integrated AQC fabricated in a high-voltage 0.18 µm CMOS technology able to provide quenching pulses up to 50 Volts with fast leading and trailing edges. Although specifically designed for optimal operation of RE-SPAD devices, the new AQC is quite versatile: it can be used with any SPAD detector, regardless its fabrication technology, reaching remarkable count rates up to 80 Mcounts/s and generating a photon detection pulse with a timing jitter as low as 119 ps full width at half maximum. The compact design of our circuit has been specifically laid out to make this IC a suitable building block for monolithically integrated AQC arrays. PMID:27505749

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

    SciTech Connect

    Birinyi-Strachan, Liesl C.; Gunning, Simon J.; Lewis, Richard J.; Nicholson, Graham M. . E-mail: Graham.Nicholson@uts.edu.au

    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, 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.

  3. Load-deflection characteristics of small bore insulated pipe clamps

    SciTech Connect

    Severud, L.K.; Clark, G.L.

    1982-01-01

    High temperature LMFBR piping is subject to rapid temperature changes during transient events. Typically, this pipe is supported by specially designed insulated pipe clamps to prevent excessive thermal stress from developing during these transients. The special insulated clamps used on both FFTF and CRBR piping utilize a Belleville spring arrangement to compensate for pipe thermal expansion. Analysis indicates that this produces a non-linear, directionally sensitive clamp spring rate. Since these spring rates influence the seismic response of a supported piping system, it was deemed necessary to evaluate them further by test. This has been accomplished for the FFTF clamps. A more standard insulated pipe clamp, which does not incorporate Belleville springs to accommodate thermal expansion, was also tested. This type clamp is simple in design, and economically attractive. It may have wide application prospects for use in LMFBR small bore auxiliary piping operating at temperatures below 427/sup 0/C. Load deflection tests were conducted on 2.54 CM and 7.62 CM diameter samples of these commercial clamps.

  4. The importance of delayed cord clamping for Aboriginal babies: a life-enhancing advantage.

    PubMed

    Weckert, Rosemary; Hancock, Heather

    2008-12-01

    Third stage management has typically focused on women and postpartum haemorrhage. Clamping and cutting the umbilical cord following the birth of the baby has continued to be a routine part of this focus. Active versus physiological management of third stage is generally accepted as an evidence-based plan for women to avoid excessive blood loss. Other considerations around this decision are rarely considered, including the baby's perspective. This paper provides a review of the literature regarding timing of clamping and cutting of the umbilical cord and related issues, and discusses the consequences for babies and in particular *Aboriginal babies. Iron stores in babies are improved (among other important advantages) if the cord is left to stop pulsating for 3 min before being clamped. Such a simple measure of patience and informed practice can make a long lasting difference to a baby's health and for Aboriginal babies this advantage can be critical in the short and the long term for their development and wellbeing. To achieve much needed reductions in infancy anaemia and essential increases in infant survival, delayed cord clamping and cutting is recommended for all Aboriginal babies.

  5. High voltage load resistor array

    DOEpatents

    Lehmann, Monty Ray

    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.

  6. BK channel activation by tungstate requires the β1 subunit extracellular loop residues essential to modulate voltage sensor function and channel gating.

    PubMed

    Fernández-Mariño, Ana I; Valverde, Miguel A; Fernández-Fernández, José M

    2014-07-01

    Tungstate, a compound with antidiabetic, antiobesity, and antihypertensive properties, activates the large-conductance voltage- and Ca(2+)-dependent K(+) (BK) channel containing either β1 or β4 subunits. The BK activation by tungstate is Mg(2+)-dependent and promotes arterial vasodilation, but only in precontracted mouse arteries expressing β1. In this study, we further explored how the β1 subunit participates in tungstate activation of BK channels. Activation of heterologously expressed human BKαβ1 channels in inside-out patches is fully dependent on the Mg(2+) sensitivity of the BK α channel subunit even at high (10 μM) cytosolic Ca(2+) concentration. Alanine mutagenesis of β1 extracellular residues Y74 or S104, which destabilize the active voltage sensor, greatly decreased the tungstate-induced left-shift of the BKαβ1 G-V curves in either the absence or presence of physiologically relevant cytosolic Ca(2+) levels (10 μM). The weakened tungstate activation of the BKαβ1Y74A and BKαβ1S104A mutant channels was not related to decreased Mg(2+) sensitivity. These results, together with previously published reports, support the idea that the putative binding site for tungstate-mediated BK channel activation is located in the pore-forming α channel subunit, around the Mg(2+) binding site. The role of β1 in tungstate-induced channel activation seems to rely on its interaction with the BK α subunit to modulate channel activity. Loop residues that are essential for the regulation of voltage sensor activation and gating of the BK channel are also relevant for BK activation by tungstate.

  7. Trimebutine maleate has inhibitory effects on the voltage-dependent Ca2+ inward current and other membrane currents in intestinal smooth muscle cells.

    PubMed

    Shimada, T; Kurachi, Y; Terano, A; Hamada, E; Sugimoto, T

    1990-04-01

    We examined effects of trimebutine maleate on the membrane currents of the intestinal smooth muscle cells by using the tight-seal whole cell clamp technique. Trimebutine suppressed the Ba2+ inward current through voltage-dependent Ca2+ channels in a dose-dependent manner. The inhibitory effect of trimebutine on the Ba2+ inward current was not use-dependent. It shifted the steady-state inactivation curve to the left along the voltage axis. Trimebutine also had inhibitory effects on the other membrane currents of the cells, such as the voltage-dependent K+ current, the Ca2(+)-activated oscillating K+ current and the acetylcholine-induced inward current. These relatively non-specific inhibitory effects of trimebutine on the membrane currents may explain, at least in part, the dual actions of the drug on the intestinal smooth muscle contractility, i.e. inhibitory as well as excitatory. PMID:2161373

  8. The influence of galvanic currents and voltage on the proliferation activity of lymphocytes and expression of cell surface molecules.

    PubMed

    Podzimek, S; Hána, K; Miksovský, M; Pousek, L; Matucha, P; Meloun, M; Procházková, J

    2008-01-01

    Release of metal ions from dental metal fillings supported by galvanism can cause local or general pathological problems in sensitive and genetically susceptible individuals. We aimed to investigate in vitro lymphocyte responses and expression of surface molecules influenced by galvanic currents and voltage. Human peripheral blood lymphocytes were influenced by galvanic currents and voltages and lymphocyte proliferation was measured. Control samples were not exposed to the influence of galvanism. We also studied the expression of surface molecules by the FACS analysis. A 15-h and shorter influence of almost all tested currents and voltages caused a significant decrease in lymphocyte proliferation and the 15-h influence of 20 microA currents significantly increased expression of surface molecules CD 19, 11a/18, 19/69 and 19/95. An influence of 10 and 3 microA currents led to a significant decrease in the expression of surface molecules CD 3, 11a/18, 3/69 and 3/95 and to a significant increase in CD 19 expression. An 80 mV voltage influence led to a significant decrease in the expression of surface molecules CD 3, 11a/18, 3/69, 3/95, 19/69 and 19/95, and 200 and 300 mV voltages significantly decreased the expression of surface molecules CD 3, 19, 11a/18, 3/95 and 19/95 and significantly increased CD 19/69 expression. A long-lasting influence of galvanism can, in sensitive and genetically susceptible individuals, influence lymphocyte proliferation and surface molecule expression. The threshold for pathological values of 5 microA for galvanic currents and 100 mV for galvanic voltage was confirmed.

  9. Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage.

    PubMed

    Brinks, Daan; Klein, Aaron J; Cohen, Adam E

    2015-09-01

    Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Förster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue.

  10. Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage.

    PubMed

    Brinks, Daan; Klein, Aaron J; Cohen, Adam E

    2015-09-01

    Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Förster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue. PMID:26331249

  11. A loudspeaker-driven system for rapid and multiple solution exchanges in patch-clamp experiments.

    PubMed

    Méry, P F; Lechêne, P; Fischmeister, R

    1992-04-01

    A new and inexpensive system allowing rapid and synchronized changes of solutions around a membrane patch or a cell under voltage-clamp conditions is described. Four plastic capillary tubings (OD 640 microns; ID 430 microns) were glued together horizontally and attached to a coil of a commercially available loudspeaker. Servo-control of the position of the coil allowed the mouth of any of the capillaries to be positioned near the pipette tip within 6 ms. A high flow speed of the test solution was crucial to achieve rapid solution exchange. At a flow speed of 5 cm/s, complete exchange of the external environment of a frog ventricular cell was achieved within 20-30 ms. The time course of solution change was found to be 3-5 times faster at the tip of an open patch pipette. To preserve the physical integrity of the cell, the cell was usually perfused by a control capillary at a slow velocity (0.2-0.4 cm/s) and test solutions flowing out of adjacent capillaries at high velocity (4-5 cm/s) were applied to the cell only for short periods. Determination of the three-dimensional contamination profile around the mouth of the control capillary allowed the optimal conditions for the use of the system to be established and possible sources of contamination to be avoided between adjacent capillaries with unmatched flow speeds. Successive and multiple changes in external solutions could be easily synchronized with voltage-clamp depolarizations to examine the time course of the effect of drugs on voltage-operated ion channels.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

    SciTech Connect

    Gaudioso, Christelle; Carlier, Edmond; Youssouf, Fahamoe; Clare, Jeffrey J.; Debanne, Dominique; Alcaraz, Gisele

    2011-07-29

    Highlights: {yields} Both Ca{sup ++}-Calmodulin (CaM) and Ca{sup ++}-free CaM bind to the C-terminal region of Nav1.1. {yields} Ca{sup ++} and CaM have both opposite and convergent effects on I{sub Nav1.1}. {yields} Ca{sup ++}-CaM modulates I{sub Nav1.1} amplitude. {yields} CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. {yields} Ca{sup ++} alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca{sup ++} depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca{sup ++} could bind the Nav1.1 C-terminal region with micromolar affinity.

  13. Dynamic Clamp Analysis of Synaptic Integration in Sympathetic Ganglia

    PubMed Central

    Horn, J. P.; Kullmann, P. H. M.

    2008-01-01

    Advances in modern neuroscience require the identification of principles that connect different levels of experimental analysis, from molecular mechanisms to explanations of cellular functions, then to circuits, and, ultimately, to systems and behavior. Here, we examine how synaptic organization of the sympathetic ganglia may enable them to function as use-dependent amplifiers of preganglionic activity and how the gain of this amplification may be modulated by metabotropic signaling mechanisms. The approach combines a general computational model of ganglionic integration together with experimental tests of the model using the dynamic clamp method. In these experiments, we recorded intracellularly from dissociated bullfrog sympathetic neurons and then mimicked physiological synapses with virtual computer-generated synapses. It thus became possible to analyze the synaptic gain by recording cellular responses to complex patterns of synaptic activity that normally arise in vivo from convergent nicotinic and muscarinic synapses. The results of these studies are significant because they illustrate how gain generated through ganglionic integration may contribute to the feedback control of important autonomic behaviors, in particular to the control of the blood pressure. We dedicate this paper to the memory of Professor Vladimir Skok, whose rich legacy in synaptic physiology helped establish the modern paradigm for connecting multiple levels of analysis in studies of the nervous system. PMID:19756262

  14. Interpretation of current-voltage relationships for "active" ion transport systems: I. Steady-state reaction-kinetic analysis of class-I mechanisms.

    PubMed

    Hansen, U P; Gradmann, D; Sanders, D; Slayman, C L

    1981-01-01

    This paper develops a simple reaction-kinetic model to describe electrogenic pumping and co- (or counter-) transport of ions. It uses the standard steady-state approach for cyclic enzyme- or carrier-mediated transport, but does not assume rate-limitation by any particular reaction step. Voltage-dependence is introduced, after the suggestion of Läuger and Stark (Biochim. Biophys. Acta 211:458-466, 1970), via a symmetric Eyring barrier, in which the charge-transit reaction constants are written as k12 = ko12 exp(zF delta psi/2RT) and k21 = ko21 exp(-zF delta psi/2RT). For interpretation of current-voltage relationships, all voltage-independent reaction steps are lumped together, so the model in its simplest form can be described as a pseudo-2-state model. It is characterized by the two voltage-dependent reaction constants, two lumped voltage-independent reaction constants (k12, k21), and two reserve factors (ri, ro) which formally take account of carrier states that are indistinguishable in the current-voltage (I-V) analysis. The model generates a wide range of I-V relationships, depending on the relative magnitudes of the four reaction constants, sufficient to describe essentially all I-V datas now available on "active" ion-transport systems. Algebraic and numerical analysis of the reserve factors, by means of expanded pseudo-3-, 4-, and 5-state models, shows them to be bounded and not large for most combinations of reaction constants in the lumped pathway. The most important exception to this rule occurs when carrier decharging immediately follows charge transit of the membrane and is very fast relative to other constituent voltage-independent reactions. Such a circumstance generates kinetic equivalence of chemical and electrical gradients, thus providing a consistent definition of ion-motive forces (e.g., proton-motive force, PMF). With appropriate restrictions, it also yields both linear and log-linear relationships between net transport velocity and either

  15. Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres

    PubMed Central

    Carbonara, Roberta; Carocci, Alessia; Roussel, Julien; Crescenzo, Giuseppe; Buonavoglia, Canio; Franchini, Carlo; Lentini, Giovanni; Camerino, Diana Conte; Desaphy, Jean-François

    2015-01-01

    Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7–1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors. PMID:26257653

  16. Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres.

    PubMed

    Carbonara, Roberta; Carocci, Alessia; Roussel, Julien; Crescenzo, Giuseppe; Buonavoglia, Canio; Franchini, Carlo; Lentini, Giovanni; Camerino, Diana Conte; Desaphy, Jean-François

    2015-01-01

    Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors. PMID:26257653

  17. Proximal clamping levels in abdominal aortic aneurysm surgery.

    PubMed Central

    Büket, S; Atay, Y; Islamoğlu, F; Yağdi, T; Posacioğlu, H; Alat, I; Cikirikçioğlu, M; Yüksel, M; Durmaz, I

    1999-01-01

    In the surgical treatment of abdominal aortic aneurysm, the single proximal cross-clamp can be placed at 3 alternative aortic levels: infrarenal, hiatal, and thoracic. We performed this retrospective study to evaluate the advantages and disadvantages of the 3 main aortic clamping locations. Eighty patients presented at our institution with abdominal aortic aneurysms from March 1993 through May 1998. Fifty of these patients had intact aneurysms and underwent elective surgery, and 30 had ruptured aneurysms that necessitated emergency surgery. Proximal aortic clamping was applied at the infrarenal level in 24 patients (22 from the intact aneurysm group, 2 from the ruptured group), at the hiatal level in 34 patients (22 intact, 12 ruptured), and at the thoracic level (descending aorta) via a limited left lateral thoracotomy in 22 patients (6 intact, 16 ruptured). Early mortality rates (within 30 days) were 4% (2 of 50 patients) among patients with intact aneurysms and 40% (12 of 30 patients) among those with ruptured aneurysms. In the 2 patients from the intact aneurysm group, proximal aortic clamps were applied at the hiatal level. In the ruptured aneurysm group, proximal aortic clamps were placed at the thoracic level in 10 patients, the infrarenal level in 1, and the hiatal level in 1. According to our study, the clinical status of the patient and the degree of operative urgency--as determined by the extent of the aneurysm--generally dictate the proximal clamp location. Patients who present with aneurysmal rupture or hypovolemic shock benefit from thoracic clamping, because it restores the blood pressure and allows time to replace the volume deficit. Infrarenal placement is advantageous in patients with intact aneurysms if there is sufficient space for the clamp between the renal arteries and the aortic aneurysm. In patients with juxtarenal aneurysms, hiatal clamping enables safe and easy anastomosis to the healthy aorta. Clamping at this level also helps prevent

  18. Zn{sup 2+} induces apoptosis in human highly metastatic SHG-44 glioma cells, through inhibiting activity of the voltage-gated proton channel Hv1

    SciTech Connect

    Wang, Yifan; Zhang, Shangrong; Li, Shu Jie

    2013-08-23

    Highlights: •Hv1 is expressed in highly metastatic glioma cell. •Zn{sup 2+} ions induces apoptosis in highly metastatic glioma cells. •Zn{sup 2+} ions markedly inhibit proton secretion. •Zn{sup 2+} ions reduce the gelatinase activity. •Inhibition of Hv1 activity via Zn{sup 2+} ions can effectively retard the cancer growth. -- Abstract: In contrast to the voltage-gated K{sup +} channels, the voltage-gated proton channel Hv1 contains a voltage-sensor domain but lacks a pore domain. Here, we showed that Hv1 is expressed in the highly metastatic glioma cell SHG-44, but lowly in the poorly metastatic glioma cell U-251. Inhibition of Hv1 activity by 140 μM zinc chloride induces apoptosis in the human highly metastatic glioma cells. Zn{sup 2+} ions markedly inhibit proton secretion, and reduce the gelatinase activity in the highly metastatic glioma cells. In vivo, the glioma tumor sizes of the implantation of the SHG-44 xenografts in nude mice that were injected zinc chloride solution, were dramatically smaller than that in the controlled groups. The results demonstrated that the inhibition of Hv1 activity via Zn{sup 2+} ions can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Our results suggest that Zn{sup 2+} ions may be used as a potential anti-glioma drug for glioma therapy.

  19. Molecular Mechanisms of COMPLEXIN Fusion Clamp Function in Synaptic Exocytosis Revealed in a New Drosophila Mutant

    PubMed Central

    Iyer, Janani; Wahlmark, Christopher J.; Kuser-Ahnert, Giselle A.; Kawasaki, Fumiko

    2013-01-01

    The COMPLEXIN (CPX) proteins play a critical role in synaptic vesicle fusion and neurotransmitter release. Previous studies demonstrated that CPX functions in both activation of evoked neurotransmitter release and inhibition/clamping of spontaneous synaptic vesicle fusion. Here we report a new cpx mutant in Drosophila melanogaster, cpx1257, revealing spatially defined and separable pools of CPX which make distinct contributions to the activation and clamping functions. In cpx1257, lack of only the last C-terminal amino acid of CPX is predicted to disrupt prenylation and membrane targeting of CPX. Immunocytochemical analysis established localization of wild-type CPX to active zone (AZ) regions containing neurotransmitter release sites as well as broader presynaptic membrane compartments including synaptic vesicles. Parallel biochemical studies confirmed CPX membrane association and demonstrated robust binding interactions of CPX with all three SNAREs. This is in contrast to the cpx1257 mutant, in which AZ localization of CPX persists but general membrane localization and, surprisingly, the bulk of CPX-SNARE protein interactions are abolished. Furthermore, electrophysiological analysis of neuromuscular synapses revealed interesting differences between cpx1257 and a cpx null mutant. The cpx null exhibited a marked decrease in the EPSC amplitude, slowed EPSC rise and decay times and an increased mEPSC frequency with respect to wild-type. In contrast, cpx1257 exhibited a wild-type EPSC with an increased mEPSC frequency and thus a selective failure to clamp spontaneous release. These results indicate that spatially distinct and separable interactions of CPX with presynaptic membranes and SNARE proteins mediate separable activation and clamping functions of CPX in neurotransmitter release. PMID:23769723

  20. Current-Sensing and Voltage-Feedback Driving Method for Large-Area High-Resolution Active Matrix Organic Light Emitting Diodes

    NASA Astrophysics Data System (ADS)

    In, Hai‑Jung; Choi, Byong‑Deok; Chung, Ho‑Kyoon; Kwon, Oh‑Kyong

    2006-05-01

    There is the problem of picture quality nonuniformity due to thin film transistor (TFT) characteristic variations throughout a panel of large-area high-resolution active matrix organic light emitting diodes. The current programming method could solve this issue, but it also requires very long charging time of a data line at low gray shades. Therefore, we propose a new driving method and a pixel circuit with emission-current sensing and feedback operation in order to resolve these problems. The proposed driving method and pixel circuit successfully compensate threshold voltage and mobility variations of TFTs and overcome the data line charging problem. Simulation results show that emission current deviations of the proposed driving method are less than 1.7% with ± 10.0% mobility and ± 0.3 V threshold voltage variations of pixel-driving TFTs, which means the proposed driving method is applicable to large-area high-resolution applications.

  1. Action potential characterization of human induced pluripotent stem cell-derived cardiomyocytes using automated patch-clamp technology.

    PubMed

    Scheel, Olaf; Frech, Stefanie; Amuzescu, Bogdan; Eisfeld, Jörg; Lin, Kun-Han; Knott, Thomas

    2014-10-01

    Recent progress in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) research led to high-purity preparations of human cardiomyocytes (CMs) differentiated from these two sources-suitable for tissue regeneration, in vitro models of disease, and cardiac safety pharmacology screening. We performed a detailed characterization of the effects of nifedipine, cisapride, and tetrodotoxin (TTX) on Cor.4U(®) human iPSC-CM, using automated whole-cell patch-clamp recordings with the CytoPatch™ 2 equipment, within a complex assay combining multiple voltage-clamp and current-clamp protocols in a well-defined sequence, and quantitative analysis of several action potential (AP) parameters. We retrieved three electrical phenotypes based on AP shape: ventricular, atrial/nodal, and S-type (with ventricular-like depolarization and lack of plateau). To suppress spontaneous firing, present in many cells, we injected continuously faint hyperpolarizing currents of -10 or -20 pA. We defined quality criteria (both seal and membrane resistance over 1 GΩ), and focused our study on cells with ventricular-like AP. Nifedipine induced marked decreases in AP duration (APD): APD90 (49.8% and 40.8% of control values at 1 and 10 μM, respectively), APD50 (16.1% and 12%); cisapride 0.1 μM increased APD90 to 176.2%; and tetrodotoxin 10 μM decreased maximum slope of phase to 33.3% of control, peak depolarization potential to 76.3% of control, and shortened APD90 on average to 80.4%. These results prove feasibility of automated voltage- and current-clamp recordings on human iPSC-CM and their potential use for in-depth drug evaluation and proarrhythmic liability assessment, as well as for diagnosis and pharmacology tests for cardiac channelopathy patients. PMID:25353059

  2. Dendrotoxin acceptor from bovine synaptic plasma membranes. Binding properties, purification and subunit composition of a putative constituent of certain voltage-activated K+ channels.

    PubMed Central

    Parcej, D N; Dolly, J O

    1989-01-01

    Dendrotoxin is a snake polypeptide that blocks selectively and potently certain voltage-sensitive, fast-activating K+ channels in the nervous system, where it binds with high affinity to membranous acceptors. Herein, the acceptor protein for dendrotoxin in bovine synaptic membranes is solubilized in active form and its complete purification achieved by affinity chromatography, involving a novel elution procedure. This putative K+-channel constituent is shown to be a large oligomeric glycoprotein containing two major subunits, with Mr values of 75,000 and 37,000. Images Fig. 2. PMID:2930493

  3. Gambierol, a toxin produced by the dinoflagellate Gambierdiscus toxicus, is a potent blocker of voltage-gated potassium channels.

    PubMed

    Cuypers, Eva; Abdel-Mottaleb, Yousra; Kopljar, Ivan; Rainier, Jon D; Raes, Adam L; Snyders, Dirk J; Tytgat, Jan

    2008-05-01

    In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Na(v)1.1-Na(v)1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian K(v)1.1-K(v)1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 microM. In contrast, K(v)1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (>97%) and an half maximal inhibitory concentration (IC(50)) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments. PMID:18313714

  4. Patterning of polystyrene by UV-laser radiation for the fabrication of devices for patch clamping

    NASA Astrophysics Data System (ADS)

    Pfleging, W.; Bruns, M.; Przybylski, M.; Welle, A.; Wilson, S.

    2008-02-01

    Two types of laser patterning are of interest for application in microsystem technology: direct ablation of polymer material for the generation of two or three dimensional shapes such as microfluidic channels, curved shapes or micro-holes and alternatively photo-induced change of chemical or physical properties. An appropriate choice of laser and process parameters enables new approaches for the fabrication of lab-on-chip devices with integrated functionalities. We will present our current research results in laser-assisted ablation and modification of polystyrene (PS) with respect to the fabrication of polymer devices for high throughput planar patch clamping. Patch clamping is a highly sensitive technique used to measure the electrical activity of a cell. It is used in applications which include drug screening where there is demand for high throughput systems (HTS). While there are a few commercially available HTS patch clamping systems on the market using traditional patch clamping materials, there are no systems on the market using novel materials, or for dealing with cell networks - a physiologically important consideration for the developing fields of tissue engineering and understanding cell to cell interactions. This paper presents potential design approaches and processes for producing a polymer based automated patch clamping system. For this purpose laser micro-drilling of PS and subsequent surface functionalization was investigated as function of laser and process parameters. A high power ArF-excimer laser radiation source with pulse length of 20 ns (repetition rate up to 40 Hz) as well as high repetition ArF- and KrF-excimer laser sources with pulse lengths of 4-6 ns (repetition rates up to 500 Hz) were used in order to study the influence of laser pulse length on laser drilling and laser-induced surface modification. Micro-drilling of PS with diameters down to 1.5 μm were demonstrated. Furthermore the localized formation of chemical structures

  5. One-channel Cell-attached Patch-clamp Recording

    PubMed Central

    Maki, Bruce A.; Cummings, Kirstie A.; Paganelli, Meaghan A.; Murthy, Swetha E.; Popescu, Gabriela K.

    2014-01-01

    Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel’s conductance properties and the temporal sequence of open-closed conformations that make up the channel’s activation mechanism, thus helping to understand their functions in health and disease. PMID:24961614

  6. Generalization of the dynamic clamp concept in neurophysiology and behavior.

    PubMed

    Chamorro, Pablo; Muñiz, Carlos; Levi, Rafael; Arroyo, David; Rodríguez, Francisco B; Varona, Pablo

    2012-01-01

    The idea of closed-loop interaction in in vitro and in vivo electrophysiology has been successfully implemented in the dynamic clamp concept strongly impacting the research of membrane and synaptic properties of neurons. In this paper we show that this concept can be easily generalized to build other kinds of closed-loop protocols beyond (or in addition to) electrical stimulation and recording in neurophysiology and behavioral studies for neuroethology. In particular, we illustrate three different examples of goal-driven real-time closed-loop interactions with drug microinjectors, mechanical devices and video event driven stimulation. Modern activity-dependent stimulation protocols can be used to reveal dynamics (otherwise hidden under traditional stimulation techniques), achieve control of natural and pathological states, induce learning, bridge between disparate levels of analysis and for a further automation of experiments. We argue that closed-loop interaction calls for novel real time analysis, prediction and control tools and a new perspective for designing stimulus-response experiments, which can have a large impact in neuroscience research.

  7. Spectral infrared hemispherical reflectance measurements for LDEF tray clamps

    NASA Technical Reports Server (NTRS)

    Wood, Bobby E.; Cromwell, Brian K.; Pender, Charles W.; Shepherd, Seth D.

    1992-01-01

    This paper describes infrared hemispherical reflectance measurements (2-15 microns) that were made on 58 chromic acid anodized tray clamps retrieved from the LDEF spacecraft. These clamps were used for maintaining the experiments in place and were located at various locations about the spacecraft. Changes in reflectance of the tray clamps at these locations were compared with atomic oxygen fluxes at the same locations. A decrease in absorption band depth was seen for the surfaces exposed to space indicating that there was some surface layer erosion. In all of the surfaces measured, little evidence of contamination was observed and none of the samples showed evidence of the brown nicotine stain that was so prominent in other experiments. Total emissivity values were calculated for both exposed and unexposed tray clamp surfaces. Only small differences, usually less than 1 percent, were observed. The spectral reflectances were measured using a hemi-ellipsoidal mirror reflectometer matched with an interferometer spectrometer. The rapid scanning capability of the interferometer allowed the reflectance measurements to be made in a timely fashion. The ellipsoidal mirror has its two foci separated by 2 inches and located on the major axis. A blackbody source was located at one focus while the tray clamp samples were located at the conjugate focus. The blackbody radiation was modulated and then focused by the ellipsoid onto the tray clamps. Radiation reflected from the tray clamp was sampled by the interferometer by viewing through a hole in the ellipsoid. A gold mirror (reflectance approximately 98 percent) was used as the reference surface.

  8. Measuring beta-cell function relative to insulin sensitivity in youth: Does the hyperglycemic clamp suffice?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To compare beta-cell function relative to insulin sensitivity, disposition index (DI), calculated from two clamps (2cDI, insulin sensitivity from the hyperinsulinemic-euglycemic clamp and first-phase insulin from the hyperglycemic clamp) with the DI calculated from the hyperglycemic clamp alone (hcD...

  9. High Voltage Insulation Technology

    NASA Astrophysics Data System (ADS)

    Scherb, V.; Rogalla, K.; Gollor, M.

    2008-09-01

    In preparation of new Electronic Power Conditioners (EPC's) for Travelling Wave Tub Amplifiers (TWTA's) on telecom satellites a study for the development of new high voltage insulation technology is performed. The initiative is mandatory to allow compact designs and to enable higher operating voltages. In a first task a market analysis was performed, comparing different materials with respect to their properties and processes. A hierarchy of selection criteria was established and finally five material candidates (4 Epoxy resins and 1 Polyurethane resin) were selected to be further investigated in the test program. Samples for the test program were designed to represent core elements of an EPC, the high voltage transformer and Printed Circuit Boards of the high voltage section. All five materials were assessed in the practical work flow of the potting process and electrical, mechanical, thermal and lifetime testing was performed. Although the lifetime tests results were overlayed by a larges scatter, finally two candidates have been identified for use in a subsequent qualification program. This activity forms part of element 5 of the ESA ARTES Programme.

  10. 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.

  11. 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.

  12. Chemical Synthesis of Tetracyclic Terpenes and Evaluation of Antagonistic Activity on Endothelin-A Receptors and Voltage-gated Calcium Channels

    PubMed Central

    Lu, Jianyu; Aguilar, Angelo; Zou, Bende; Bao, Weier; Koldas, Serkan; Aibin, Shi; Desper, John; Wangemann, Philine; Xie, Xinmin Simon; Hua, Duy H.

    2015-01-01

    A class of tetracyclic terpenes was synthesized and evaluated for antagonistic activity of endothelin-1 (ET-1) induced vasoconstriction and inhibitory activity of voltage-activated Ca2+ channels. Three repeated Robinson annulation reactions were utilized to construct the tetracyclic molecules. A stereoselective reductive Robinson annulation was discovered for the formation of optically pure tricyclic terpenes. Stereoselective addition of cyanide to the hindered α-face of tetracyclic enone (-)-18 was found and subsequent transformation into the aldehyde function was affected by the formation of bicyclic hemiiminal (-)-4. Six selected synthetic tetracyclic terpenes show inhibitory activities in ET-1 induced vasoconstriction in the gerbil spiral modiolar artery with putative affinity constants ranging between 93 and 319 nM. Moreover, one compound, (-)-3, was evaluated further and found to inhibit voltage-activated Ca2+ currents but not to affect Na+ or K+ currents in dorsal root ganglion cells under similar concentrations. These observations imply a dual mechanism of action. In conclusion, tetracyclic terpenes represent a new class of hit molecules for the discovery of new drugs for the treatment of pulmonary hypertension and vascular related diseases. PMID:26190460

  13. Releasing the peri-neuronal net to patch-clamp neurons in adult CNS.

    PubMed

    Morales, Ezequiel; Fernandez, Fernando R; Sinclair, Suzanne; Molineux, Michael L; Mehaffey, W Hamish; Turner, Ray W

    2004-05-01

    The extracellular matrix of adult neural tissue contains chondroitin sulphated proteogylcans that form a dense peri-neuronal net surrounding the cell body and proximal dendrites of many neuronal classes. Development of the peri-neuronal net beyond approximately postnatal day 17 obscures visualization and often access by patch electrodes to neuronal membranes with the result that patch clamp recordings are most readily obtained from early postnatal animals. We describe a technique in which the surface tension of a sucrose-based medium promotes partial dissociation of thin tissue slices from adult tissue. Surface tension spreads the tissue and loosens the peri-neuronal net from neuronal membranes within minutes and in the absence of proteolytic enzymes. Furthermore, the extent of dissociation can be controlled so as to maintain the overall slice structure and allow identification of specific cell classes. Excellent structural preservation of neurons and dendrites can be obtained and full access by patch electrodes made possible for current- or voltage-clamp recordings in tissue well beyond the development of peri-neuronal nets. We demonstrate the feasibility of using this approach through patch recordings from neurons in the brainstem and cerebellum of adult gymnotiform fish and in deep cerebellar nuclei of rats as old as 6 months.

  14. 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.

  15. 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.

  16. Design of an integrated thermoelectric generator power converter for ultra-low power and low voltage body energy harvesters aimed at ExG active electrodes

    NASA Astrophysics Data System (ADS)

    Ataei, Milad; Robert, Christian; Boegli, Alexis; Farine, Pierre-André

    2015-10-01

    This paper describes a detailed design procedure for an efficient thermal body energy harvesting integrated power converter. The procedure is based on the examination of power loss and power transfer in a converter for a self-powered medical device. The efficiency limit for the system is derived and the converter is optimized for the worst case scenario. All optimum system parameters are calculated respecting the transducer constraints and the application form factor. Circuit blocks including pulse generators are implemented based on the system specifications and optimized converter working frequency. At this working condition, it has been demonstrated that the wide area capacitor of the voltage doubler, which provides high voltage switch gating, can be eliminated at the expense of wider switches. With this method, measurements show that 54% efficiency is achieved for just a 20 mV transducer output voltage and 30% of the chip area is saved. The entire electronic board can fit in one EEG or ECG electrode, and the electronic system can convert the electrode to an active electrode.

  17. Ca2+-activated Cl− current in rabbit sinoatrial node cells

    PubMed Central

    Verkerk, Arie O; Wilders, Ronald; Zegers, Jan G; van Borren, Marcel M G J; Ravesloot, Jan H; Verheijck, E Etienne

    2002-01-01

    The Ca2+-activated Cl− current (ICl(Ca)) has been identified in atrial, Purkinje and ventricular cells, where it plays a substantial role in phase-1 repolarization and delayed after-depolarizations. In sinoatrial (SA) node cells, however, the presence and functional role of ICl(Ca) is unknown. In the present study we address this issue using perforated patch-clamp methodology and computer simulations. Single SA node cells were enzymatically isolated from rabbit hearts. ICl(Ca) was measured, using the perforated patch-clamp technique, as the current sensitive to the anion blocker 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS). Voltage clamp experiments demonstrate the presence of ICl(Ca) in one third of the spontaneously active SA node cells. The current was transient outward with a bell-shaped current-voltage relationship. Adrenoceptor stimulation with 1 μm noradrenaline doubled the ICl(Ca) density. Action potential clamp measurements demonstrate that ICl(Ca) is activate late during the action potential upstroke. Current clamp experiments show, both in the absence and presence of 1 μm noradrenaline, that blockade of ICl(Ca) increases the action potential overshoot and duration, measured at 20 % repolarization. However, intrinsic interbeat interval, upstroke velocity, diastolic depolarization rate and the action potential duration measured at 50 and 90 % repolarization were not affected. Our experimental data are supported by computer simulations, which additionally demonstrate that ICl(Ca) has a limited role in pacemaker synchronization or action potential conduction. In conclusion, ICl(Ca) is present in one third of SA node cells and is activated during the pacemaker cycle. However, ICl(Ca) does not modulate intrinsic interbeat interval, pacemaker synchronization or action potential conduction. PMID:11927673

  18. 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

  19. Automated ion channel screening: patch clamping made easy.

    PubMed

    Farre, Cecilia; Stoelzle, Sonja; Haarmann, Claudia; George, Michael; Brüggemann, Andrea; Fertig, Niels

    2007-04-01

    Efficient high resolution techniques are required for screening efforts and research targeting ion channels. The conventional patch clamp technique, a high resolution but low efficiency technique, has been established for 25 years. Recent advances have opened up new possibilities for automated patch clamping. This new technology meets the need of drug developers for higher throughput and facilitates new experimental approaches in ion channel research. Specifically, Nanion's electrophysiology workstations, the Port-a-Patch and the Patchliner, have been successfully introduced as high-quality automated patch clamp platforms for industry as well as academic users. Both platforms give high quality patch clamp recordings, capable of true giga-seals and stable recordings, accessible to the user without the need for years of practical training. They also offer sophisticated experimental possibilities, such as accurate and fast ligand application, temperature control and internal solution exchange. This article describes the chip-based patch clamp technology and its usefulness in ion channel drug screening and academic research.

  20. Ultra-fast force-clamp laser trapping of single molecular motors and DNA binding proteins

    NASA Astrophysics Data System (ADS)

    Capitanio, Marco; Monico, Carina; Vanzi, Francesco; Pavone, Francesco S.

    2013-09-01

    Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay of only ˜10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  1. Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

    PubMed Central

    Py, Christophe; Martina, Marzia; Monette, Robert; Comas, Tanya; Denhoff, Mike W.; Luk, Collin; Syed, Naweed I.; Mealing, Geoff

    2012-01-01

    Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by

  2. 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. PMID:27270880

  3. 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.

  4. Novel Full-bridge ZVS DC-DC Converter with an Clamp Diodes

    NASA Astrophysics Data System (ADS)

    Guangqun, Nan; Xue, Hu

    The two arms of this converter all work at zero-voltage switching condition, that bring many advantages, such as little loss of power, simple control, etc. But these also exists some problems, such as parasitic oscillation on the output rectifier diodes. A novel full-bridge ZVS PWM DC-DC converter which adopts two clamping diodes in the first side of the transformer is proposed to reduce the parasitic oscillation in this paper. In this paper, It heavily analyzes the realization process of their soft-switching and the reason of related issues and solution, we establishes simulation model and simulates using orCAD. Finally,a 5 kW ZVS PWM DC-DC converter Prototype in that TMS320F2812 is the core controlled chip has been set up, and the experiments showed us the validation of the correlative theory.

  5. Reversible, voltage-activated formation of biomimetic membranes between triblock copolymer-coated aqueous droplets in good solvents.

    PubMed

    Tamaddoni, Nima; Taylor, Graham; Hepburn, Trevor; Michael Kilbey, S; Sarles, Stephen A

    2016-06-21

    Biomimetic membranes assembled from block copolymers attract considerable interest because they exhibit greater stability and longetivity compared to lipid bilayers, and some enable the reconstitution of functional transmembrane biomolecules. Yet to-date, block copolymer membranes have not been achieved using the droplet interface bilayer (DIB) method, which uniquely allows assembling single- and multi-membrane networks between water droplets in oil. Herein, we investigate the formation of poly(ethylene oxide)-b-poly(dimethyl siloxane)-b-poly(ethylene oxide) triblock copolymer-stabilized interfaces (CSIs) between polymer-coated aqueous droplets in solutions comprising combinations of decane, hexadecane and AR20 silicone oil. We demonstrate that triblock-coated droplets do not spontaneously adhere in these oils because all are thermodynamically good solvents for the hydrophobic PDMS middle block. However, thinned planar membranes are reversibly formed at the interface between droplets upon the application of a sufficient transmembrane voltage, which removes excess solvent from between droplets through electrocompression. At applied voltages above the threshold required to initiate membrane thinning, electrowetting causes the area of the CSI between droplets to increase while thickness remains constant; the CSI electrowetting response is similar to that encountered with lipid-based DIBs. In combination, these results reveal that stable membranes can be assembled in a manner that is completely reversible when an external pressure is used to overcome a barrier to adhesion caused by solvent-chain interactions, and they demonstrate new capability for connecting and disconnecting aqueous droplets via polymer-stabilized membranes.

  6. A biophysical model examining the role of low-voltage-activated potassium currents in shaping the responses of vestibular ganglion neurons.

    PubMed

    Hight, Ariel E; Kalluri, Radha

    2016-08-01

    The vestibular nerve is characterized by two broad groups of neurons that differ in the timing of their interspike intervals; some fire at highly regular intervals, whereas others fire at highly irregular intervals. Heterogeneity in ion channel properties has been proposed as shaping these firing patterns (Highstein SM, Politoff AL. Brain Res 150: 182-187, 1978; Smith CE, Goldberg JM. Biol Cybern 54: 41-51, 1986). Kalluri et al. (J Neurophysiol 104: 2034-2051, 2010) proposed that regularity is controlled by the density of low-voltage-activated potassium currents (IKL). To examine the impact of IKL on spike timing regularity, we implemented a single-compartment model with three conductances known to be present in the vestibular ganglion: transient sodium (gNa), low-voltage-activated potassium (gKL), and high-voltage-activated potassium (gKH). Consistent with in vitro observations, removing gKL depolarized resting potential, increased input resistance and membrane time constant, and converted current step-evoked firing patterns from transient (1 spike at current onset) to sustained (many spikes). Modeled neurons were driven with a time-varying synaptic conductance that captured the random arrival times and amplitudes of glutamate-driven synaptic events. In the presence of gKL, spiking occurred only in response to large events with fast onsets. Models without gKL exhibited greater integration by responding to the superposition of rapidly arriving events. Three synaptic conductance were modeled, each with different kinetics to represent a variety of different synaptic processes. In response to all three types of synaptic conductance, models containing gKL produced spike trains with irregular interspike intervals. Only models lacking gKL when driven by rapidly arriving small excitatory postsynaptic currents were capable of generating regular spiking. PMID:27121577

  7. Cd(2+) sensitivity and permeability of a low voltage-activated Ca(2+) channel with CatSper-like selectivity filter.

    PubMed

    Garza-López, Edgar; Chávez, Julio César; Santana-Calvo, Carmen; López-González, Ignacio; Nishigaki, Takuya

    2016-07-01

    CatSper is a sperm-specific Ca(2+) channel that plays an essential role in the male fertility. However, its biophysical properties have been poorly characterized mainly due to its deficient heterologous expression. As other voltage-gated Ca(2+) channels (CaVs), CatSper possesses a conserved Ca(2+)-selective filter motif ([T/S]x[D/E]xW) in the pore region. Interestingly, CatSper conserves four aspartic acids (DDDD) as the negatively charged residues in this motif while high voltage-activated CaVs have four glutamic acids (EEEE) and low voltage-activated CaVs possess two glutamic acids and two aspartic acids (EEDD). Previous studies based on site-directed mutagenesis of L- and T-type channels showed that the number of D seems to have a negative correlation with their cadmium (Cd(2+)) sensitivity. These results suggest that CatSper (DDDD) would have low sensitivity to Cd(2+). To explore Cd(2+)-sensitivity and -permeability of CatSper, we performed two types of experiments: 1) Electrophysiological analysis of heterologously expressed human CaV3.1 channel and three pore mutants (DEDD, EDDD and DDDD), 2) Cd(2+) imaging of human spermatozoa with FluoZin-1. Electrophysiological studies showed a significant increase in Cd(2+) and manganese (Mn(2+)) currents through the CaV3.1 mutants as well as a reduction in the inhibitory effect of Cd(2+) on the Ca(2+) current. In fluorescence imaging with human sperm, we observed an increase in Cd(2+) influx potentiated by progesterone, a potent activator of CatSper. These results support our hypothesis, namely that Cd(2+)-sensitivity and -permeability are related to the absolute number of D in the Ca(2+)-selective filter independently to the type of the Cav channels.

  8. The tarantula toxin jingzhaotoxin-XI (κ-theraphotoxin-Cj1a) regulates the activation and inactivation of the voltage-gated sodium channel Nav1.5.

    PubMed

    Tang, Cheng; Zhou, Xi; Huang, Yin; Zhang, Yunxiao; Hu, Zhaotun; Wang, Meichi; Chen, Ping; Liu, Zhonghua; Liang, Songping

    2014-12-15

    Specific peptide toxins interact with voltage-gated sodium channels by regulating the activation or inactivation of targeted channels. However, few toxins possessing dual effects have been identified. In the present study, we showed that jingzhaotoxin-XI/κ-theraphotoxin-Cj1a (JZTX-XI), a 34-residue peptide from the venom of the Chinese spider Chilobrachys jingzhao, inhibits the sodium conductance (IC50 = 124 ± 26 nM) and slows the fast inactivation (EC50 = 1.18 ± 0.2 μM) of Nav1.5 expressed in Chinese hamster ovary (CHO-K1) cells. JZTX-XI significantly shifted the activation to more depolarized voltages and decreased the deactivation of Nav1.5 currents upon extreme depolarization, but only slightly affected voltage-dependence of steady-state inactivation. In addition, JZTX-XI caused an approximately five-fold decrease in the rate of recovery from inactivation and an approximately 1.9-fold reduction in the closed-state inactivation rate. Our data suggest that JZTX-XI integrates the functions of site 3 toxins (α-scorpion toxins) with site 4 toxins (β-scorpion and spider toxins) by targeting multiple sites on Nav1.5. The unique properties displayed by JZTX-XI in its inhibitory activity on Nav1.5 suggest that its mechanism of action is distinct from those of site 3 and site 4 toxins, making JZTX-XI a useful probe for investigating the gating mechanism of Nav1.5 and toxin-channel interactions. PMID:25240294

  9. Hysteresis modeling of clamp band joint with macro-slip

    NASA Astrophysics Data System (ADS)

    Qin, Zhaoye; Cui, Delin; Yan, Shaoze; Chu, Fulei

    2016-01-01

    Clamp band joints are commonly used to connect spacecrafts with launch vehicles. Due to the frictional slippage between the joint components, hysteresis behavior might occur at joint interfaces under cyclic loading. The joint hysteresis will bring friction damping into the launching systems. In this paper, a closed-form hysteresis model for the clamp band joint is developed based on theoretical and numerical analyses of the interactions of the joint components. Then, the hysteresis model is applied to investigating the dynamic response of a payload fastened by the clamp band joint, where the nonlinearity and friction damping effects of the joint is evaluated. The proposed analytical model, which is validated by both finite element analyses and quasi-static experiments, has a simple form with sound accuracy and can be incorporated into the dynamic models of launching systems conveniently.

  10. Umbilical cord clamping. An analysis of a usual neonatological conduct.

    PubMed

    Papagno, L

    1998-01-01

    Here we described a critical analysis of the neonatological procedure of early cord clamping, meaning this, within 40 seconds after birth. Fifty three cases are here analysed, in which this practice was not performed, but instead a late umbilical cord clamping was done after birth or after the cord had stopped beating. Variations in hematocrito values within 24 to 36 hours after birth were studied. A transitory polycithemia, with a maximum peak 12 hours post-delivery was observed. These values returned to normal levels between 24 and 36 hours after birth. K vitamin was not administered to any of the newborns. No pathology appeared related to this transitory polycithemia. In can be concluded that the late umbilical cord clamping represents no risk to the new-born and that the pathological phenomena described under these circumstances may be attributed to the increase in K vitamin dependent coagulation factors that are induced by the routinary administration of phitonadione to all normal newborns.

  11. Resveratrol: inhibitory effects on metastatic cell behaviors and voltage-gated Na⁺ channel activity in rat prostate cancer in vitro.

    PubMed

    Fraser, Scott Paton; Peters, Alex; Fleming-Jones, Sian; Mukhey, Dev; Djamgoz, Mustafa Bilgin Ali

    2014-01-01

    Resveratrol, a natural plant phenolic found at high concentration in red grapes, has been suggested to have a range of health benefits. Here, we tested its effects on metastatic cell behaviors. The strongly metastatic rat prostate MAT-LyLu cells were used as a model. At 20 μM, resveratrol had no effect on cellular proliferation or viability. However, it suppressed significantly 1) lateral motility by up to 25%; 2) transverse motility by 31%; and invasion by 37%. It also increased the cells' adhesion to substrate by 55%. Electrophysiologically, resveratrol inhibited voltage-gated Na(+) channel (VGSC) activity that has been shown previously to promote metastatic cell behaviors. This effect was dose-dependent with an IC50 of ∼50 μM. Voltage dependencies of current activation and peak were not affected but steady-state inactivation was shifted to more hyperpolarized potentials and recovery from inactivation was slowed. Coapplication of resveratrol with the highly specific VGSC blocker tetrodotoxin did not result in any additive effect on inhibition of both 1) VGSC activity and 2) metastatic cell behaviors. These results suggest 1) that a significant mode of action of resveratrol is VGSC blockage and 2) that resveratrol has promise as a natural antimetastatic agent. PMID:25102135

  12. Plasma temperature clamping in filamentation laser induced breakdown spectroscopy

    SciTech Connect

    Harilal, Sivanandan S.; Yeak, J.; Phillips, Mark C.

    2015-10-19

    Ultrafast laser filament induced breakdown spectroscopy is a very promising method for remote material detection. We present characteristics of plasmas generated in a metal target by laser filaments in air. Our measurements show that the temperature of the ablation plasma is clamped along the filamentation channel due to intensity clamping in a filament. Nevertheless, significant changes in radiation intensity are noticeable, and this is essentially due to variation in the number density of emitting atoms. The present results also partly explains the reason for the occurrence of atomic plume during fs LIBS in air compared to long-pulse ns LIBS.

  13. Static DC to DC Power Conditioning-Active Ripple Filter, 1 MHZ DC to DC Conversion, and Nonlinear Analysis. Ph.D. Thesis; [voltage regulation and conversion circuitry for spacecraft power supplies

    NASA Technical Reports Server (NTRS)

    Sander, W. A., III

    1973-01-01

    Dc to dc static power conditioning systems on unmanned spacecraft have as their inputs highly fluctuating dc voltages which they condition to regulated dc voltages. These input voltages may be less than or greater than the desired regulated voltages. The design of two circuits which address specific problems in the design of these power conditioning systems and a nonlinear analysis of one of the circuits are discussed. The first circuit design is for a nondissipative active ripple filter which uses an operational amplifier to amplify and cancel the sensed ripple voltage. A dc to dc converter operating at a switching frequency of 1 MHz is the second circuit discussed. A nonlinear analysis of the type of dc to dc converter utilized in designing the 1 MHz converter is included.

  14. Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects.

    PubMed

    Pall, Martin L

    2013-08-01

    The direct targets of extremely low and microwave frequency range electromagnetic fields (EMFs) in producing non-thermal effects have not been clearly established. However, studies in the literature, reviewed here, provide substantial support for such direct targets. Twenty-three studies have shown that voltage-gated calcium channels (VGCCs) produce these and other EMF effects, such that the L-type or other VGCC blockers block or greatly lower diverse EMF effects. Furthermore, the voltage-gated properties of these channels may provide biophysically plausible mechanisms for EMF biological effects. Downstream responses of such EMF exposures may be mediated through Ca(2+) /calmodulin stimulation of nitric oxide synthesis. Potentially, physiological/therapeutic responses may be largely as a result of nitric oxide-cGMP-protein kinase G pathway stimulation. A well-studied example of such an apparent therapeutic response, EMF stimulation of bone growth, appears to work along this pathway. However, pathophysiological responses to EMFs may be as a result of nitric oxide-peroxynitrite-oxidative stress pathway of action. A single such well-documented example, EMF induction of DNA single-strand breaks in cells, as measured by alkaline comet assays, is reviewed here. Such single-strand breaks are known to be produced through the action of this pathway. Data on the mechanism of EMF induction of such breaks are limited; what data are available support this proposed mechanism. Other Ca(2+) -mediated regulatory changes, independent of nitric oxide, may also have roles. This article reviews, then, a substantially supported set of targets, VGCCs, whose stimulation produces non-thermal EMF responses by humans/higher animals with downstream effects involving Ca(2+) /calmodulin-dependent nitric oxide increases, which may explain therapeutic and pathophysiological effects.

  15. Electromagnetic fields act via activation of voltage-gated calcium channels to produce beneficial or adverse effects

    PubMed Central

    Pall, Martin L

    2013-01-01

    The direct targets of extremely low and microwave frequency range electromagnetic fields (EMFs) in producing non-thermal effects have not been clearly established. However, studies in the literature, reviewed here, provide substantial support for such direct targets. Twenty-three studies have shown that voltage-gated calcium channels (VGCCs) produce these and other EMF effects, such that the L-type or other VGCC blockers block or greatly lower diverse EMF effects. Furthermore, the voltage-gated properties of these channels may provide biophysically plausible mechanisms for EMF biological effects. Downstream responses of such EMF exposures may be mediated through Ca2+/calmodulin stimulation of nitric oxide synthesis. Potentially, physiological/therapeutic responses may be largely as a result of nitric oxide-cGMP-protein kinase G pathway stimulation. A well-