Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

PubMed

Behera, Rabindra K; Torres, Rodrigo; Tosha, Takehiko; Bradley, Justin M; Goulding, Celia W; Theil, Elizabeth C

2015-09-01

Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP λ max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.

Fe2+ Substrate Transport through Ferritin Protein Cage Ion Channels Influences Enzyme Activity and Biomineralization

PubMed Central

Behera, Rabindra K.; Torres, Rodrigo; Tosha, Takehiko; Bradley, Justin M.; Goulding, Celia W.; Theil, Elizabeth C.

2015-01-01

Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3.H2O), by moving cytoplasmic Fe2+ through intracage ion channels to cage-embedded enzyme (2Fe2+/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe2+ movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one – CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650nm (DFP λmax). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe3+-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: 1. narrower interior ion channel openings/pores, 2. increased numbers of ion channel protein-metal binding sites, and 3. a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells. PMID:26202907

Validity of the Electrodiffusion Model for Calculating Conductance of Simple Ion Channels.

PubMed

Pohorille, Andrew; Wilson, Michael A; Wei, Chenyu

2017-04-20

We examine the validity and utility of the electrodiffusion (ED) equation, i.e., the generalized Nernst-Planck equation, to characterize, in combination with molecular dynamics, the electrophysiological behavior of simple ion channels. As models, we consider three systems-two naturally occurring channels formed by α-helical bundles of peptaibols, trichotoxin, and alamethicin, and a synthetic, hexameric channel, formed by a peptide that contains only leucine and serine. All these channels mediate transport of potassium and chloride ions. Starting with equilibrium properties, such as the potential of mean force experienced by an ion traversing the channel and diffusivity, obtained from molecular dynamics simulations, the ED equation can be used to determine the full current-voltage dependence with modest or no additional effort. The potential of mean force can be obtained not only from equilibrium simulations, but also, with comparable accuracy, from nonequilibrium simulations at a single voltage. The main assumptions underlying the ED equation appear to hold well for the channels and voltages studied here. To expand the utility of the ED equation, we examine what are the necessary and sufficient conditions for Ohmic and nonrectifying behavior and relate deviations from this behavior to the shape of the ionic potential of mean force.

Ion Transport through Membrane-Spanning Nanopores Studied by Molecular Dynamics Simulations and Continuum Electrostatics Calculations

PubMed Central

Peter, Christine; Hummer, Gerhard

2005-01-01

Narrow hydrophobic regions are a common feature of biological channels, with possible roles in ion-channel gating. We study the principles that govern ion transport through narrow hydrophobic membrane pores by molecular dynamics simulation of model membranes formed of hexagonally packed carbon nanotubes. We focus on the factors that determine the energetics of ion translocation through such nonpolar nanopores and compare the resulting free-energy barriers for pores with different diameters corresponding to the gating regions in closed and open forms of potassium channels. Our model system also allows us to compare the results from molecular dynamics simulations directly to continuum electrostatics calculations. Both simulations and continuum calculations show that subnanometer wide pores pose a huge free-energy barrier for ions, but a small increase in the pore diameter to ∼1 nm nearly eliminates that barrier. We also find that in those wider channels the ion mobility is comparable to that in the bulk phase. By calculating local electrostatic potentials, we show that the long range Coulomb interactions of ions are strongly screened in the wide water-filled channels. Whereas continuum calculations capture the overall energetics reasonably well, the local water structure, which is not accounted for in this model, leads to interesting effects such as the preference of hydrated ions to move along the pore wall rather than through the center of the pore. PMID:16006629

Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

PubMed Central

Ryu, Shin-Young; Jhun, Bong Sook; Hurst, Stephen

2014-01-01

Abstract Significance: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. Recent Advances: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. Critical Issues: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. Future Directions: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters. Antioxid. Redox Signal. 21, 987–1006. PMID:24180309

Trafficking Mechanisms Underlying Neuronal Voltage-gated Ion Channel Localization at the Axon Initial Segment

PubMed Central

Vacher, Helene; Trimmer, James S.

2012-01-01

Summary Voltage-gated ion channels are diverse and fundamental determinants of neuronal intrinsic excitability. Voltage-gated K+ (Kv) and Na+ (Nav) channels play complex yet fundamentally important roles in determining intrinsic excitability. The Kv and Nav channels located at the axon initial segment (AIS) play a unique and especially important role in generating neuronal output in the form of anterograde axonal and backpropagating action potentials, Aberrant intrinsic excitability in individual neurons within networks contributes to synchronous neuronal activity leading to seizures. Mutations in ion channel genes gives rise to a variety of seizure-related “Channelopathies”, and many of the ion channel subunits associated with epilepsy mutations are localized at the AIS, making this a hotspot for epileptogenesis. Here we review the cellular mechanisms that underlie the trafficking of Kv and Nav channels found at the AIS, and how Kv and Nav channel mutations associated with epilepsy can alter these processes. PMID:23216576

Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis

PubMed Central

Nieto-Torres, Jose L.; Verdiá-Báguena, Carmina; Castaño-Rodriguez, Carlos; Aguilella, Vicente M.; Enjuanes, Luis

2015-01-01

Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology. PMID:26151305

The Earliest Ion Channels in Protocellular Membranes

NASA Technical Reports Server (NTRS)

Mijajlovic, Milan; Pohorille, Andrew; Wilson, Michael; Wei, Chenyu

2010-01-01

Cellular membranes with their hydrophobic interior are virtually impermeable to ions. Bulk of ion transport through them is enabled through ion channels. Ion channels of contemporary cells are complex protein molecules which span the membrane creating a cylindrical pore filled with water. Protocells, which are widely regarded as precursors to modern cells, had similarly impermeable membranes, but the set of proteins in their disposal was much simpler and more limited. We have been, therefore, exploring an idea that the first ion channels in protocellular membranes were formed by much smaller peptide molecules that could spontaneously selfassemble into short-lived cylindrical bundles in a membrane. Earlier studies have shown that a group of peptides known as peptaibols is capable of forming ion channels in lipid bilayers when they are exposed to an electric field. Peptaibols are small, non-genetically encoded peptides produced by some fungi as a part of their system of defense against bacteria. They are usually only 14-20 residues long, which is just enough to span the membrane. Their sequence is characterized by the presence of non-standard amino acids which, interestingly, are also expected to have existed on the early earth. In particular, the presence of 2-aminoisobutyric acid (AIB) gives peptaibols strong helix forming propensities. Association of the helices inside membranes leads to the formation of cylindrical bundles, typically containing 4 to 10 monomers. Although peptaibols are excellent candidates for models of the earliest ion channels their structures, which are stabilized only by van der Waals forces and occasional hydrogen bonds between neighboring helices, are not very stable. Although it might properly reflect protobiological reality, it is also a major obstacle in studying channel behavior. For this reason we focused on two members of the peptaibol family, trichotoxin and antiamoebin, which are characterized by a single conductance level. This indicates that their structures are unique and stable. In addition, it is also believed that the trichotoxin channel displays some selectivity between potassium and chloride ions. This makes trichotoxin and antiamoebin ideal models of the earliest ion channels that could provide insight into the origins of ion conductance and selectivity. In the absence of crystal structure of the trichotoxin and antiamoebin channels, we propose their molecular models based on experimentally determined number of monomers forming the bundles. We use molecular dynamics simulations to validate the models in terms of their conductance and selectivity. On the basis of our simulations we show that the emergence of channels built of small, alpha-helical peptides was protobiologically plausible and did not require highly specific amino acid sequences, which is a convenient evolutionary trait. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. To this end, we will discuss how the amino acid sequence and structure of primitive channels give rise to the phenomena of ionic conductance and selectivity across the earliest cell walls, which were essential functions for the emergence and early evolution of protocells. Furthermore, we will argue that even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

The earliest ion channels in protocellular membranes

NASA Astrophysics Data System (ADS)

Mijajlovic, Milan; Pohorille, Andrew; Wilson, Michael; Wei, Chenyu

Cellular membranes with their hydrophobic interior are virtually impermeable to ions. Bulk of ion transport through them is enabled through ion channels. Ion channels of contemporary cells are complex protein molecules which span the membrane creating a cylindrical pore filled with water. Protocells, which are widely regarded as precursors to modern cells, had similarly impermeable membranes, but the set of proteins in their disposal was much simpler and more limited. We have been, therefore, exploring an idea that the first ion channels in protocellular membranes were formed by much smaller peptide molecules that could spontaneously self-assemble into short-lived cylindrical bundles in a membrane. Earlier studies have shown that a group of peptides known as peptaibols is capable of forming ion channels in lipid bilayers when they are exposed to an electric field. Peptaibols are small, non-genetically encoded peptides produced by some fungi as a part of their system of defense against bacteria. They are usually only 14-20 residues long, which is just enough to span the membrane. Their sequence is characterized by the presence of non-standard amino acids which, interestingly, are also expected to have existed on the early earth. In particular, the presence of 2-aminoisobutyric acid (AIB) gives peptaibols strong helix forming propensities. Association of the helices inside membranes leads to the formation of cylindrical bundles, typically containing 4 to 10 monomers. Although peptaibols are excellent candidates for models of the earliest ion channels their struc-tures, which are stabilized only by van der Waals forces and occasional hydrogen bonds between neighboring helices, are not very stable. Although it might properly reflect protobiological real-ity, it is also a major obstacle in studying channel behavior. For this reason we focused on two members of the peptaibol family, trichotoxin and antiamoebin, which are characterized by a single conductance level. This indicates that their structures are unique and stable. In addition, it is also believed that the trichotoxin channel displays some selectivity between potassium and chloride ions. This makes trichotoxin and antiamoebin ideal models of the earliest ion channels that could provide insight into the origins of ion conductance and selectivity. In the absence of crystal structure of the trichotoxin and antiamoebin channels, we propose their molecular models based on experimentally determined number of monomers forming the bundles. We use molecular dynamics simulations to validate the models in terms of their conductance and selectivity. On the basis of our simulations we show that the emergence of channels built of small, α-helical peptides was protobiologically plausible and did not require highly specific amino acid sequences, which is a convenient evolutionary trait. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. To this end, we will discuss how the amino acid sequence and structure of primitive channels give rise to the phenomena of ionic conductance and selectivity across the earliest cell walls, which were essential functions for the emergence and early evolution of protocells. Furthermore, we will argue that even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel

PubMed Central

Zhao, Juan; Blunck, Rikard

2016-01-01

Domains in macromolecular complexes are often considered structurally and functionally conserved while energetically coupled to each other. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). Here, the energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the proximal C-terminus. In order to characterize the intrinsic gating of the voltage sensing domain in the absence of the pore domain, the Shaker Kv channel was truncated after the fourth transmembrane helix S4 (Shaker-iVSD). Shaker-iVSD showed significantly altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain. Shaker-iVSD also displays pronounced 'relaxation'. Closing of the pore correlates with entry into relaxation suggesting that the two processes are energetically related. DOI: http://dx.doi.org/10.7554/eLife.18130.001 PMID:27710769

Electric Field-Controlled Ion Transport In TiO2 Nanochannel.

PubMed

Li, Dan; Jing, Wenheng; Li, Shuaiqiang; Shen, Hao; Xing, Weihong

2015-06-03

On the basis of biological ion channels, we constructed TiO2 membranes with rigid channels of 2.3 nm to mimic biomembranes with flexible channels; an external electric field was employed to regulate ion transport in the confined channels at a high ionic strength in the absence of electrical double layer overlap. Results show that transport rates for both Na+ and Mg2+ were decreased irrespective of the direction of the electric field. Furthermore, a voltage-gated selective ion channel was formed, the Mg2+ channel closed at -2 V, and a reversed relative electric field gradient was at the same order of the concentration gradient, whereas the Na+ with smaller Stokes radius and lower valence was less sensitive to the electric field and thus preferentially occupied and passed the channel. Thus, when an external electric field is applied, membranes with larger nanochannels have promising applications in selective separation of mixture salts at a high concentration.

Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels

NASA Astrophysics Data System (ADS)

Lau, Carus H. Y.; King, Glenn F.; Mobli, Mehdi

2016-09-01

Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.

Atomistic Detailed Mechanism and Weak Cation-Conducting Activity of HIV-1 Vpu Revealed by Free Energy Calculations

PubMed Central

Padhi, Siladitya; Burri, Raghunadha Reddy; Jameel, Shahid; Priyakumar, U. Deva

2014-01-01

The viral protein U (Vpu) encoded by HIV-1 has been shown to assist in the detachment of virion particles from infected cells. Vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. An understanding of the mechanism of ion transport through Vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. Using a structure of the pentameric form of Vpu – modeled and validated based on available experimental data – umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in Vpu. Free energy profiles corresponding to the permeation of Na+ and K+ were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. The Ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. A two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. It is shown that only one ion can pass through the channel at a time. This, along with a stretch of hydrophobic residues in the transmembrane domain of Vpu, explains the slow kinetics of ion conduction. The results are consistent with previous conductance studies that showed Vpu to be a weakly conducting ion channel. PMID:25392993

Self-Assembling Organic Nanopores as Synthetic Transmembrane Channels with Tunable Functions

NASA Astrophysics Data System (ADS)

Wei, Xiaoxi

A long-standing goal in the area of supramolecular self-assembly involves the development of synthetic ion/water channels capable of mimicking the mass-transport characteristics of biological channels and pores. Few examples of artificial transmembrane channels with large lumen, high conductivity and selectivity are known. A review of pronounced biological transmembrane protein channels and some representative synthetic models have been provided in Chapter 1, followed by our discovery and initial investigation of shape-persistent oligoamide and phenylene ethynylene macrocycles as synthetic ion/water channels. In Chapter 2, the systematic structural modification of oligoamide macrocycles 1, the so-called first-generation of these shape-persistent macrocycles, has led to third-generation macrocycles 3. The third generation was found to exhibit unprecedented, strong intermolecular association in both the solid state and solution via multiple techniques including X-ray diffraction (XRD), SEM, and 1H NMR. Fluorescence spectroscopy paired with dynamic light scattering (DLS) revealed that macrocycles 3 can assemble into a singly dispersed nanotubular structure in solution. The resultant self-assembling pores consisting of 3 were examined by HPTS-LUVs assays and BLM studies (Chapter 3) and found to form cation-selective (PK+/PCl- = 69:1) transmembrane ion channels with large conductance (200 ˜ 2000 pS for alkali cations) and high stability with open times reaching to 103 seconds. Tuning the aggregation state of macrocycles by choosing an appropriate polar solvent mixture (i.e., 3:1, THF:DMF, v/v) and concentration led to the formation of ion channels with well-defined square top behavior. A parallel study using DLS to examine the size of aggregates was used in conjunction with channel activity assays (LUVs/BLM) to reveal the effects of the aggregation state on channel activity. Empirical evidence now clearly indicates that a preassembled state, perhaps that of a nanotubular assembly, rather than the individual molecules of 3, is required to partition into the lipid bilayer in order for these macrocycles to act as channels. Further structural modification has led to fourth-generation macrocycles 4 having readily-tunable cavities (Chapter 4). Macrocycles 4 , with a hybrid backbone composed half of the oligoamide and half of the phenylene ethynylene moieties, exhibits similar self-assembling behavior by forming nanotubular stacks. The results of a preliminary study based on LUVs-assays and BLM single channel recording experiments are summarized and clearly indicate that ion channels formed by this fourth-generation exhibit high stability and differing ion selectivity largely consistent with the corresponding structural modification of the interior cavity. Especially, the increased anion conductance observed for 4d indicates that our strategy of tuning supramolecular function based on synthetic modification of the backbone and pore is effective. In Chapter 5, our four-residue tetraurea macrocycles 5 have shown significant potency to selectively interact with the G-quadruplex, leading to a strong stabilization effect for G-quadruplex without binding to duplex DNA as observed by UV-melt assays. The ready synthetic availability of these macrocycles makes them amenable to future chemical modification, which allows systematic improvement of binding affinity and specificity. Moreover, it has been discovered that these macrocycles can partition into lipid bilayers and form very stable transmembrane ion channels with a pore size of ˜5 A. Preliminary data shows that this smaller ion channel may lead to exceptional ion conducting selectivity, which is rarely seen in the field of synthetic ion pores. These molecules may serve as a unique platform for the rational development of potent and versatile therapeutic agents. The exceptional ion conducting properties of these channels place aromatic oligoamide macrocycles 3 and 4 at a unique position with both high conductance and long channel-opening duration. These results demonstrate that oligoamide macrocycles provide us a reliable platform based on which further development of highly conducting and selective synthetic mass-transporting channels, with functions that are comparable to or even rival those of natural channels and pores, may be developed. Further improvement of these synthetic channels could lead to numerous applications, such as those for complementing ion channel deficiency in clinical medicine, designing biosensors, and the development of new materials, as well as their use in separation and purifications.

Structure of conducting channel of lightning

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

Alanakyan, Yu. R.

2013-08-15

The spatial distribution of the plasma density in a lightning channel is studied theoretically. It is shown that the electric-field double layer is formed at the channel boundary. In this case, the electron temperature changes abruptly and ions are accelerated by the electric field of the double layer. The ion momentum flux density is close to the surrounding gas pressure. Cleaning of the channel from heavy particles occurs in particle-exchange processes between the plasma channel and the surrounding air. Hydrogen ions are accumulated inside the expanding channel from the surrounding air, which is enriched by hydrogen-contained molecules. In this case,more » the plasma channel is unstable and splits to a chain of equidistant bunches of plasma. The hydrogen-enrich bunches burn diffusely after recombination exhibiting the bead lightning behavior.« less

Ion Permeation and Mechanotransduction Mechanisms of Mechanosensitive Piezo Channels.

PubMed

Zhao, Qiancheng; Wu, Kun; Geng, Jie; Chi, Shaopeng; Wang, Yanfeng; Zhi, Peng; Zhang, Mingmin; Xiao, Bailong

2016-03-16

Piezo proteins have been proposed as the long-sought-after mechanosensitive cation channels in mammals that play critical roles in various mechanotransduction processes. However, the molecular bases that underlie their ion permeation and mechanotransduction have remained functionally undefined. Here we report our finding of the miniature pore-forming module of Piezo1 that resembles the pore architecture of other trimeric channels and encodes the essential pore properties. We further identified specific residues within the pore module that determine unitary conductance, pore blockage and ion selectivity for divalent and monovalent cations and anions. The non-pore-containing region of Piezo1 confers mechanosensitivity to mechano-insensitive trimeric acid-sensing ion channels, demonstrating that Piezo1 channels possess intrinsic mechanotransduction modules separate from their pore modules. In conclusion, this is the first report on the bona fide pore module and mechanotransduction components of Piezo channels, which define their ion-conducting properties and gating by mechanical stimuli, respectively. Copyright © 2016 Elsevier Inc. All rights reserved.

Potential Roles of Amiloride-Sensitive Sodium Channels in Cancer Development.

PubMed

Xu, Siguang; Liu, Cui; Ma, Yana; Ji, Hong-Long; Li, Xiumin

2016-01-01

The ENaC/degenerin ion channel superfamily includes the amiloride-sensitive epithelial sodium channel (ENaC) and acid sensitive ionic channel (ASIC). ENaC is a multimeric ion channel formed by heteromultimeric membrane glycoproteins, which participate in a multitude of biological processes by mediating the transport of sodium (Na(+)) across epithelial tissues such as the kidney, lungs, bladder, and gut. Aberrant ENaC functions contribute to several human disease states including pseudohypoaldosteronism, Liddle syndrome, cystic fibrosis, and salt-sensitive hypertension. Increasing evidence suggests that ion channels not only regulate ion homeostasis and electric signaling in excitable cells but also play important roles in cancer cell behaviors such as proliferation, apoptosis, invasion, and migration. Indeed, ENaCs/ASICs had been reported to be associated with cancer characteristics. Given their cell surface localization and pharmacology, pharmacological strategies to target ENaC/ASIC family members may be promising cancer therapeutics.

Design and synthesis of guest-host nanostructures to enhance ionic conductivity across nanocomposite membranes

DOEpatents

Hu, Michael Z [Knoxville, TN; Kosacki, Igor [Oak Ridge, TN

2010-01-05

An ion conducting membrane has a matrix including an ordered array of hollow channels and a nanocrystalline electrolyte contained within at least some or all of the channels. The channels have opposed open ends, and a channel width of 1000 nanometers or less, preferably 60 nanometers or less, and most preferably 10 nanometers or less. The channels may be aligned perpendicular to the matrix surface, and the length of the channels may be 10 nanometers to 1000 micrometers. The electrolyte has grain sizes of 100 nanometers or less, and preferably grain sizes of 1 to 50 nanometers. The electrolyte may include grains with a part of the grain boundaries aligned with inner walls of the channels to form a straight oriented grain-wall interface or the electrolyte may be a single crystal. In one form, the electrolyte conducts oxygen ions, the matrix is silica, and the electrolyte is yttrium doped zirconia.

Computational study of a calcium release-activated calcium channel

NASA Astrophysics Data System (ADS)

Talukdar, Keka; Shantappa, Anil

2016-05-01

The naturally occurring proteins that form hole in membrane are commonly known as ion channels. They play multiple roles in many important biological processes. Deletion or alteration of these channels often leads to serious problems in the physiological processes as it controls the flow of ions through it. The proper maintenance of the flow of ions, in turn, is required for normal health. Here we have investigated the behavior of a calcium release-activated calcium ion channel with pdb entry 4HKR in Drosophila Melanogaster. The equilibrium energy as well as molecular dynamics simulation is performed first. The protein is subjected to molecular dynamics simulation to find their energy minimized value. Simulation of the protein in the environment of water and ions has given us important results too. The solvation energy is also found using Charmm potential.

Molecular Dynamics Simulation of the Antiamoebin Ion Channel: Linking Structure and Conductance

NASA Technical Reports Server (NTRS)

Wilson, Michael A.; Wei, Chenyu; Bjelkmar, Paer; Wallace, B. A.; Pohorille, Andrew

2011-01-01

Molecular dynamics simulations were carried out in order to ascertain which of the potential multimeric forms of the transmembrane peptaibol channel, antiamoebin, is consistant with its measured conductance. Estimates of the conductance obtained through counting ions that cross the channel and by solving the Nernst-Planck equation yield consistent results, indicating that the motion of ions inside the channel can be satisfactorily described as diffusive.The calculated conductance of octameric channels is markedly higher than the conductance measured in single channel recordings, whereas the tetramer appears to be non-conducting. The conductance of the hexamer was estimated to be 115+/-34 pS and 74+/-20 pS, at 150 mV and 75 mV, respectively, in satisfactory agreement with the value of 90 pS measured at 75 mV. On this basis we propose that the antiamoebin channel consists of six monomers. Its pore is large enough to accommodate K(+) and Cl(-) with their first solvation shells intact. The free energy barrier encountered by K(+) is only 2.2 kcal/mol whereas Cl(-) encounters a substantially higher barrier of nearly 5 kcal/mol. This difference makes the channel selective for cations. Ion crossing events are shown to be uncorrelated and follow Poisson statistics. keywords: ion channels, peptaibols, channel conductance, molecular dynamics

Free-Energy Calculations. A Mathematical Perspective

NASA Technical Reports Server (NTRS)

Pohorille, Andrzej

2015-01-01

Ion channels are pore-forming assemblies of transmembrane proteins that mediate and regulate ion transport through cell walls. They are ubiquitous to all life forms. In humans and other higher organisms they play the central role in conducting nerve impulses. They are also essential to cardiac processes, muscle contraction and epithelial transport. Ion channels from lower organisms can act as toxins or antimicrobial agents, and in a number of cases are involved in infectious diseases. Because of their important and diverse biological functions they are frequent targets of drug action. Also, simple natural or synthetic channels find numerous applications in biotechnology. For these reasons, studies of ion channels are at the forefront of biophysics, structural biology and cellular biology. In the last decade, the increased availability of X-ray structures has greatly advanced our understanding of ion channels. However, their mechanism of action remains elusive. This is because, in order to assist controlled ion transport, ion channels are dynamic by nature, but X-ray crystallography captures the channel in a single, sometimes non-native state. To explain how ion channels work, X-ray structures have to be supplemented with dynamic information. In principle, molecular dynamics (MD) simulations can aid in providing this information, as this is precisely what MD has been designed to do. However, MD simulations suffer from their own problems, such as inability to access sufficiently long time scales or limited accuracy of force fields. To assess the reliability of MD simulations it is only natural to turn to the main function of channels - conducting ions - and compare calculated ionic conductance with electrophysiological data, mainly single channel recordings, obtained under similar conditions. If this comparison is satisfactory it would greatly increase our confidence that both the structures and our computational methodologies are sufficiently accurate. Channel conductance, defined as the ratio of ionic current through the channel to applied voltage, can be calculated in MD simulations by way of applying an external electric field to the system and counting the number of ions that traverse the channel per unit time. If the current is small, a voltage significantly higher than the experimental one needs to be applied to collect sufficient statistics of ion crossing events. Then, the calculated conductance has to be extrapolated to the experimental voltage using procedures of unknown accuracy. Instead, we propose an alternative approach that applies if ion transport through channels can be described with sufficient accuracy by the one-dimensional diffusion equation in the potential given by the free energy profile and applied voltage. Then, it is possible to test the assumptions of the equation, recover the full voltage/current dependence, determine the reliability of the calculated conductance and reconstruct the underlying (equilibrium) free energy profile, all from MD simulations at a single voltage. We will present the underlying theory, model calculations that test this theory and simulations on ion conductance through a channel that has been extensively studied experimentally. To our knowledge this is the first case in which the complete, experimentally measured dependence of the current on applied voltage has been reconstructed from MD simulations.

A hydrophobic gate in an ion channel: the closed state of the nicotinic acetylcholine receptor

NASA Astrophysics Data System (ADS)

Beckstein, Oliver; Sansom, Mark S. P.

2006-06-01

The nicotinic acetylcholine receptor (nAChR) is the prototypic member of the 'Cys-loop' superfamily of ligand-gated ion channels which mediate synaptic neurotransmission, and whose other members include receptors for glycine, γ-aminobutyric acid and serotonin. Cryo-electron microscopy has yielded a three-dimensional structure of the nAChR in its closed state. However, the exact nature and location of the channel gate remains uncertain. Although the transmembrane pore is constricted close to its center, it is not completely occluded. Rather, the pore has a central hydrophobic zone of radius about 3 Å. Model calculations suggest that such a constriction may form a hydrophobic gate, preventing movement of ions through a channel. We present a detailed and quantitative simulation study of the hydrophobic gating model of the nicotinic receptor, in order to fully evaluate this hypothesis. We demonstrate that the hydrophobic constriction of the nAChR pore indeed forms a closed gate. Potential of mean force (PMF) calculations reveal that the constriction presents a barrier of height about 10 kT to the permeation of sodium ions, placing an upper bound on the closed channel conductance of 0.3 pS. Thus, a 3 Å radius hydrophobic pore can form a functional barrier to the permeation of a 1 Å radius Na+ ion. Using a united-atom force field for the protein instead of an all-atom one retains the qualitative features but results in differing conductances, showing that the PMF is sensitive to the detailed molecular interactions.

Understanding the conductive channel evolution in Na:WO3-x-based planar devices

NASA Astrophysics Data System (ADS)

Shang, Dashan; Li, Peining; Wang, Tao; Carria, Egidio; Sun, Jirong; Shen, Baogen; Taubner, Thomas; Valov, Ilia; Waser, Rainer; Wuttig, Matthias

2015-03-01

An ion migration process in a solid electrolyte is important for ion-based functional devices, such as fuel cells, batteries, electrochromics, gas sensors, and resistive switching systems. In this study, a planar sandwich structure is prepared by depositing tungsten oxide (WO3-x) films on a soda-lime glass substrate, from which Na+ diffuses into the WO3-x films during the deposition. The entire process of Na+ migration driven by an alternating electric field is visualized in the Na-doped WO3-x films in the form of conductive channel by in situ optical imaging combined with infrared spectroscopy and near-field imaging techniques. A reversible change of geometry between a parabolic and a bar channel is observed with the resistance change of the devices. The peculiar channel evolution is interpreted by a thermal-stress-induced mechanical deformation of the films and an asymmetric Na+ mobility between the parabolic and the bar channels. These results exemplify a typical ion migration process driven by an alternating electric field in a solid electrolyte with a low ion mobility and are expected to be beneficial to improve the controllability of the ion migration in ion-based functional devices, such as resistive switching devices.An ion migration process in a solid electrolyte is important for ion-based functional devices, such as fuel cells, batteries, electrochromics, gas sensors, and resistive switching systems. In this study, a planar sandwich structure is prepared by depositing tungsten oxide (WO3-x) films on a soda-lime glass substrate, from which Na+ diffuses into the WO3-x films during the deposition. The entire process of Na+ migration driven by an alternating electric field is visualized in the Na-doped WO3-x films in the form of conductive channel by in situ optical imaging combined with infrared spectroscopy and near-field imaging techniques. A reversible change of geometry between a parabolic and a bar channel is observed with the resistance change of the devices. The peculiar channel evolution is interpreted by a thermal-stress-induced mechanical deformation of the films and an asymmetric Na+ mobility between the parabolic and the bar channels. These results exemplify a typical ion migration process driven by an alternating electric field in a solid electrolyte with a low ion mobility and are expected to be beneficial to improve the controllability of the ion migration in ion-based functional devices, such as resistive switching devices. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07545e

Charging the quantum capacitance of graphene with a single biological ion channel.

PubMed

Wang, Yung Yu; Pham, Ted D; Zand, Katayoun; Li, Jinfeng; Burke, Peter J

2014-05-27

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitance of the graphene. This approach combines nanotechnology with electrophysiology to demonstrate qualitatively new ways of measuring ion channel currents.

Charging the Quantum Capacitance of Graphene with a Single Biological Ion Channel

PubMed Central

2015-01-01

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitance of the graphene. This approach combines nanotechnology with electrophysiology to demonstrate qualitatively new ways of measuring ion channel currents. PMID:24754625

Artificial transmembrane ion channels from self-assembling peptide nanotubes

NASA Astrophysics Data System (ADS)

Ghadiri, M. Reza; Granja, Juan R.; Buehler, Lukas K.

1994-05-01

NATURALLY occurring membrane channels and pores are formed from a large family of diverse proteins, peptides and organic secon-dary metabolites whose vital biological functions include control of ion flow, signal transduction, molecular transport and produc-tion of cellular toxins. But despite the availability of a large amount of biochemical information about these molecules1, the design and synthesis of artificial systems that can mimic the bio-logical function of natural compounds remains a formidable task2-12. Here we present a simple strategy for the design of artifi-cial membrane ion channels based on a self-assembled cylindrical β-sheet peptide architecture13. Our systems-essentially stacks of peptide rings-display good channel-mediated ion-transport activ-ity with rates exceeding 107 ions s-1, rivalling the performance of many naturally occurring counterparts. Such molecular assemblies should find use in the design of novel cytotoxic agents, membrane transport vehicles and drug-delivery systems.

Fatty acids and related Kv2 channel blockers: electrophysiology and toxicity on mosquitoes

USDA-ARS?s Scientific Manuscript database

Ligand-gated ion channels form an important superfamily of proteins involved in many biological processes. Among them, the potassium channels constitute a very diverse group involved in neural signaling, neuronal activity and action potential. Among the different types of channel activation, voltage...

Expression and distribution of voltage-gated ion channels in ferret sinoatrial node.

PubMed

Brahmajothi, Mulugu V; Morales, Michael J; Campbell, Donald L; Steenbergen, Charles; Strauss, Harold C

2010-10-01

Spontaneous diastolic depolarization in the sinoatrial (SA) node enables it to serve as pacemaker of the heart. The variable cell morphology within the SA node predicts that ion channel expression would be heterogeneous and different from that in the atrium. To evaluate ion channel heterogeneity within the SA node, we used fluorescent in situ hybridization to examine ion channel expression in the ferret SA node region and atrial appendage. SA nodal cells were distinguished from surrounding cardiac myocytes by expression of the slow (SA node) and cardiac (surrounding tissue) forms of troponin I. Nerve cells in the sections were identified by detection of GAP-43 and cytoskeletal middle neurofilament. Transcript expression was characterized for the 4 hyperpolarization-activated cation channels, 6 voltage-gated Na(+) channels, 3 voltage-gated Ca(2+) channels, 24 voltage-gated K(+) channel α-subunits, and 3 ancillary subunits. To ensure that transcript expression was representative of protein expression, immunofluorescence was used to verify localization patterns of voltage-dependent K(+) channels. Colocalizations were performed to observe any preferential patterns. Some overlapping and nonoverlapping binding patterns were observed. Measurement of different cation channel transcripts showed heterogeneous expression with many different patterns of expression, attesting to the complexity of electrical activity in the SA node. This study provides insight into the possible role ion channel heterogeneity plays in SA node pacemaker activity.

Identification and characterization of a bacterial hydrosulphide ion channel

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

Czyzewski, Bryan K.; Wang, Da-Neng

2012-10-26

The hydrosulphide ion (HS{sup -}) and its undissociated form, hydrogen sulphide (H{sub 2}S), which are believed to have been critical to the origin of life on Earth, remain important in physiology and cellular signalling. As a major metabolite in anaerobic bacterial growth, hydrogen sulphide is a product of both assimilatory and dissimilatory sulphate reduction. These pathways can reduce various oxidized sulphur compounds including sulphate, sulphite and thiosulphate. The dissimilatory sulphate reduction pathway uses this molecule as the terminal electron acceptor for anaerobic respiration, in which process it produces excess amounts of H{sub 2}S. The reduction of sulphite is a keymore » intermediate step in all sulphate reduction pathways. In Clostridium and Salmonella, an inducible sulphite reductase is directly linked to the regeneration of NAD{sup +}, which has been suggested to have a role in energy production and growth, as well as in the detoxification of sulphite. Above a certain concentration threshold, both H{sub 2}S and HS{sup -} inhibit cell growth by binding the metal centres of enzymes and cytochrome oxidase, necessitating a release mechanism for the export of this toxic metabolite from the cell. Here we report the identification of a hydrosulphide ion channel in the pathogen Clostridium difficile through a combination of genetic, biochemical and functional approaches. The HS{sup -} channel is a member of the formate/nitrite transport family, in which about 50 hydrosulphide ion channels form a third subfamily alongside those for formate (FocA) and for nitrite (NirC). The hydrosulphide ion channel is permeable to formate and nitrite as well as to HS{sup -} ions. Such polyspecificity can be explained by the conserved ion selectivity filter observed in the channel's crystal structure. The channel has a low open probability and is tightly regulated, to avoid decoupling of the membrane proton gradient.« less

Role of TRP ion channels in cancer and tumorigenesis.

PubMed

Shapovalov, George; Ritaine, Abigael; Skryma, Roman; Prevarskaya, Natalia

2016-05-01

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

Harnessing the Flow of Excitation: TRP, Voltage-Gated Na(+), and Voltage-Gated Ca(2+) Channels in Contemporary Medicine.

PubMed

Frolov, Roman V; Weckström, Matti

2016-01-01

Cellular signaling in both excitable and nonexcitable cells involves several classes of ion channels. Some of them are of minor importance, with very specialized roles in physiology, but here we concentrate on three major channel classes: TRP (transient receptor potential channels), voltage-gated sodium channels (Nav), and voltage-gated calcium channels (Cav). Here, we first propose a conceptual framework binding together all three classes of ion channels, a "flow-of-excitation model" that takes into account the inputs mediated by TRP and other similar channels, the outputs invariably provided by Cav channels, and the regenerative transmission of signals in the neural networks, for which Nav channels are responsible. We use this framework to examine the function, structure, and pharmacology of these channel classes both at cellular and also at whole-body physiological level. Building on that basis we go through the pathologies arising from the direct or indirect malfunction of the channels, utilizing ion channel defects, the channelopathies. The pharmacological interventions affecting these channels are numerous. Part of those are well-established treatments, like treatment of hypertension or some forms of epilepsy, but many other are deeply problematic due to poor drug specificity, ion channel diversity, and widespread expression of the channels in tissues other than those actually targeted. © 2016 Elsevier Inc. All rights reserved.

Piezo1 forms mechanosensitive ion channels in the human MCF-7 breast cancer cell line

NASA Astrophysics Data System (ADS)

Li, Chouyang; Rezania, Simin; Kammerer, Sarah; Sokolowski, Armin; Devaney, Trevor; Gorischek, Astrid; Jahn, Stephan; Hackl, Hubert; Groschner, Klaus; Windpassinger, Christian; Malle, Ernst; Bauernhofer, Thomas; Schreibmayer, Wolfgang

2015-02-01

Mechanical interaction between cells - specifically distortion of tensional homeostasis-emerged as an important aspect of breast cancer genesis and progression. We investigated the biophysical characteristics of mechanosensitive ion channels (MSCs) in the malignant MCF-7 breast cancer cell line. MSCs turned out to be the most abundant ion channel species and could be activated by negative pressure at the outer side of the cell membrane in a saturable manner. Assessing single channel conductance (GΛ) for different monovalent cations revealed an increase in the succession: Li+ < Na+ < K+ ~Rb+ ~ Cs+. Divalent cations permeated also with the order: Ca2+ < Ba2+. Comparison of biophysical properties enabled us to identify MSCs in MCF-7 as ion channels formed by the Piezo1 protein. Using patch clamp technique no functional MSCs were observed in the benign MCF-10A mammary epithelial cell line. Blocking of MSCs by GsMTx-4 resulted in decreased motility of MCF-7, but not of MCF-10A cells, underscoring a possible role of Piezo1 in invasion and metastatic propagation. The role of Piezo1 in biology and progression of breast cancer is further substantiated by markedly reduced overall survival in patients with increased Piezo1 mRNA levels in the primary tumor.

Atomic Force Microscopy and MD Simulations Reveal Pore-Like Structures of All-D-Enantiomer of Alzheimer’s β-Amyloid Peptide: Relevance to the Ion Channel Mechanism of AD Pathology

PubMed Central

Connelly, Laura; Arce, Fernando Teran; Jang, Hyunbum; Capone, Ricardo; Kotler, Samuel A.; Ramachandran, Srinivasan; Kagan, Bruce L.; Nussinov, Ruth; Lal, Ratnesh

2012-01-01

Alzheimer’s disease (AD) is a protein misfolding disease characterized by a build-up of β-amyloid (Aβ) peptide as senile plaques, uncontrolled neurodegeneration, and memory loss. AD pathology is linked to the destabilization of cellular ionic homeostasis and involves Aβ peptide-plasma membrane interactions. In principle, there are two possible ways through which disturbance of the ionic homeostasis can take place: directly, where the Aβ peptide either inserts into the membrane and creates ion-conductive pores or destabilizes the membrane organization; or, indirectly, where the Aβ peptide interacts with existing cell membrane receptors. To distinguish between these two possible types of Aβ-membrane interactions, we took advantage of the biochemical tenet that ligand-receptor interactions are stereospecific; L-amino acid peptides, but not their D-counterparts, bind to cell membrane receptors. However, with respect to the ion channel-mediated mechanism, like L-amino acids, D-amino acid peptides will also form ion channel-like structures. Using atomic force microscopy (AFM) we imaged the structures of both D- and L-enantiomers of the full length Aβ1-42 when reconstituted in lipid bilayers. AFM imaging shows that both L- and D-Aβ isomers form similar channel-like structures. Molecular dynamics (MD) simulations support the AFM imaged 3D structures. Earlier we have shown that D-Aβ1-42 channels conduct ions similarly to their L-counter parts. Taken together, our results support the direct mechanism of Aβ ion channel-mediated destabilization of ionic homeostasis rather than the indirect mechanism through Aβ interaction with membrane receptors. PMID:22217000

Potential therapeutic targets for ATP-gated P2X receptor ion channels.

PubMed

Li, Zhiyuan; Liang, Dong; Chen, Ling

2008-04-01

P2X receptors make up a novel family of ligand-gated ion channels that are activated by binding of extracellular ATP. These receptors can form a number of homomeric and heteromeric ion channels, which are widely distributed throughout the human body. They are thought to play an important role in many cellular processes, including synaptic transmission and thrombocyte aggregation. These ion channels are also involved in the pathology of several disease states, including chronic inflammation and neuropathic pain, and thus are the potential targets for drug development. The recent discovery of potent and highly selective antagonists for P2X(7) receptors, through the use of high-throughput screening, has helped to further understand the P2X receptor pharmacology and provided new evidence that P2X(7) receptors play a specific role in chronic pain states. In this review, we discuss how the P2X family of ion channels has distinguished itself as a potential new drug target. We are optimistic that safe and effective candidate drugs will be suitable for progression into clinical development.

Understanding the conductive channel evolution in Na:WO(3-x)-based planar devices.

PubMed

Shang, Dashan; Li, Peining; Wang, Tao; Carria, Egidio; Sun, Jirong; Shen, Baogen; Taubner, Thomas; Valov, Ilia; Waser, Rainer; Wuttig, Matthias

2015-04-14

An ion migration process in a solid electrolyte is important for ion-based functional devices, such as fuel cells, batteries, electrochromics, gas sensors, and resistive switching systems. In this study, a planar sandwich structure is prepared by depositing tungsten oxide (WO(3-x)) films on a soda-lime glass substrate, from which Na(+) diffuses into the WO(3-x) films during the deposition. The entire process of Na(+) migration driven by an alternating electric field is visualized in the Na-doped WO(3-x) films in the form of conductive channel by in situ optical imaging combined with infrared spectroscopy and near-field imaging techniques. A reversible change of geometry between a parabolic and a bar channel is observed with the resistance change of the devices. The peculiar channel evolution is interpreted by a thermal-stress-induced mechanical deformation of the films and an asymmetric Na(+) mobility between the parabolic and the bar channels. These results exemplify a typical ion migration process driven by an alternating electric field in a solid electrolyte with a low ion mobility and are expected to be beneficial to improve the controllability of the ion migration in ion-based functional devices, such as resistive switching devices.

Radio-frequency response of single pores and artificial ion channels

NASA Astrophysics Data System (ADS)

Kim, H. S.; Ramachandran, S.; Stava, E.; van der Weide, D. W.; Blick, R. H.

2011-09-01

Intercellular communication relies on ion channels and pores in cell membranes. These protein-formed channels enable the exchange of ions and small molecules to electrically and/or chemically interact with the cells. Traditionally, recordings on single-ion channels and pores are performed in the dc regime, due to the extremely high impedance of these molecular junctions. This paper is intended as an introduction to radio-frequency (RF) recordings of single-molecule junctions in bilipid membranes. First, we demonstrate how early approaches to using microwave circuitry as readout devices for ion channel formation were realized. The second step will then focus on how to engineer microwave coupling into the high-impedance channel by making use of bio-compatible micro-coaxial lines. We then demonstrate integration of an ultra-broadband microwave circuit for the direct sampling of single α-hemolysin pores in a suspended bilipid membrane. Simultaneous direct current recordings reveal that we can monitor and correlate the RF transmission signal. This enables us to relate the open-close states of the direct current to the RF signal. Altogether, our experiments lay the ground for an RF-readout technique to perform real-time in vitro recordings of pores. The technique thus holds great promise for research and drug screening applications. The possible enhancement of sampling rates of single channels and pores by the large recording bandwidth will allow us to track the passage of single ions.

Role of Protein Flexibility in Ion Permeation: A Case Study in Gramicidin A

PubMed Central

Baştuğ, Turgut; Gray-Weale, Angus; Patra, Swarna M.; Kuyucak, Serdar

2006-01-01

Proteins have a flexible structure, and their atoms exhibit considerable fluctuations under normal operating conditions. However, apart from some enzyme reactions involving ligand binding, our understanding of the role of flexibility in protein function remains mostly incomplete. Here we investigate this question in the realm of membrane proteins that form ion channels. Specifically, we consider ion permeation in the gramicidin A channel, and study how the energetics of ion conduction changes as the channel structure is progressively changed from completely flexible to a fixed one. For each channel structure, the potential of mean force for a permeating potassium ion is determined from molecular dynamics (MD) simulations. Using the same molecular dynamics data for completely flexible gramicidin A, we also calculate the average densities and fluctuations of the peptide atoms and investigate the correlations between these fluctuations and the motion of a permeating ion. Our results show conclusively that peptide flexibility plays an important role in ion permeation in the gramicidin A channel, thus providing another reason—besides the well-known problem with the description of single file pore water—why this channel cannot be modeled using continuum electrostatics with a fixed structure. The new method developed here for studying the role of protein flexibility on its function clarifies the contributions of the fluctuations to energy and entropy, and places limits on the level of detail required in a coarse-grained model. PMID:16415054

Dynamic hybrid materials for constitutional self-instructed membranes.

PubMed

Cazacu, Adinela; Legrand, Yves-Marie; Pasc, Andreea; Nasr, Gihane; Van der Lee, Arie; Mahon, Eugene; Barboiu, Mihail

2009-05-19

Constitutional self-instructed membranes were developed and used for mimicking the adaptive structural functionality of natural ion-channel systems. These membranes are based on dynamic hybrid materials in which the functional self-organized macrocycles are reversibly connected with the inorganic silica through hydrophobic noncovalent interactions. Supramolecular columnar ion-channel architectures can be generated by reversible confinement within scaffolding hydrophobic silica mesopores. They can be structurally determined by using X-ray diffraction and morphologically tuned by alkali-salts templating. From the conceptual point of view, these membranes express a synergistic adaptive behavior: the simultaneous binding of the fittest cation and its anion would be a case of "homotropic allosteric interactions," because in time it increases the transport efficiency of the pore-contained superstructures by a selective evolving process toward the fittest ion channel. The hybrid membranes presented here represent dynamic constitutional systems evolving over time to form the fittest ion channels from a library of molecular and supramolecular components, or selecting the fittest ion pairs from a mixture of salts demonstrating flexible adaptation.

Structure-function study on a de novo synthetic hydrophobic ion channel.

PubMed Central

Qi, Z; Sokabe, M; Donowaki, K; Ishida, H

1999-01-01

Ion conduction properties of a de novo synthesized channel, formed from cyclic octa-peptides consisting of four alternate L-alanine (Ala) and N'-acylated 3-aminobenzoic acid (Aba) moieties, were studied in bilayer membranes. The single-channel conductance was 9 pS in symmetrical 500 mM KCl. The channel favored permeation of cations over anions with a permeability ratio (PCl-/PK+) of 0.15. The selectivity sequence among monovalent cations based on permeability ratio (PX+/PK+) fell into an order: NH4+(1.4) > Cs+(1. 1) >/= K+(1.0) > Na+(0.4) >> Li+(0). The conductance-activity relationship of the channel in K+ solutions followed simple Michaelis-Menten kinetics with a half-maximal saturating activity of 8 mM and a maximal conductance of 9 pS. The permeability ratio PNa+/PK+ remained constant ( approximately 0.40) under biionic concentrations from 10 to 500 mM. These results suggests that the channel is a one-ion channel. The pore diameter probed by a set of organic cations was approximately 6 A. The single-channel current was blocked by Ca2+ in a dose-dependent manner that followed a single-site titration curve with a voltage-dependent dissociation constant of 0.6 mM at 100 mV. The electric distance of the binding site for Ca2+ was 0.07 from both entrances of the channel, indicating the presence of two symmetrical binding sites in each vicinity of the channel entrance. Correlations between conduction properties and structural aspects of the channel are discussed in terms of a three-barrier and two-binding-site (3B2S) model of Eyring rate theory. All available structural information supported an idea that the channel was formed from a tail-to-tail associated dimer of the molecule, the pore of which was lined with hydrophobic acyl chains. This is the first report to have made a systematic analysis of ion permeation through a hydrophobic pore. PMID:9929469

Laser-Driven Ion Acceleration from Plasma Micro-Channel Targets

PubMed Central

Zou, D. B.; Pukhov, A.; Yi, L. Q.; Zhou, H. B.; Yu, T. P.; Yin, Y.; Shao, F. Q.

2017-01-01

Efficient energy boost of the laser-accelerated ions is critical for their applications in biomedical and hadron research. Achiev-able energies continue to rise, with currently highest energies, allowing access to medical therapy energy windows. Here, a new regime of simultaneous acceleration of ~100 MeV protons and multi-100 MeV carbon-ions from plasma micro-channel targets is proposed by using a ~1020 W/cm2 modest intensity laser pulse. It is found that two trains of overdense electron bunches are dragged out from the micro-channel and effectively accelerated by the longitudinal electric-field excited in the plasma channel. With the optimized channel size, these “superponderomotive” energetic electrons can be focused on the front surface of the attached plastic substrate. The much intense sheath electric-field is formed on the rear side, leading to up to ~10-fold ionic energy increase compared to the simple planar geometry. The analytical prediction of the optimal channel size and ion maximum energies is derived, which shows good agreement with the particle-in-cell simulations. PMID:28218247

Laser-Driven Ion Acceleration from Plasma Micro-Channel Targets

NASA Astrophysics Data System (ADS)

Zou, D. B.; Pukhov, A.; Yi, L. Q.; Zhou, H. B.; Yu, T. P.; Yin, Y.; Shao, F. Q.

2017-02-01

Efficient energy boost of the laser-accelerated ions is critical for their applications in biomedical and hadron research. Achiev-able energies continue to rise, with currently highest energies, allowing access to medical therapy energy windows. Here, a new regime of simultaneous acceleration of ~100 MeV protons and multi-100 MeV carbon-ions from plasma micro-channel targets is proposed by using a ~1020 W/cm2 modest intensity laser pulse. It is found that two trains of overdense electron bunches are dragged out from the micro-channel and effectively accelerated by the longitudinal electric-field excited in the plasma channel. With the optimized channel size, these “superponderomotive” energetic electrons can be focused on the front surface of the attached plastic substrate. The much intense sheath electric-field is formed on the rear side, leading to up to ~10-fold ionic energy increase compared to the simple planar geometry. The analytical prediction of the optimal channel size and ion maximum energies is derived, which shows good agreement with the particle-in-cell simulations.

Probing membrane permeabilization by the antimicrobial peptide distinctin in mercury-supported biomimetic membranes.

PubMed

Becucci, Lucia; Papini, Martina; Mullen, Daniel; Scaloni, Andrea; Veglia, Gianluigi; Guidelli, Rolando

2011-11-01

The mechanism of membrane permeabilization by the antimicrobial peptide distinctin was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). Incorporation of distinctin into a lipid monolayer from its aqueous solution yields rapidly ion channels selective toward inorganic cations, such as Tl(+) and Cd(2+). Conversely, its incorporation in a tBLM allows the formation of ion channels permeable to potassium ions only at non-physiological transmembrane potentials, more negative than -340mV. These channels, once formed, are unstable at less negative transmembrane potentials. The kinetics of their formation is consistent with the disruption of distinctin clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth. Comparing the behavior of distinctin in tBLMs with that in conventional black lipid membranes strongly suggests that distinctin channel formation in lipid bilayer requires the partitioning of distinctin molecules between the two sides of the lipid bilayer. We can tentatively hypothesize that an ion channel is formed when one distinctin cluster on one side of the lipid bilayer matches another one on the opposite side. Copyright © 2011 Elsevier B.V. All rights reserved.

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

PubMed Central

Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

1992-01-01

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola

NASA Astrophysics Data System (ADS)

Dhakshnamoorthy, Balasundaresan; Rohaim, Ahmed; Rui, Huan; Blachowicz, Lydia; Roux, Benoît

2016-09-01

The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel.

Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.

PubMed

Desuzinges Mandon, Elodie; Traversier, Aurélien; Champagne, Anne; Benier, Lorraine; Audebert, Stéphane; Balme, Sébastien; Dejean, Emmanuel; Rosa Calatrava, Manuel; Jawhari, Anass

2017-03-01

Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Electron-impact Ionization of P-like Ions Forming Si-like Ions

NASA Astrophysics Data System (ADS)

Kwon, D.-H.; Savin, D. W.

2014-03-01

We have calculated electron-impact ionization (EII) for P-like systems from P to Zn15 + forming Si-like ions. The work was performed using the flexible atomic code (FAC) which is based on a distorted-wave approximation. All 3l → nl' (n = 3-35) excitation-autoionization (EA) channels near the 3p direct ionization threshold and 2l → nl' (n = 3-10) EA channels at the higher energies are included. Close attention has been paid to the detailed branching ratios. Our calculated total EII cross sections are compared both with previous FAC calculations, which omitted many of these EA channels, and with the available experimental results. Moreover, for Fe11 +, we find that part of the remaining discrepancies between our calculations and recent measurements can be accounted for by the inclusion of the resonant excitation double autoionization process. Lastly, at the temperatures where each ion is predicted to peak in abundances in collisional ionization equilibrium, the Maxwellian rate coefficients derived from our calculations differ by 50%-7% from the previous FAC rate coefficients, with the difference decreasing with increasing charge.

Control of Internal Transport Barriers in Magnetically Confined Fusion Plasmas

NASA Astrophysics Data System (ADS)

Panta, Soma; Newman, David; Sanchez, Raul; Terry, Paul

2016-10-01

In magnetic confinement fusion devices the best performance often involves some sort of transport barriers to reduce the energy and particle flow from core to edge. Those barriers create gradients in the temperature and density profiles. If gradients in the profiles are too steep that can lead to instabilities and the system collapses. Control of these barriers is therefore an important challenge for fusion devices (burning plasmas). In this work we focus on the dynamics of internal transport barriers. Using a simple 7 field transport model, extensively used for barrier dynamics and control studies, we explore the use of RF heating to control the local gradients and therefore the growth rates and shearing rates for barrier initiation and control in self-heated fusion plasmas. Ion channel barriers can be formed in self-heated plasmas with some NBI heating but electron channel barriers are very sensitive. They can be formed in self-heated plasmas with additional auxiliary heating i.e. NBI and radio-frequency(RF). Using RF heating on both electrons and ions at proper locations, electron channel barriers along with ion channel barriers can be formed and removed demonstrating a control technique. Investigating the role of pellet injection in controlling the barriers is our next goal. Work supported by DOE Grant DE-FG02-04ER54741.

Single-Walled Carbon Nanotubes: Mimics of Biological Ion Channels.

PubMed

Amiri, Hasti; Shepard, Kenneth L; Nuckolls, Colin; Hernández Sánchez, Raúl

2017-02-08

Here we report on the ion conductance through individual, small diameter single-walled carbon nanotubes. We find that they are mimics of ion channels found in natural systems. We explore the factors governing the ion selectivity and permeation through single-walled carbon nanotubes by considering an electrostatic mechanism built around a simplified version of the Gouy-Chapman theory. We find that the single-walled carbon nanotubes preferentially transported cations and that the cation permeability is size-dependent. The ionic conductance increases as the absolute hydration enthalpy decreases for monovalent cations with similar solid-state radii, hydrated radii, and bulk mobility. Charge screening experiments using either the addition of cationic or anionic polymers, divalent metal cations, or changes in pH reveal the enormous impact of the negatively charged carboxylates at the entrance of the single-walled carbon nanotubes. These observations were modeled in the low-to-medium concentration range (0.1-2.0 M) by an electrostatic mechanism that mimics the behavior observed in many biological ion channel-forming proteins. Moreover, multi-ion conduction in the high concentration range (>2.0 M) further reinforces the similarity between single-walled carbon nanotubes and protein ion channels.

Conditional fast expression and function of multimeric TRPV5 channels using Shield-1.

PubMed

Schoeber, Joost P H; van de Graaf, Stan F J; Lee, Kyu Pil; Wittgen, Hanneke G M; Hoenderop, Joost G J; Bindels, René J M

2009-01-01

A recently described novel controllable method to regulate protein expression is based on a mutated FK506-binding protein-12 (mtFKBP) that is unstable and rapidly degraded in mammalian cells. This instability can be conferred to other proteins directly fused to mtFKBP. Binding of a synthetic cell-permeant ligand (Shield-1) to mtFKBP reverses the instability, allowing conditional expression of mtFKBP-fused proteins. We adapted this strategy to study multimeric plasma membrane proteins using the ion channel TRPV5 as model protein. mtFKBP-TRPV5 forms functional ion channels and its expression can be controlled in a time- and dose-dependent fashion using Shield-1. Moreover, in the presence of Shield-1, mtFKBP-TRPV5 formed heteromultimeric channels with untagged TRPV5, which were codegraded upon washout of Shield-1, providing a strategy to study multimeric plasma membrane protein complexes without the need to destabilize all individual subunits.

Nanometer-scale water- and proton-diffusion heterogeneities across water channels in polymer electrolyte membranes.

PubMed

Song, Jinsuk; Han, Oc Hee; Han, Songi

2015-03-16

Nafion, the most widely used polymer for electrolyte membranes (PEMs) in fuel cells, consists of a fluorocarbon backbone and acidic groups that, upon hydration, swell to form percolated channels through which water and ions diffuse. Although the effects of the channel structures and the acidic groups on water/ion transport have been studied before, the surface chemistry or the spatially heterogeneous diffusivity across water channels has never been shown to directly influence water/ion transport. By the use of molecular spin probes that are selectively partitioned into heterogeneous regions of the PEM and Overhauser dynamic nuclear polarization relaxometry, this study reveals that both water and proton diffusivity are significantly faster near the fluorocarbon and the acidic groups lining the water channels than within the water channels. The concept that surface chemistry at the (sub)nanometer scale dictates water and proton diffusivity invokes a new design principle for PEMs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Influence of proline position upon the ion channel activity of alamethicin.

PubMed Central

Kaduk, C; Duclohier, H; Dathe, M; Wenschuh, H; Beyermann, M; Molle, G; Bienert, M

1997-01-01

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable. PMID:9129817

The Transcription Factors Islet and Lim3 Combinatorially Regulate Ion Channel Gene Expression

PubMed Central

Wolfram, Verena; Southall, Tony D.; Günay, Cengiz; Prinz, Astrid A.; Brand, Andrea H.

2014-01-01

Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Our previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. In this study, we use the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K+ channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. We used body wall muscle as a test tissue because in conditions of low Ca2+, the fast K+ current is carried solely by Sh channels (unlike neurons in which a second fast K+ current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression. PMID:24523544

Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

PubMed Central

Fujiwara, Yuichiro; Arrigoni, Cristina; Domigan, Courtney; Ferrara, Giuseppina; Pantoja, Carlos; Thiel, Gerhard; Moroni, Anna; Minor, Daniel L.

2009-01-01

Background Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs. Methodology/Principal Findings We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T→S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding. Conclusions/Significance The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features. PMID:19834614

A family of fluoride-specific ion channels with dual-topology architecture.

PubMed

Stockbridge, Randy B; Robertson, Janice L; Kolmakova-Partensky, Ludmila; Miller, Christopher

2013-08-27

Fluoride ion, ubiquitous in soil, water, and marine environments, is a chronic threat to microorganisms. Many prokaryotes, archea, unicellular eukaryotes, and plants use a recently discovered family of F(-) exporter proteins to lower cytoplasmic F(-) levels to counteract the anion's toxicity. We show here that these 'Fluc' proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F(-) over Cl(-). The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation. This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family, and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins. DOI:http://dx.doi.org/10.7554/eLife.01084.001.

Dynamic hybrid materials for constitutional self-instructed membranes

PubMed Central

Cazacu, Adinela; Legrand, Yves-Marie; Pasc, Andreea; Nasr, Gihane; Van der Lee, Arie; Mahon, Eugene; Barboiu, Mihail

2009-01-01

Constitutional self-instructed membranes were developed and used for mimicking the adaptive structural functionality of natural ion-channel systems. These membranes are based on dynamic hybrid materials in which the functional self-organized macrocycles are reversibly connected with the inorganic silica through hydrophobic noncovalent interactions. Supramolecular columnar ion-channel architectures can be generated by reversible confinement within scaffolding hydrophobic silica mesopores. They can be structurally determined by using X-ray diffraction and morphologically tuned by alkali-salts templating. From the conceptual point of view, these membranes express a synergistic adaptive behavior: the simultaneous binding of the fittest cation and its anion would be a case of “homotropic allosteric interactions,” because in time it increases the transport efficiency of the pore-contained superstructures by a selective evolving process toward the fittest ion channel. The hybrid membranes presented here represent dynamic constitutional systems evolving over time to form the fittest ion channels from a library of molecular and supramolecular components, or selecting the fittest ion pairs from a mixture of salts demonstrating flexible adaptation. PMID:19416909

The Basal Thermal Sensitivity of the TRPV1 Ion Channel Is Determined by PKCβII

PubMed Central

Li, Lin; Hasan, Raquibul

2014-01-01

Peripheral nociceptors are excited by the activation of membrane receptors and ion channels. The heat-sensitive TRPV1 ion channel responds to various noxious chemical and thermal stimuli, causing pain and itch. Here, we show that TRPV1 is coexpressed with PKCβII in a subset of mouse sensory neurons and that, in these neurons, TRPV1 binds directly to PKCβII, leading to the activation and translocation of PKCβII. Activated PKCβII, in turn, significantly increases the responsiveness of TRPV1 by phosphorylating Thr705. The heat sensitivity of TRPV1 is almost eliminated by either knocking down PKCβII or mutating Thr705; however, neither of these manipulations affects the potentiation of TRPV1 caused by the activation of PKCε. PKCβII thus acts as an auxiliary subunit of TRPV1 by forming a population-dependent TRPV1 ion channel complex controlling the sensitivity of TRPV1 and setting the threshold for pain and itch. PMID:24920628

Dielectric boundary force and its crucial role in gramicidin

NASA Astrophysics Data System (ADS)

Nadler, Boaz; Hollerbach, Uwe; Eisenberg, R. S.

2003-08-01

In an electrostatic problem with nonuniform geometry, a charge Q in one region induces surface charges [called dielectric boundary charges (DBC)] at boundaries between different dielectrics. These induced surface charges, in return, exert a force [called dielectric boundary force (DBF)] on the charge Q that induced them. The DBF is often overlooked. It is not present in standard continuum theories of (point) ions in or near membranes and proteins, such as Gouy-Chapman, Debye-Huckel, Poisson-Boltzmann or Poisson-Nernst- Planck. The DBF is important when a charge Q is near dielectric interfaces, for example, when ions permeate through protein channels embedded in biological membranes. In this paper, we define the DBF and calculate it explicitly for a planar dielectric wall and for a tunnel geometry resembling the ionic channel gramicidin. In general, we formulate the DBF in a form useful for continuum theories, namely, as a solution of a partial differential equation with boundary conditions. The DBF plays a crucial role in the permeation of ions through the gramicidin channel. A positive ion in the channel produces a DBF of opposite sign to that of the fixed charge force (FCF) produced by the permanent charge of the gramicidin polypeptide, and so the net force on the positive ion is reduced. A negative ion creates a DBF of the same sign as the FCF and so the net (repulsive) force on the negative ion is increased. Thus, a positive ion can permeate the channel, while a negative ion is excluded from it. In gramicidin, it is this balance between the FCF and DBF that allows only singly charged positive ions to move into and through the channel. The DBF is not directly responsible, however, for selectivity between the alkali metal ions (e.g., Li+, Na+, K+): we prove that the DBF on a mobile spherical ion is independent of the ion’s radius.

Environmental sensing with optical fiber sensors processed with focused ion beam and atomic layer deposition

NASA Astrophysics Data System (ADS)

Flores, Raquel; Janeiro, Ricardo; Dahlem, Marcus; Viegas, Jaime

2015-03-01

We report an optical fiber chemical sensor based on a focused ion beam processed optical fiber. The demonstrated sensor is based on a cavity formed onto a standard 1550 nm single-mode fiber by either chemical etching, focused ion beam milling (FIB) or femtosecond laser ablation, on which side channels are drilled by either ion beam milling or femtosecond laser irradiation. The encapsulation of the cavity is achieved by optimized fusion splicing onto a standard single or multimode fiber. The empty cavity can be used as semi-curved Fabry-Pérot resonator for gas or liquid sensing. Increased reflectivity of the formed cavity mirrors can be achieved with atomic layer deposition (ALD) of alternating metal oxides. For chemical selective optical sensors, we demonstrate the same FIB-formed cavity concept, but filled with different materials, such as polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA) which show selective swelling when immersed in different solvents. Finally, a reducing agent sensor based on a FIB formed cavity partially sealed by fusion splicing and coated with a thin ZnO layer by ALD is presented and the results discussed. Sensor interrogation is achieved with spectral or multi-channel intensity measurements.

TMEM16 proteins: unknown structure and confusing functions.

PubMed

Picollo, Alessandra; Malvezzi, Mattia; Accardi, Alessio

2015-01-16

The TMEM16 family of membrane proteins, also known as anoctamins, plays key roles in a variety of physiological functions that range from ion transport to phospholipid scrambling and to regulating other ion channels. The first two family members to be functionally characterized, TMEM16A (ANO1) and TMEM16B (ANO2), form Ca(2+)-activated Cl(-) channels and are important for transepithelial ion transport, olfaction, phototransduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The roles of other family members, such as TMEM16C (ANO3), TMEM16D (ANO4), TMEM16F (ANO6), TMEM16G (ANO7) and TMEM16J (ANO9), remain poorly understood and controversial. These homologues were reported to be phospholipid scramblases, ion channels, to have both functions or to be regulatory subunits of other channels. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca(2+)-dependent externalization of phosphatidylserine in activated platelets, suggesting that this homologue might be a scramblase. However, overexpression of TMEM16F has also been associated with a remarkable number of different ion channel types, raising the possibility that this protein might be involved in both ion and lipid transports. The recent identification of an ancestral TMEM16 homologue with intrinsic channel and scramblase activities supports this hypothesis. Thus, the TMEM16 family might have diverged in two or three different subclasses, channels, scramblases and dual-function channel/scramblases. The structural bases and functional implication of such a functional diversity within a single protein family remain to be elucidated and the links between TMEM16 functions and human physiology and pathologies need to be investigated. Copyright © 2014 Elsevier Ltd. All rights reserved.

Excitation of a nonlinear plasma ion wake by intense energy sources with applications to the crunch-in regime

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

Sahai, Aakash A.

We show the excitation of a nonlinear ion-wake mode by plasma electron modes in the bubble regime driven by intense energy sources, using analytical theory and simulations. The ion wake is shown to be a driven nonlinear ion-acoustic wave in the form of a long-lived cylindrical ion soliton which limits the repetition rate of a plasma-based particle accelerator in the bubble regime. We present the application of this evacuated and radially outwards propagating ion-wake channel with an electron skin-depth scale radius for the “crunch-in” regime of hollow-channel plasma. It is shown that the time-asymmetric focusing force phases in the bubblemore » couple to ion motion significantly differently than in the linear electron mode. The electron compression in the back of the bubble sucks in the ions whereas the space charge within the bubble cavity expels them, driving a cylindrical ion-soliton structure at the bubble radius. Once formed, the soliton is sustained and driven radially outwards by the thermal pressure of the wake energy in electrons. Particle-in-cell simulations are used to study the ion-wake soliton structure, its driven propagation and its use for positron acceleration in the crunch-in regime.« less

Excitation of a nonlinear plasma ion wake by intense energy sources with applications to the crunch-in regime

DOE PAGES

Sahai, Aakash A.

2017-08-23

We show the excitation of a nonlinear ion-wake mode by plasma electron modes in the bubble regime driven by intense energy sources, using analytical theory and simulations. The ion wake is shown to be a driven nonlinear ion-acoustic wave in the form of a long-lived cylindrical ion soliton which limits the repetition rate of a plasma-based particle accelerator in the bubble regime. We present the application of this evacuated and radially outwards propagating ion-wake channel with an electron skin-depth scale radius for the “crunch-in” regime of hollow-channel plasma. It is shown that the time-asymmetric focusing force phases in the bubblemore » couple to ion motion significantly differently than in the linear electron mode. The electron compression in the back of the bubble sucks in the ions whereas the space charge within the bubble cavity expels them, driving a cylindrical ion-soliton structure at the bubble radius. Once formed, the soliton is sustained and driven radially outwards by the thermal pressure of the wake energy in electrons. Particle-in-cell simulations are used to study the ion-wake soliton structure, its driven propagation and its use for positron acceleration in the crunch-in regime.« less

Electron-impact ionization of P-like ions forming Si-like ions

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

Kwon, D.-H.; Savin, D. W., E-mail: hkwon@kaeri.re.kr

2014-03-20

We have calculated electron-impact ionization (EII) for P-like systems from P to Zn{sup 15+} forming Si-like ions. The work was performed using the flexible atomic code (FAC) which is based on a distorted-wave approximation. All 3ℓ → nℓ' (n = 3-35) excitation-autoionization (EA) channels near the 3p direct ionization threshold and 2ℓ → nℓ' (n = 3-10) EA channels at the higher energies are included. Close attention has been paid to the detailed branching ratios. Our calculated total EII cross sections are compared both with previous FAC calculations, which omitted many of these EA channels, and with the available experimentalmore » results. Moreover, for Fe{sup 11+}, we find that part of the remaining discrepancies between our calculations and recent measurements can be accounted for by the inclusion of the resonant excitation double autoionization process. Lastly, at the temperatures where each ion is predicted to peak in abundances in collisional ionization equilibrium, the Maxwellian rate coefficients derived from our calculations differ by 50%-7% from the previous FAC rate coefficients, with the difference decreasing with increasing charge.« less

A family of fluoride-specific ion channels with dual-topology architecture

PubMed Central

Stockbridge, Randy B; Robertson, Janice L; Kolmakova-Partensky, Ludmila; Miller, Christopher

2013-01-01

Fluoride ion, ubiquitous in soil, water, and marine environments, is a chronic threat to microorganisms. Many prokaryotes, archea, unicellular eukaryotes, and plants use a recently discovered family of F− exporter proteins to lower cytoplasmic F− levels to counteract the anion’s toxicity. We show here that these ‘Fluc’ proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F− over Cl−. The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation. This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family, and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins. DOI: http://dx.doi.org/10.7554/eLife.01084.001 PMID:23991286

Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels.

PubMed

Elinder, Fredrik; Liin, Sara I

2017-01-01

Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (Na V ), potassium (K V ), calcium (Ca V ), and proton (H V ) channels, as well as calcium-activated potassium (K Ca ), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1 : The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2 : The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3 : The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4 : The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5 : The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels.

Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels

PubMed Central

Elinder, Fredrik; Liin, Sara I.

2017-01-01

Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (NaV), potassium (KV), calcium (CaV), and proton (HV) channels, as well as calcium-activated potassium (KCa), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1: The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2: The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3: The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4: The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5: The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels. PMID:28220076

Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel

PubMed Central

Sauguet, Ludovic; Howard, Rebecca J.; Malherbe, Laurie; Lee, Ui S.; Corringer, Pierre-Jean; Harris, R. Adron; Delarue, Marc

2014-01-01

Ethanol alters nerve signalling by interacting with proteins in the central nervous system, particularly pentameric ligand-gated ion channels. A recent series of mutagenesis experiments on Gloeobacter violaceus ligand-gated ion channel, a prokaryotic member of this family, identified a single-site variant that is potentiated by pharmacologically relevant concentrations of ethanol. Here we determine crystal structures of the ethanol-sensitized variant in the absence and presence of ethanol and related modulators, which bind in a transmembrane cavity between channel subunits and may stabilize the open form of the channel. Structural and mutagenesis studies defined overlapping mechanisms of potentiation by alcohols and anaesthetics via the inter-subunit cavity. Furthermore, homology modelling show this cavity to be conserved in human ethanol-sensitive glycine and GABA(A) receptors, and to involve residues previously shown to influence alcohol and anaesthetic action on these proteins. These results suggest a common structural basis for ethanol potentiation of an important class of targets for neurological actions of ethanol. PMID:23591864

Elucidating distinct ion channel populations on the surface of hippocampal neurons via single-particle tracking recurrence analysis

NASA Astrophysics Data System (ADS)

Sikora, Grzegorz; Wyłomańska, Agnieszka; Gajda, Janusz; Solé, Laura; Akin, Elizabeth J.; Tamkun, Michael M.; Krapf, Diego

2017-12-01

Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it has been recently recognized that ion channels assemble into surface nanoclusters in the soma of cultured neurons. However, the interactions of these molecules with surface nanodomains display a considerable degree of heterogeneity. Here, we investigate this heterogeneity and develop statistical tools based on the recurrence of individual trajectories to identify subpopulations within ion channels in the neuronal surface. We specifically study the dynamics of the K+ channel Kv1.4 and the Na+ channel Nav1.6 on the surface of cultured hippocampal neurons at the single-molecule level. We find that both these molecules are expressed in two different forms with distinct kinetics with regards to surface interactions, emphasizing the complex proteomic landscape of the neuronal surface. Further, the tools presented in this work provide new methods for the analysis of membrane nanodomains, transient confinement, and identification of populations within single-particle trajectories.

Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.

PubMed

Snyder, P M; Cheng, C; Prince, L S; Rogers, J C; Welsh, M J

1998-01-09

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero- and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na+ channel (hENaC), we expressed the three wild-type hENaC subunits (alpha, beta, and gamma) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three alpha, three beta, and three gamma subunits. Sucrose gradient sedimentation of alphahENaC translated in vitro, as well as alpha-, beta-, and gammahENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.

CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane.

PubMed

Gupta, Ankit; Balabaskaran-Nina, Praveen; Nguitragool, Wang; Saggu, Gagandeep S; Schureck, Marc A; Desai, Sanjay A

2018-05-08

Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. These proteins lack the multiple transmembrane domains typically present in channel-forming proteins, raising doubts about their precise roles. Using the virulent human Plasmodium falciparum parasite, we report that CLAG3 undergoes self-association and that this protein's expression determines channel phenotype quantitatively. We overcame epigenetic silencing of clag3 paralogs and engineered parasites that express two CLAG3 isoforms simultaneously. Stoichiometric expression of these isoforms yielded intermediate channel phenotypes, in agreement with observed trafficking of both proteins to the host membrane. Coimmunoprecipitation and surface labeling revealed formation of CLAG3 oligomers. In vitro selections applied to these transfectant lines yielded distinct mutants with correlated changes in channel activity. These findings support involvement of the identified oligomers in PSAC formation and parasite nutrient acquisition. IMPORTANCE Malaria parasites are globally important pathogens that evade host immunity by replicating within circulating erythrocytes. To facilitate intracellular growth, these parasites increase erythrocyte nutrient uptake through an unusual ion channel. The parasite CLAG3 protein is a key determinant of this channel, but its lack of homology to known ion channels has raised questions about possible mechanisms. Using a new method that allows simultaneous expression of two different CLAG3 proteins, we identify self-association of CLAG3. The two expressed isoforms faithfully traffic to and insert in the host membrane, while remaining associated with two unrelated parasite proteins. Both the channel phenotypes and molecular changes produced upon selections with a highly specific channel inhibitor are consistent with a multiprotein complex that forms the nutrient pore. These studies support direct involvement of the CLAG3 protein in channel formation and are relevant to antimalarial drug discovery projects targeting parasite nutrient acquisition.

S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

PubMed Central

Shipston, Michael J.

2014-01-01

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel.

PubMed

Held, Katharina; Gruss, Fabian; Aloi, Vincenzo Davide; Janssens, Annelies; Ulens, Chris; Voets, Thomas; Vriens, Joris

2018-03-31

Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na + , K + and Ca 2+ channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K + channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K + channels, in which mutations in S4 can create an analogous 'omega' pore, we performed site-directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non-canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway. © 2018 KU Leuven The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Nano-channels in the spider fang for the transport of Zn ions to cross-link His-rich proteins pre-deposited in the cuticle matrix.

PubMed

Politi, Yael; Pippel, Eckhard; Licuco-Massouh, Ana C J; Bertinetti, Luca; Blumtritt, Horst; Barth, Friedrich G; Fratzl, Peter

2017-01-01

We identify the presence of multiple vascular channels within the spider fang. These channels seem to serve the transport of zinc to the tip of the fang to cross-link the protein matrix by binding to histidine residues. According to amino acid and elemental analysis of fangs extracted shortly after ecdysis, His-rich proteins are deposited before Zn is incorporated into the cuticle. Microscopic and spectroscopic investigations in the electron microscope and synchrotron radiation experiments suggest that Zn ions are transported through these channels in a liable (yet unidentified) form, and then form stable complexes upon His binding. The resulting cross-linking through the Zn-His complexes is conferring hardness to the fang. Our observations of nano-channels serving the Zn-transport within the His-rich protein matrix of the fibre reinforced spider fang may also support recent bio-inspired attempts to design artificial polymeric vascular materials for self-healing and in-situ curing. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

[Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane].

PubMed

Venediktova, N I; Kuznetsov, K V; Gritsenko, E N; Gulidova, G P; Mironova, G D

2012-01-01

Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age

PubMed Central

Oiki, Shigetoshi

2015-01-01

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the ‘frozen’ crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. PMID:25833254

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age.

PubMed

Oiki, Shigetoshi

2015-06-15

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the 'frozen' crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states.

PubMed

Bargiello, Thaddeus A; Oh, Seunghoon; Tang, Qingxiu; Bargiello, Nicholas K; Dowd, Terry L; Kwon, Taekyung

2018-01-01

Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating. Each hemichannel displays two distinct voltage-gating mechanisms that are primarily sensitive to a voltage gradient formed along the length of the channel pore (the transjunctional voltage) rather than sensitivity to the absolute membrane potential (V m or V i-o ). These transjunctional voltage dependent processes have been termed V j - or fast-gating and loop- or slow-gating. Understanding the mechanism of voltage-gating, defined as the sequence of voltage-driven transitions that connect open and closed states, first and foremost requires atomic resolution models of the end states. Although ion channels formed by connexins were among the first to be characterized structurally by electron microscopy and x-ray diffraction in the early 1980's, subsequent progress has been slow. Much of the current understanding of the structure-function relations of connexin channels is based on two crystal structures of Cx26 gap junction channels. Refinement of crystal structure by all-atom molecular dynamics and incorporation of charge changing protein modifications has resulted in an atomic model of the open state that arguably corresponds to the physiologic open state. Obtaining validated atomic models of voltage-dependent closed states is more challenging, as there are currently no methods to solve protein structure while a stable voltage gradient is applied across the length of an oriented channel. It is widely believed that the best approach to solve the atomic structure of a voltage-gated closed ion channel is to apply different but complementary experimental and computational methods and to use the resulting information to derive a consensus atomic structure that is then subjected to rigorous validation. In this paper, we summarize our efforts to obtain and validate atomic models of the open and voltage-driven closed states of undocked connexin hemichannels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

Easily Accessible Polycyclic Amines that Inhibit the Wild-Type and Amantadine-Resistant Mutants of the M2 Channel of Influenza A Virus

PubMed Central

2015-01-01

Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogues has revolved around the synthesis of novel adamantane derivatives, we have recently found that other polycyclic scaffolds effectively block the M2 proton channel, including amantadine-resistant mutant channels. In this work, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogues of amantadine. Inhibition of the wild-type M2 channel and the A/M2-S31N, A/M2-V27A, and A/M2-L26F mutant forms of the channel were measured in Xenopus oocytes using two-electrode voltage clamp assays. Most of the novel compounds inhibited the wild-type ion channel in the low micromolar range. Of note, two of the compounds inhibited the amantadine-resistant A/M2-V27A and A/M2-L26F mutant ion channels with submicromolar and low micromolar IC50, respectively. None of the compounds was found to inhibit the S31N mutant ion channel. PMID:24941437

Isolated pores dissected from human two-pore channel 2 are functional

PubMed Central

Penny, Christopher J.; Rahman, Taufiq; Sula, Altin; Miles, Andrew J.; Wallace, B. A.; Patel, Sandip

2016-01-01

Multi-domain voltage-gated ion channels appear to have evolved through sequential rounds of intragenic duplication from a primordial one-domain precursor. Whereas modularity within one-domain symmetrical channels is established, little is known about the roles of individual regions within more complex asymmetrical channels where the domains have undergone substantial divergence. Here we isolated and characterised both of the divergent pore regions from human TPC2, a two-domain channel that holds a key intermediate position in the evolution of voltage-gated ion channels. In HeLa cells, each pore localised to the ER and caused Ca2+ depletion, whereas an ER-targeted pore mutated at a residue that inactivates full-length TPC2 did not. Additionally, one of the pores expressed at high levels in E. coli. When purified, it formed a stable, folded tetramer. Liposomes reconstituted with the pore supported Ca2+ and Na+ uptake that was inhibited by known blockers of full-length channels. Computational modelling of the pore corroborated cationic permeability and drug interaction. Therefore, despite divergence, both pores are constitutively active in the absence of their partners and retain several properties of the wild-type pore. Such symmetrical ‘pore-only’ proteins derived from divergent channel domains may therefore provide tractable tools for probing the functional architecture of complex ion channels. PMID:27941820

Isolated pores dissected from human two-pore channel 2 are functional.

PubMed

Penny, Christopher J; Rahman, Taufiq; Sula, Altin; Miles, Andrew J; Wallace, B A; Patel, Sandip

2016-12-12

Multi-domain voltage-gated ion channels appear to have evolved through sequential rounds of intragenic duplication from a primordial one-domain precursor. Whereas modularity within one-domain symmetrical channels is established, little is known about the roles of individual regions within more complex asymmetrical channels where the domains have undergone substantial divergence. Here we isolated and characterised both of the divergent pore regions from human TPC2, a two-domain channel that holds a key intermediate position in the evolution of voltage-gated ion channels. In HeLa cells, each pore localised to the ER and caused Ca 2+ depletion, whereas an ER-targeted pore mutated at a residue that inactivates full-length TPC2 did not. Additionally, one of the pores expressed at high levels in E. coli. When purified, it formed a stable, folded tetramer. Liposomes reconstituted with the pore supported Ca 2+ and Na + uptake that was inhibited by known blockers of full-length channels. Computational modelling of the pore corroborated cationic permeability and drug interaction. Therefore, despite divergence, both pores are constitutively active in the absence of their partners and retain several properties of the wild-type pore. Such symmetrical 'pore-only' proteins derived from divergent channel domains may therefore provide tractable tools for probing the functional architecture of complex ion channels.

Fragmentation mechanisms for methane induced by 55 eV, 75 eV, and 100 eV electron impact.

PubMed

Wei, B; Zhang, Y; Wang, X; Lu, D; Lu, G C; Zhang, B H; Tang, Y J; Hutton, R; Zou, Y

2014-03-28

The fragmentation of CH4 (2+) dications following 55 eV, 75 eV, and 100 eV electron impact double ionization of methane was studied using a cold target recoil-ion momentum spectroscopy. From the measured momentum of each recoil ion, the momentum of the neutral particles has been deduced and the kinetic energy release distribution for the different fragmentation channels has been obtained. The doubly charged molecular ions break up into three or more fragments in one or two-step processes, resulting in different signatures in the data. We observed the fragmentation of CH4 (2+) dications through different mechanisms according to the momentum of the neutral particles. For example, our result shows that there are three reaction channels to form CH2 (+), H(+), and H, one synchronous concerted reaction channel and two two-step reaction channels. For even more complicated fragmentation processes of CH4 (2+) dications, the fragmentation mechanism can still be identified in the present measurements. The slopes of the peak in the ion-ion coincidence spectra were also estimated here, as they are also related to the fragmentation mechanism.

Dynamics of the EAG1 K+ channel selectivity filter assessed by molecular dynamics simulations.

PubMed

Bernsteiner, Harald; Bründl, Michael; Stary-Weinzinger, Anna

2017-02-26

EAG1 channels belong to the KCNH family of voltage gated potassium channels. They are expressed in several brain regions and increased expression is linked to certain cancer types. Recent cryo-EM structure determination finally revealed the structure of these channels in atomic detail, allowing computational investigations. In this study, we performed molecular dynamics simulations to investigate the ion binding sites and the dynamical behavior of the selectivity filter. Our simulations suggest that sites S2 and S4 form stable ion binding sites, while ions placed at sites S1 and S3 rapidly switched to sites S2 and S4. Further, ions tended to dissociate away from S0 within less than 20 ns, due to increased filter flexibility. This was followed by water influx from the extracellular side, leading to a widening of the filter in this region, and likely non-conductive filter configurations. Simulations with the inactivation-enhancing mutant Y464A or Na + ions lead to trapped water molecules behind the SF, suggesting that these simulations captured early conformational changes linked to C-type inactivation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Transportation behavior of alkali ions through a cell membrane ion channel. A quantum chemical description of a simplified isolated model.

PubMed

Billes, Ferenc; Mohammed-Ziegler, Ildikó; Mikosch, Hans

2012-08-01

Quantum chemical model calculations were carried out for modeling the ion transport through an isolated ion channel of a cell membrane. An isolated part of a natural ion channel was modeled. The model channel was a calixarene derivative, hydrated sodium and potassium ions were the models of the transported ion. The electrostatic potential of the channel and the energy of the channel-ion system were calculated as a function of the alkali ion position. Both attractive and repulsive ion-channel interactions were found. The calculations - namely the dependence of the system energy and the atomic charges of the water molecules with respect to the position of the alkali ion in the channel - revealed the molecular-structural background of the potassium selectivity of this artificial ion channel. It was concluded that the studied ion channel mimics real biological ion channel quite well.

Crystal structure and cation exchanging properties of a novel open framework phosphate of Ce (IV)

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

Bevara, Samatha; Achary, S. N., E-mail: sachary@barc.gov.in; Tyagi, A. K.

2016-05-23

Herein we report preparation, crystal structure and ion exchanging properties of a new phosphate of tetravalent cerium, K{sub 2}Ce(PO{sub 4}){sub 2}. A monoclinic structure having framework type arrangement of Ce(PO{sub 4}){sub 6} units formed by C2O{sub 8} square-antiprism and PO{sub 4} tetrahedra is assigned for K{sub C}e(PO{sub 4}){sub 2}. The K{sup +} ions are occupied in the channels formed by the Ce(PO{sub 4})6 and provide overall charge neutrality. The unique channel type arrangements of the K+ make them exchangeable with other cations. The ion exchanging properties of K2Ce(PO4)2 has been investigated by equilibrating with solution of 90Sr followed by radiometricmore » analysis. In optimum conditions, significant exchange of K+ with Sr2+ with Kd ~ 8000 mL/g is observed. The details of crystal structure and ion exchange properties are explained and a plausible mechanism for ion exchange is presented.« less

Mapping of Residues Forming the Voltage Sensor of the Voltage-Dependent Anion-Selective Channel

NASA Astrophysics Data System (ADS)

Thomas, Lorie; Blachly-Dyson, Elizabeth; Colombini, Marco; Forte, Michael

1993-06-01

Voltage-gated ion-channel proteins contain "voltage-sensing" domains that drive the conformational transitions between open and closed states in response to changes in transmembrane voltage. We have used site-directed mutagenesis to identify residues affecting the voltage sensitivity of a mitochondrial channel, the voltage-dependent anion-selective channel (VDAC). Although charge changes at many sites had no effect, at other sites substitutions that increased positive charge also increased the steepness of voltage dependance and substitutions that decreased positive charge decreased voltage dependance by an appropriate amount. In contrast to the plasma membrane K^+ and Na^+ channels, these residues are distributed over large parts of the VDAC protein. These results have been used to define the conformational transitions that accompany voltage gating of an ion channel. This gating mechanism requires the movement of large portions of the VDAC protein through the membrane.

Voltage gated sodium channels as drug discovery targets

PubMed Central

Bagal, Sharan K; Marron, Brian E; Owen, Robert M; Storer, R Ian; Swain, Nigel A

2015-01-01

Voltage-gated sodium (NaV) channels are a family of transmembrane ion channel proteins. They function by forming a gated, water-filled pore to help establish and control cell membrane potential via control of the flow of ions between the intracellular and the extracellular environments. Blockade of NaVs has been successfully accomplished in the clinic to enable control of pathological firing patterns that occur in a diverse range of conditions such as chronic pain, epilepsy, and cardiac arrhythmias. First generation sodium channel modulator drugs, despite low inherent subtype selectivity, preferentially act on over-excited cells which reduces undesirable side effects in the clinic. However, the limited therapeutic indices observed with the first generation demanded a new generation of sodium channel inhibitors. The structure, function and the state of the art in sodium channel modulator drug discovery are discussed in this chapter. PMID:26646477

Identification of a unique Ca2+-binding site in rat acid-sensing ion channel 3.

PubMed

Zuo, Zhicheng; Smith, Rachel N; Chen, Zhenglan; Agharkar, Amruta S; Snell, Heather D; Huang, Renqi; Liu, Jin; Gonzales, Eric B

2018-05-25

Acid-sensing ion channels (ASICs) evolved to sense changes in extracellular acidity with the divalent cation calcium (Ca 2+ ) as an allosteric modulator and channel blocker. The channel-blocking activity is most apparent in ASIC3, as removing Ca 2+ results in channel opening, with the site's location remaining unresolved. Here we show that a ring of rat ASIC3 (rASIC3) glutamates (Glu435), located above the channel gate, modulates proton sensitivity and contributes to the formation of the elusive Ca 2+ block site. Mutation of this residue to glycine, the equivalent residue in chicken ASIC1, diminished the rASIC3 Ca 2+ block effect. Atomistic molecular dynamic simulations corroborate the involvement of this acidic residue in forming a high-affinity Ca 2+ site atop the channel pore. Furthermore, the reported observations provide clarity for past controversies regarding ASIC channel gating. Our findings enhance understanding of ASIC gating mechanisms and provide structural and energetic insights into this unique calcium-binding site.

Understanding carbon nanotube channel formation in the lipid membrane

NASA Astrophysics Data System (ADS)

Choi, Moon-ki; Kim, Hyunki; Lee, Byung Ho; Kim, Teayeop; Rho, Junsuk; Kim, Moon Ki; Kim, Kyunghoon

2018-03-01

Carbon nanotubes (CNTs) have been considered a prominent nano-channel in cell membranes because of their prominent ion-conductance and ion-selectivity, offering agents for a biomimetic channel platform. Using a coarse-grained molecular dynamics simulation, we clarify a construction mechanism of vertical CNT nano-channels in a lipid membrane for a long period, which has been difficult to observe in previous CNT-lipid interaction simulations. The result shows that both the lipid coating density and length of CNT affect the suitable fabrication condition for a vertical and stable CNT channel. Also, simulation elucidated that a lipid coating on the surface of the CNT prevents the CNT from burrowing into the lipid membrane and the vertical channel is stabilized by the repulsion force between the lipids in the coating and membrane. Our study provides an essential understanding of how CNTs can form stable and vertical channels in the membrane, which is important for designing new types of artificial channels as biosensors for bio-fluidic studies.

Standardizing the atomic description, axis and centre of biological ion channels.

PubMed

Kaats, Adrian J; Galiana, Henrietta L; Nadeau, Jay L

2007-09-15

A general representation of the atomic co-ordinates of a biological ion channel is obtained from a definition of channel axis and centre. Through rotation and translation of the channel, its centre becomes the origin of the standard co-ordinate system, and the channel axis becomes the system's z-axis. A method for determining the channel axis and centre based on the concepts of mass centre and mass moment of inertia is presented. The method for determining the channel axis can be directly applied to channels that adhere to two specific conditions regarding their geometry and mass distribution. Specific examples are given for Gramicidin A (GA), and the mammalian potassium channel Kv 1.2. For channels that do not adhere to these conditions, minor modifications of these procedures can be applied in determining the channel axis. Specific examples are given for the outer membrane bacterial porin OmpF, and for the staphylococcal pore-forming toxin alpha-hemolysin (alpha HL). The definitions and procedures presented are made in an effort to establish a standard basis for performing, sharing, and comparing computations in a consistent manner.

Beltless translocation domain of botulinum neurotoxin A embodies a minimum ion-conductive channel.

PubMed

Fischer, Audrey; Sambashivan, Shilpa; Brunger, Axel T; Montal, Mauricio

2012-01-13

Botulinum neurotoxin, the causative agent of the paralytic disease botulism, is an endopeptidase composed of a catalytic domain (or light chain (LC)) and a heavy chain (HC) encompassing the translocation domain (TD) and receptor-binding domain. Upon receptor-mediated endocytosis, the LC and TD are proposed to undergo conformational changes in the acidic endocytic environment resulting in the formation of an LC protein-conducting TD channel. The mechanism of channel formation and the conformational changes in the toxin upon acidification are important but less well understood aspects of botulinum neurotoxin intoxication. Here, we have identified a minimum channel-forming truncation of the TD, the "beltless" TD, that forms transmembrane channels with ion conduction properties similar to those of the full-length TD. At variance with the holotoxin and the HC, channel formation for both the TD and the beltless TD occurs independent of a transmembrane pH gradient. Furthermore, acidification in solution induces moderate secondary structure changes. The subtle nature of the conformational changes evoked by acidification on the TD suggests that, in the context of the holotoxin, larger structural rearrangements and LC unfolding occur preceding or concurrent to channel formation. This notion is consistent with the hypothesis that although each domain of the holotoxin functions individually, each domain serves as a chaperone for the others.

Material from the Internal Surface of Squid Axon Exhibits Excess Noise

PubMed Central

Fishman, Harvey M.

1981-01-01

A fluid material from a squid (Loligo pealei) axon was isolated by mechanical application of two types of microcapillary (1-3-μm Diam) to the internal surface of intact and cut-axon preparations. Current noise in the isolated material exceeded thermal levels and power spectra were 1/f in form in the frequency range 1.25-500 Hz with voltage-dependent intensities that were unrelated to specific ion channels. Whether conduction in this material is a significant source of excess noise during axon conduction remains to be determined. Nevertheless, a source of excess noise external to or within an ion channel may not be properly represented solely as an additive term to the spectrum of ion channel noise; a deconvolution of these spectral components may be required for modeling purposes. PMID:6266542

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels.

PubMed

Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L

2014-01-23

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. © 2013. Published by Elsevier Ltd. All rights reserved.

Varieties of Dioptase: Their Magnetic Structure and Collective Excitations

NASA Astrophysics Data System (ADS)

Prisk, T.; Hoffmann, C.; Kolesnikov, A.; Anovitz, L. M.; Matsuda, M.; Podlesnyak, A.; Yan, J.

2017-12-01

Diopase [Cu6Si6O18 * 6 H2O] is a copper cyclosilicate mineral that exists within three different polymorphic forms. In the naturally occurring green phase, micropore channels are formed by corrugated rings of Si6O18 connected by Cu2+ ions. These channels contain rings of water molecules H2O which are co-ordinated to the Cu2+ ions, forming Jahn-Teller distorted octahedra. This results in the polymorph's green color. When the mineral is annealed at 400 C, it transforms into a blue polymorph where the positions of the the water molecules H2O become disordered within the channels. If annealed at 600 C, the water molecules are expelled from crystal structure altogether, leaving intact a framework where where the copper atom has quasi-planar co-ordination with neighboring oxygen atoms. The Cu2+ ions are responsible for the appearance of long-range antiferromagnetic order in green dioptase at 14 K. Comparative magnetic susceptibility measurements of these polymorphs demonstrate that altering the hydration state of the mineral leads to profound changes in its magnetic properties. This is presumably due to the fact that the local environment of the Cu2+ ions is modified by changes in the Cu atom's co-ordination with the water oxygen. Green, blue, and black dioptase together provide a case study in the coupling of a mineral's hydration state to its magnetic properties. In this presentation, we report upon a comparative study of dioptase polymorphs. We have applied heat capacity, magnetic susceptibility, single-crystal diffraction, and inelastic neutron scattering techniques. Magnetic diffraction patterns were obtained using the Polarized Triple Axis Spectrometer at the High Flux Isotope Reactor, while measurements of the spin wave dispersion were performed using the Cold Neutron Chopper Spectrometer at the Spallation Neutron Source. We find that, in green dioptase, the Cu2+ ions form spiral antiferromagnetic chains parallel to the micropore channels. However, in blue dioptase, long-range antiferromagnetic order is completely suppressed down to at least 2 K. Black dioptase undergoes a phase transition at 7 K into a low dimensional antiferromagnet, apparently having a different magnetic structure from its green counterpart.

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

PubMed

Iwamoto, T; Grove, A; Montal, M O; Montal, M; Tomich, J M

1994-06-01

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture.

PubMed

Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong; Kim, Sunghoon; She, Ji; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

2017-07-27

TMEM175 is a lysosomal K + channel that is important for maintaining the membrane potential and pH stability in lysosomes. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K + channels and lacks the TVGYG selectivity filter motif found in these channels. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K + channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K + channel family.

Progress in Development of Improved Ion-Channel Biosensors

NASA Technical Reports Server (NTRS)

Nadeau, Jay L.; White, Victor E.; Maurer, Joshua A.; Dougherty, Dennis A.

2008-01-01

Further improvements have recently been made in the development of the devices described in Improved Ion-Channel Biosensors (NPO-30710), NASA Tech Briefs, Vol. 28, No. 10 (October 2004), page 30. As discussed in more detail in that article, these sensors offer advantages of greater stability, greater lifetime, and individual electrical addressability, relative to prior ion-channel biosensors. In order to give meaning to a brief description of the recent improvements, it is necessary to recapitulate a substantial portion of the text of the cited previous article. The figure depicts one sensor that incorporates the recent improvements, and can be helpful in understanding the recapitulated text, which follows: These sensors are microfabricated from silicon and other materials compatible with silicon. Typically, the sensors are fabricated in arrays in silicon wafers on glass plates. Each sensor in the array can be individually electrically addressed, without interference with its neighbors. Each sensor includes a well covered by a thin layer of silicon nitride, in which is made a pinhole for the formation of a lipid bilayer membrane. In one stage of fabrication, the lower half of the well is filled with agarose, which is allowed to harden. Then the upper half of the well is filled with a liquid electrolyte (which thereafter remains liquid) and a lipid bilayer is painted over the pinhole. The liquid contains a protein that forms an ion channel on top of the hardened agarose. The combination of enclosure in the well and support by the hardened agarose provides the stability needed to keep the membrane functional for times as long as days or even weeks. An electrode above the well, another electrode below the well, and all the materials between the electrodes together constitute a capacitor. What is measured is the capacitive transient current in response to an applied voltage pulse. One notable feature of this sensor, in comparison with prior such sensors, is a relatively thick dielectric layer between the top of the well and the top electrode. This layer greatly reduces the capacitance of an aperture across which the ion channels are formed, thereby increasing the signal-to-noise ratio. The use of a relatively large aperture with agarose support makes it possible to form many ion channels instead of only one, thereby further increasing the signal-to-noise ratio and effectively increasing the size of the available ionic reservoir. The relatively large reservoir makes it possible to measure AC rather than DC. This concludes the recapitulation from the cited previous article.

Carbons for lithium batteries prepared using sepiolite as an inorganic template

DOEpatents

Sandi, Giselle; Winans, Randall E.; Gregar, K. Carrado

2000-01-01

A method of preparing an anode material using sepiolite clay having channel-like interstices in its lattice structure. Carbonaceous material is deposited in the channel-like interstices of the sepiolite clay and then the sepiolite clay is removed leaving the carbonaceous material. The carbonaceous material is formed into an anode. The anode is combined with suitable cathode and electrolyte materials to form a battery of the lithium-ion type.

Structural Basis for Xenon Inhibition in a Cationic Pentameric Ligand-Gated Ion Channel

PubMed Central

Sauguet, Ludovic; Fourati, Zeineb; Prangé, Thierry; Delarue, Marc; Colloc'h, Nathalie

2016-01-01

GLIC receptor is a bacterial pentameric ligand-gated ion channel whose action is inhibited by xenon. Xenon has been used in clinical practice as a potent gaseous anaesthetic for decades, but the molecular mechanism of interactions with its integral membrane receptor targets remains poorly understood. Here we characterize by X-ray crystallography the xenon-binding sites within both the open and “locally-closed” (inactive) conformations of GLIC. Major binding sites of xenon, which differ between the two conformations, were identified in three distinct regions that all belong to the trans-membrane domain of GLIC: 1) in an intra-subunit cavity, 2) at the interface between adjacent subunits, and 3) in the pore. The pore site is unique to the locally-closed form where the binding of xenon effectively seals the channel. A putative mechanism of the inhibition of GLIC by xenon is proposed, which might be extended to other pentameric cationic ligand-gated ion channels. PMID:26910105

Structural Basis for Xenon Inhibition in a Cationic Pentameric Ligand-Gated Ion Channel.

PubMed

Sauguet, Ludovic; Fourati, Zeineb; Prangé, Thierry; Delarue, Marc; Colloc'h, Nathalie

2016-01-01

GLIC receptor is a bacterial pentameric ligand-gated ion channel whose action is inhibited by xenon. Xenon has been used in clinical practice as a potent gaseous anaesthetic for decades, but the molecular mechanism of interactions with its integral membrane receptor targets remains poorly understood. Here we characterize by X-ray crystallography the xenon-binding sites within both the open and "locally-closed" (inactive) conformations of GLIC. Major binding sites of xenon, which differ between the two conformations, were identified in three distinct regions that all belong to the trans-membrane domain of GLIC: 1) in an intra-subunit cavity, 2) at the interface between adjacent subunits, and 3) in the pore. The pore site is unique to the locally-closed form where the binding of xenon effectively seals the channel. A putative mechanism of the inhibition of GLIC by xenon is proposed, which might be extended to other pentameric cationic ligand-gated ion channels.

mTor Regulates Lysosomal ATP-sensitive Two-Pore Na+ Channel to Adapt to Metabolic State

PubMed Central

Navarro, Betsy; Seo, Young-jun; Aranda, Kimberly; Shi, Lucy; Battaglia-Hsu, Shyuefang; Nissim, Itzhak; Clapham, David E.; Ren, Dejian

2014-01-01

SUMMARY Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here we identify an endolysosomal ATP-sensitive Na+ channel (lysoNaATP). The channel is a complex formed by Two-Pore Channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosome's membrane potential, pH stability, and the amino acid homeostasis. Mutant mice lacking lysoNaATP have much reduced exercise endurance after fasting. Thus, TPCs are a new ion channel family that couple the cell's metabolic state to endolysosomal function and are crucial for physical endurance during food restriction. PMID:23394946

Structure of a eukaryotic cyclic nucleotide-gated channel

PubMed Central

Li, Minghui; Zhou, Xiaoyuan; Wang, Shu; Michailidis, Ioannis; Gong, Ye; Su, Deyuan; Li, Huan; Li, Xueming; Yang, Jian

2018-01-01

Summary Cyclic nucleotide-gated (CNG) channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5 Å-resolution single-particle electron cryomicroscopy structure of a CNG channel from C. elegans in the cGMP-bound open state. The channel has an unusual voltage-sensor-like domain (VSLD), accounting for its deficient voltage dependence. A C-terminal linker connecting S6 and the cyclic nucleotide-binding domain interacts directly with both the VSLD and pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of CNG channels and cyclic nucleotide modulation of related channels. PMID:28099415

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

PubMed

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

2016-01-01

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

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

PubMed Central

Hille, Bertil

1972-01-01

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na+ ≈ Li+ > Tl+ > K+. The ratio P K/P Na is 1/12. The permeabilities to Rb+, Cs+, Ca++, and Mg++ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E Na differs significantly from the Nernst potential for Na+ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria. PMID:5025743

Effects of protein-protein interactions and ligand binding on the ion permeation in KCNQ1 potassium channel.

PubMed

Jalily Hasani, Horia; Ganesan, Aravindhan; Ahmed, Marawan; Barakat, Khaled H

2018-01-01

The voltage-gated KCNQ1 potassium ion channel interacts with the type I transmembrane protein minK (KCNE1) to generate the slow delayed rectifier (IKs) current in the heart. Mutations in these transmembrane proteins have been linked with several heart-related issues, including long QT syndromes (LQTS), congenital atrial fibrillation, and short QT syndrome. Off-target interactions of several drugs with that of KCNQ1/KCNE1 ion channel complex have been known to cause fatal cardiac irregularities. Thus, KCNQ1/KCNE1 remains an important avenue for drug-design and discovery research. In this work, we present the structural and mechanistic details of potassium ion permeation through an open KCNQ1 structural model using the combined molecular dynamics and steered molecular dynamics simulations. We discuss the processes and key residues involved in the permeation of a potassium ion through the KCNQ1 ion channel, and how the ion permeation is affected by (i) the KCNQ1-KCNE1 interactions and (ii) the binding of chromanol 293B ligand and its derivatives into the complex. The results reveal that interactions between KCNQ1 with KCNE1 causes a pore constriction in the former, which in-turn forms small energetic barriers in the ion-permeation pathway. These findings correlate with the previous experimental reports that interactions of KCNE1 dramatically slows the activation of KCNQ1. Upon ligand-binding onto the complex, the energy-barriers along ion permeation path are more pronounced, as expected, therefore, requiring higher force in our steered-MD simulations. Nevertheless, pulling the ion when a weak blocker is bound to the channel does not necessitate high force in SMD. This indicates that our SMD simulations have been able to discern between strong and week blockers and reveal their influence on potassium ion permeation. The findings presented here will have some implications in understanding the potential off-target interactions of the drugs with the KCNQ1/KCNE1 channel that lead to cardiotoxic effects.

Regulation of aldosterone production by ion channels: From basal secretion to primary aldosteronism.

PubMed

Yang, Tingting; He, Min; Hu, Changlong

2018-03-01

Aldosterone is produced by zona glomerulosa (ZG) cells of the adrenal cortex and plays a key role in balancing water and electrolytes levels. Autonomous overproduction of aldosterone leads to primary aldosteronism (PA), which is the most common form of secondary endocrine hypertension. Recently, significant progress has been made towards understanding the genetic basis of PA, where increasing clinical evidence suggests that mutations in ion channels appear to be the major cause of aldosterone-producing adenomas. In this review, we focused on potassium and calcium channels that regulate aldosterone secretion, and their roles in the pathology of PA. Because potassium and calcium channels are differentially expressed in ZG cells in different species of mammals, the limitations of published studies are also discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

The Small Hydrophobic Protein of the Human Respiratory Syncytial Virus Forms Pentameric Ion Channels*

PubMed Central

Gan, Siok-Wan; Tan, Edward; Lin, Xin; Yu, Dejie; Wang, Juejin; Tan, Gregory Ming-Yeong; Vararattanavech, Ardcharaporn; Yeo, Chiew Ying; Soon, Cin Huang; Soong, Tuck Wah; Pervushin, Konstantin; Torres, Jaume

2012-01-01

The small hydrophobic (SH) protein is encoded by the human respiratory syncytial virus. Its absence leads to viral attenuation in the context of whole organisms, and it prevents apoptosis in infected cells. Herein, we have examined the structure of SH protein in detergent micelles and in lipid bilayers, by solution NMR and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. We found that SH protein has a single α-helical transmembrane domain and forms homopentamers in several detergents. In detergent micelles, the transmembrane domain is flanked N-terminally by an α-helix that forms a ring around the lumen of the pore and C-terminally by an extended β-turn. SH protein was found in the plasma membrane of transiently expressing HEK 293 cells, which showed pH-dependent (acid-activated) channel activity. Channel activity was abolished in mutants lacking both native His residues, His22 and His51, but not when either His was present. Herein, we propose that the pentameric model of SH protein presented is a physiologically relevant conformation, albeit probably not the only one, in which SH contributes to RSV infection and replication. Viroporins are short (∼100 amino acids) viral membrane proteins that form oligomers of a defined size, act as proton or ion channels, and in general enhance membrane permeability in the host. However, with some exceptions, their precise biological role of their channel activity is not understood. In general, viroporins resemble poorly specialized proteins but are nevertheless critical for viral fitness. In vivo, viruses lacking viroporins usually exhibit an attenuated or weakened phenotype, altered tropism, and diminished pathological effects. We have chosen to study the SH protein, 64 amino acids long, found in the human respiratory syncytial virus because of the effect of RSV on human health and the lack of adequate antivirals. We show that SH protein forms oligomers that behave as ion channels when activated at low pH. This study adds SH protein to a growing group of viroporins that have been structurally characterized. Although the precise biological role of this pentameric channel is still unknown, this report is nevertheless essential to fill some of the many gaps that exist in the understanding of SH protein function. PMID:22621926

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels

PubMed Central

Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L.

2013-01-01

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail ‘neck’, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the ‘outer ion’ site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies shows that this site forms a previously unknown determinant of CaV high affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. PMID:24120938

Epithelial Sodium and Acid-Sensing Ion Channels

NASA Astrophysics Data System (ADS)

Kellenberger, Stephan

The epithelial Na+ channel (ENaC) and acid-sensing ion channels (ASICs) are non-voltage-gated Na+ channels that form their own subfamilies within the ENaC/degenerin ion channel family. ASICs are sensors of extracellular pH, and ENaC, whose main function is trans-epithelial Na+ transport, can sense extra- and intra-cellular Na+. In aldosterone-responsive epithelial cells of the kidney, ENaC plays a critical role in the control of sodium balance, blood volume and blood pressure. In airway epithelia, ENaC has a distinct role in controlling fluid reabsorption at the air-liquid interface, thereby determining the rate of mucociliary transport. In taste receptor cells of the tongue, ENaC is involved in salt taste sensation. ASICs have emerged as key sensors for extracellular protons in central and peripheral neurons. Although not all of their physiological and pathological functions are firmly established yet, there is good evidence for a role of ASICs in the brain in learning, expression of fear, and in neurodegeneration after ischaemic stroke. In sensory neurons, ASICs are involved in nociception and mechanosensation. ENaC and ASIC subunits share substantial sequence homology and the conservation of several functional domains. This chapter summarises our current understanding of the physiological functions and of the mechanisms of ion permeation, gating and regulation of ENaC and ASICs.

Peptides and genes coding for scorpion toxins that affect ion-channels.

PubMed

Possani, L D; Merino, E; Corona, M; Bolivar, F; Becerril, B

2000-01-01

Most scorpion toxins are ligand peptides that recognize and bind to integral membrane proteins known as ion-channels. To date there are at least 202 distinct sequences described, obtained from 30 different species of scorpions, 27 from the family Buthidae and three from the family Scorpionidae. Toxins that recognize potassium and chloride channels are usually from 29 to 41 amino acids long, stabilized by three or four disulfide bridges, whereas those that recognize sodium channels are longer, 60 to 76 amino acid residues, compacted by four disulfide bridges. Toxins specific for calcium channels are scarcely known and have variable amino acid lengths. The entire repertoire of toxins, independently of their specificity, was analyzed together by computational programs and a phylogenetic tree was built showing two separate branches. The K(+) and Cl(-) channel specific toxins are clustered into 14 subfamilies, whereas those of Na(+) and Ca(2+) specific toxins comprise at least 12 subfamilies. There are clear similarities among them, both in terms of primary sequence and the main three-dimensional folding pattern. A dense core formed by a short alpha helix segment and several antiparallel beta-sheet stretches, maintained by disulfide pairing, seems to be a common structural feature present in all toxins. The physiological function of these peptides is manifested by a blockage of ion passage through the channels or by a modification of the gating mechanism that controls opening and closing of the ion pore.

Two-sided block of a dual-topology F- channel.

PubMed

Turman, Daniel L; Nathanson, Jacob T; Stockbridge, Randy B; Street, Timothy O; Miller, Christopher

2015-05-05

The Fluc family is a set of small membrane proteins forming F(-)-specific electrodiffusive ion channels that rescue microorganisms from F(-) toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.

Influence of membrane thickness and ion concentration on the properties of the gramicidin a channel. Autocorrelation, spectral power density, relaxation and single-channel studies.

PubMed

Kolb, H A; Bamberg, E

1977-01-04

The properties of the gramicidin A channel in membranes made from a series of monoglycerides have been studied. In agreement with previous studies, the dissociation rate constant kD of the dimeric channel was found to increase strongly with increasing chain length of the monoglyceride, corresponding to a decrease of the mean life-time of the channel. The value of kD, however, was not strictly correlated with the membrane thickness, as seen from a comparison of membranes with different solvent content. Furthermore, the life-time of the channel increased with the concentration of the permeable ion. This effect was tentatively explained by an electrostatic stabilization of the channel. The single-channel conductance lambda was found to decrease with increasing membrane thickness d, if d was varied by increasing the chain length of the lipid. On the other hand, if d was changed by varying the solvent content of the membranes formed from one and the same lipid, lambda remained constant. These observations were explained by the assumption of local inhomogeneities in the membrane thickness. A striking difference between the lambda values obtained from autocorrelation analysis in the presence of many presence of many channels (lambda a) and those obtained from single-channel experiments (lambda sc) occurred with membranes from longer chain-length monoglycerides. This difference disappeared at low ion concentrations. Electrostatic interactions between channels in local clusters were proposed for an interpretation of these findings.

Characterization of Rat Meibomian Gland Ion and Fluid Transport

PubMed Central

Yu, Dongfang; Davis, Richard M.; Aita, Megumi; Burns, Kimberlie A.; Clapp, Phillip W.; Gilmore, Rodney C.; Chua, Michael; O'Neal, Wanda K.; Schlegel, Richard; Randell, Scott H.; C. Boucher, Richard

2016-01-01

Purpose We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. Methods Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of βENaC, the Na+/K+/Cl− cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays. Results Expression of multiple ion channel/transporter genes was detected in rat MG tissues. β-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels. Conclusions This study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established. PMID:27127933

Fragmentation mechanisms for methane induced by 55 eV, 75 eV, and 100 eV electron impact

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

Wei, B.; Zhang, Y.; Wang, X., E-mail: xinchengwang@fudan.edu.cn

2014-03-28

The fragmentation of CH{sub 4}{sup 2+} dications following 55 eV, 75 eV, and 100 eV electron impact double ionization of methane was studied using a cold target recoil-ion momentum spectroscopy. From the measured momentum of each recoil ion, the momentum of the neutral particles has been deduced and the kinetic energy release distribution for the different fragmentation channels has been obtained. The doubly charged molecular ions break up into three or more fragments in one or two-step processes, resulting in different signatures in the data. We observed the fragmentation of CH{sub 4}{sup 2+} dications through different mechanisms according to themore » momentum of the neutral particles. For example, our result shows that there are three reaction channels to form CH{sub 2}{sup +}, H{sup +}, and H, one synchronous concerted reaction channel and two two-step reaction channels. For even more complicated fragmentation processes of CH{sub 4}{sup 2+} dications, the fragmentation mechanism can still be identified in the present measurements. The slopes of the peak in the ion-ion coincidence spectra were also estimated here, as they are also related to the fragmentation mechanism.« less

Maximizing Ion Transmission from Atmospheric Pressure into the Vacuum of Mass Spectrometers with a Novel Electrospray Interface

PubMed Central

Krutchinsky, Andrew N.; Padovan, Júlio C.; Cohen, Herbert; Chait, Brian T.

2015-01-01

We have discovered that an electrode containing a conical channel with a small angular divergence can transmit into the vacuum almost 100% of an electrospray ion current produced at atmospheric pressure. Our first implementation of such a conical duct, which we term “ConDuct”, uses a conductive plastic pipette tip containing a ≈1.6° divergent channel at its entrance. We observed that the beam formed by the ConDuct electrode has a very low divergence (< 1°) and persisted for long distances in vacuum. Intrigued by these properties, we incorporated this electrode into a novel atmosphere-to-vacuum ion transmission interface, and devised a technique for evaluating its performance relative to commercial reference interfaces that contain heated metal capillaries. We determined that our new interface transmits at least 400 times more ions than the commercial Thermo LCQ DECA XP atmosphere-to-vacuum interface and 2–3 times more than the commercial interface in the Thermo Velos Orbitrap and the Q Exactive mass spectrometers. We conclude that it might be possible to optimize the properties of the transmitted ions further by manufacturing ConDuct inlet electrodes from metal rather than conductive plastic and by determining the optimum angle of channel divergence and channel length. PMID:25588722

Silicon-ion-implanted PMMA with nanostructured ultrathin layers for plastic electronics

NASA Astrophysics Data System (ADS)

Hadjichristov, G. B.; Ivanov, Tz E.; Marinov, Y. G.

2014-12-01

Being of interest for plastic electronics, ion-beam produced nanostructure, namely silicon ion (Si+) implanted polymethyl-methacrylate (PMMA) with ultrathin nanostructured dielectric (NSD) top layer and nanocomposite (NC) buried layer, is examined by electric measurements. In the proposed field-effect organic nanomaterial structure produced within the PMMA network by ion implantation with low energy (50 keV) Si+ at the fluence of 3.2 × 1016 cm-2 the gate NSD is ion-nanotracks-modified low-conductive surface layer, and the channel NC consists of carbon nanoclusters. In the studied ion-modified PMMA field-effect configuration, the gate NSD and the buried NC are formed as planar layers both with a thickness of about 80 nm. The NC channel of nano-clustered amorphous carbon (that is an organic semiconductor) provides a huge increase in the electrical conduction of the material in the subsurface region, but also modulates the electric field distribution in the drift region. The field effect via the gate NSD is analyzed. The most important performance parameters, such as the charge carrier field-effect mobility and amplification of this particular type of PMMA- based transconductance device with NC n-type channel and gate NSD top layer, are determined.

Redesigning Channel-Forming Peptides: Amino Acid Substitutions that Enhance Rates of Supramolecular Self-Assembly and Raise Ion Transport Activity

PubMed Central

Shank, Lalida P.; Broughman, James R.; Takeguchi, Wade; Cook, Gabriel; Robbins, Ashley S.; Hahn, Lindsey; Radke, Gary; Iwamoto, Takeo; Schultz, Bruce D.; Tomich, John M.

2006-01-01

Three series of 22-residue peptides derived from the transmembrane M2 segment of the glycine receptor α1-subunit (M2GlyR) have been designed, synthesized, and tested to determine the plasticity of a channel-forming sequence and to define whether channel pores with enhanced conductive properties could be created. Sixteen sequences were examined for aqueous solubility, solution-association tendency, secondary structure, and half-maximal concentration for supramolecular assembly, channel activity, and ion transport properties across epithelial monolayers. All peptides interact strongly with membranes: associating with, inserting across, and assembling to form homooligomeric bundles when in micromolar concentrations. Single and double amino acid replacements involving arginine and/or aromatic amino acids within the final five C-terminal residues of the peptide cause dramatic effects on the concentration dependence, yielding a range of K1/2 values from 36 ± 5 to 390 ± 220 μM for transport activity. New water/lipid interfacial boundaries were established for the transmembrane segment using charged or aromatic amino acids, thus limiting the peptides' ability to move perpendicularly to the plane of the bilayer. Formation of discrete water/lipid interfacial boundaries appears to be necessary for efficient supramolecular assembly and high anion transport activity. A peptide sequence is identified that may show efficacy in channel replacement therapy for channelopathies such as cystic fibrosis. PMID:16387776

Electron microscopic and ion scattering studies of heteroepitaxial tin-doped indium oxide films

NASA Astrophysics Data System (ADS)

Kamei, Masayuki; Shigesato, Yuzo; Takaki, Satoru; Hayashi, Yasuo; Sasaki, Mikio; Haynes, Tony E.

1994-08-01

The microstructure of heteroepitaxial tin-doped indium oxide (ITO) films were studied in detail. The surface morphology of the heteroepitaxial ITO film consisted of square-shaped, in-plane oriented subgrains (˜300 Å) in contrast to that of the polycrystalline film (characteristic grain-subgrain structure). The subgrain boundaries were predominantly formed along the {110} planes in the ITO film and dislocations were observed primarily along the subgrain boundaries. Ion channeling measurements showed the dislocation density of this film to be approximately 3×1010/cm2, and the angular distribution of the ion channeling yield showed that the subgrains are aligned to within better than 0.3° (standard deviation).

The N-terminal domain of GluR6-subtype glutamate receptor ion channels

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

Kumar, Janesh; Schuck, Peter; Jin, Rongsheng

2009-09-25

The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs.more » This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.« less

Synthetic ion channels via self-assembly: a route for embedding porous polyoxometalate nanocapsules in lipid bilayer membranes.

PubMed

Carr, Rogan; Weinstock, Ira A; Sivaprasadarao, Asipu; Müller, Achim; Aksimentiev, Aleksei

2008-11-01

Porous polyoxometalate nanocapsules of Keplerate type are known to exhibit the functionality of biological ion channels; however, their use as an artificial ion channel is tempered by the high negative charge of the capsules, which renders their spontaneous incorporation into a lipid bilayer membrane unlikely. In this Letter we report coarse-grained molecular dynamics simulations that demonstrate a route for embedding negatively charged nanocapsules into lipid bilayer membranes via self-assembly. A homogeneous mixture of water, cationic detergent, and phospholipid was observed to spontaneously self-assemble around the nanocapsule into a layered, liposome-like structure, where the nanocapsule was enveloped by a layer of cationic detergent followed by a layer of phospholipid. Fusion of such a layered liposome with a lipid bilayer membrane was observed to embed the nanocapsule into the lipid bilayer. The resulting assembly was found to remain stable even after the surface of the capsule was exposed to electrolyte. In the latter conformation, water was observed to flow into and out of the capsule as Na(+) cations entered, suggesting that a polyoxometalate nanocapsule can form a functional synthetic ion channel in a lipid bilayer membrane.

Synthetic Ion Channels via Self-Assembly: a Route for Embedding Porous Polyoxometalate Nanocapsules in Lipid Bilayer Membranes

PubMed Central

Carr, Rogan; Weinstock, Ira A.; Sivaprasadarao, Asipu; Müller, Achim; Aksimentiev, Aleksei

2010-01-01

Porous polyoxometalate nanocapsules of Keplerate type are known to exhibit the functionality of biological ion channels, however, their use as artificial ion channel is tempered by the high negative charge of the capsules, which renders their spontaneous incorporation into a lipid bilayer membrane unlikely. In this letter we report coarse-grained molecular dynamics simulations that demonstrate a route for embedding negatively charged nanocapsules into lipid bilayer membranes via self-assembly. A homogeneous mixture of water, cationic detergent, and phospholipid was observed to spontaneously self-assemble around the nanocapsule into a layered, liposome-like structure, where the nanocapsule was enveloped by a layer of cationic detergent followed by a layer of phospholipid. Fusion of such a layered liposome with a lipid bilayer membrane was observed to embed the nanocapsule into the lipid bilayer. The resulting assembly was found to remain stable even after the surface of the capsule was exposed to electrolyte. In the latter conformation, water was observed to flow into and out of the capsule as Na+ cations entered, suggesting that a polyoxometalate nanocapsule can form a functional synthetic ion channel in a lipid bilayer membrane. PMID:18844424

Exciplexes versus Loose Ion Pairs: How Does the Driving Force Impact the Initial Product Ratio of Photoinduced Charge Separation Reactions?

PubMed Central

2014-01-01

Many donor–acceptor systems can undergo a photoinduced charge separation reaction, yielding loose ion pairs (LIPs). LIPs can be formed either directly via (distant) electron transfer (ET) or indirectly via the dissociation of an initially formed exciplex or tight ion pair. Establishing the prevalence of one of the reaction pathways is challenging because differentiating initially formed exciplexes from LIPs is difficult due to similar spectroscopic footprints. Hence, no comprehensive reaction model has been established for moderately polar solvents. Here, we employ an approach based on the time-resolved magnetic field effect (MFE) of the delayed exciplex luminescence to distinguish the two reaction channels. We focus on the effects of the driving force of ET and the solvent permittivity. We show that, surprisingly, the exciplex channel is significant even for an exergonic ET system with a free energy of ET of −0.58 eV and for the most polar solutions studied (butyronitrile). Our findings demonstrate that exciplexes play a crucial role even in polar solvents and at moderate driving forces, contrary to what is usually assumed. PMID:25243054

Na Diffusion in Quasi One-Dimensional Ion Conductor NaMn2O4 Observed by μ+SR

NASA Astrophysics Data System (ADS)

Umegaki, Izumi; Nozaki, Hiroshi; Harada, Masashi; Månsson, Martin; Sakurai, Hiroya; Kawasaki, Ikuto; Watanabe, Isao; Sugiyama, Jun

A quasi one-dimensional (1D) compound, NaMn2O4, in which Mn2O4 zigzag chains form a 1D channel along the b-axis and Na ions locate at the center of the channel, is thought to be a good Na ionic conductor. In order to study Na-ion diffusion, we have measured μ+SR spectra using a powder sample in the temperature range between 100 and 500 K. A diffusive behavior was clearly observed above 325 K. Assuming a thermal activate process for jump diffusion of Na-ion between two nearest neighboring sites, a self diffusion coefficient of Na ion (DNa) and its activation energy (Ea) were estimated as DNa = (3.1 ± 0.2) × 10 - 11 cm2/s at 350 K and Ea = 180(9) meV.

Near-membrane electric field calcium ion dehydration.

PubMed

Barger, James P; Dillon, Patrick F

2016-12-01

The dehydration of ion-water complexes prior to ion channel transit has focused on channel protein-mediated dissociation of water. Ion dehydration by the membrane electric field has not previously been considered. Near membrane electric fields have previously been shown to cause the disassociation of non-covalently bound small molecule-small molecule, small molecule-protein, and protein-protein complexes. It is well known that cosmotropic, structure making ions such as calcium and sodium significantly bind multiple water ions in solution. It is also known that these ions are often not hydrated as they pass through membrane ion channels. Using capillary electrophoresis, the range of electric fields needed to strip water molecules from calcium ions has been measured. Ion migration velocity is a linear function of the electric field. At low electric fields, the migration rate of calcium ion was shown to be linearly related to the applied electric field. Using a form of the Stoke's equation applicable to ion migration, the hydrated calcium radius was found to be 0.334nm, corresponding to a water hydration shell of 5.09 water molecules. At higher electric fields, the slope of the calcium migration velocity as a function of the electric field increased, which was modeled as a decrease in the radius of the migrating ion as the water was removed. Using a tanh function to model the transition of the ion from a hydrated to a stripped state, the transition had a midpoint at 446V/cm, and was 88% complete at 587V/cm with a correlation coefficient of 0.9996. The migration velocity of the stripped calcium ion was found to be a function of both the decrease in radius and an increase in the effective, electronic viscosity of the dipole medium through which the dehydrated ion moved. The size of the electric field needed to dehydrate calcium occurs 6-7nm from the cell membrane. Calcium ions within this distance from the membrane will be devoid of water molecules when they reach the calcium selective channel pore entrances, all known to be approximately 1-2nm from the membrane. No matter what the calcium pore structure, calcium ions reaching the channel entrance will be devoid of a water shell. Copyright © 2016 Elsevier Ltd. All rights reserved.

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

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

Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong

TMEM175 is a lysosomal K+ channel that is important for maintaining the membrane potential and pH stability in lysosomes1. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif found in these channels2, 3, 4. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completelymore » different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K+ channel family.« less

Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

PubMed Central

Siroy, Axel; Molle, Virginie; Lemaître-Guillier, Christelle; Vallenet, David; Pestel-Caron, Martine; Cozzone, Alain J.; Jouenne, Thierry; Dé, Emmanuelle

2005-01-01

It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (Mw and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels. PMID:16304148

Exploring the intrinsic polar [4+2(+)] cycloaddition reactivity of gaseous carbosulfonium and carboxonium ions.

PubMed

Basher, Muftah M; Corilo, Yuri E; Sparrapan, Regina; Benassi, Mario; Augusti, Rodinei; Eberlin, Marcos N; Riveros, José M

2012-11-01

Gas-phase reactions of model carbosulfonium ions (CH(3)-S(+)=CH(2;) CH(3)CH(2)-S(+)=CH(2) and Ph-S(+)=CH(2)) and an O-analogue carboxonium ion (CH(3)-O(+)=CH(2)) with acyclic (isoprene, 1,3-butadiene, methyl vinyl ketone) and cyclic (1,3-cyclohexadiene, thiophene, furan) conjugated dienes were systematically investigated by pentaquadrupole mass spectrometry. As corroborated by B3LYP/6-311 G(d,p) calculations, the carbosulfonium ions first react at large extents with the dienes forming adducts via simple addition. The nascent adducts, depending on their stability and internal energy, react further via two competitive channels: (1) in reactions with acyclic dienes via cyclization that yields formally [4+2(+)] cycloadducts, or (2) in reactions with the cyclic dienes via dissociation by HSR loss that yields methylenation (net CH(+) transfer) products. In great contrast to its S-analogues, CH(3)-O(+)=CH(2) (as well as C(2)H(5)-O(+)=CH(2) and Ph-O(+)=CH(2) in reactions with isoprene) forms little or no adduct and proton transfer is the dominant reaction channel. Isomerization to more acidic protonated aldehydes in the course of reaction seems to be the most plausible cause of the contrasting reactivity of carboxonium ions. The CH(2)=CH-O(+)=CH(2) ion forms an abundant [4+2(+)] cycloadduct with isoprene, but similar to the behavior of such α,β-unsaturated carboxonium ions in solution, seems to occur across the C=C bond. Copyright © 2012 John Wiley & Sons, Ltd.

Is there a role for voltage-gated Na+ channels in the aggressiveness of breast cancer?

PubMed

Rhana, P; Trivelato, R R; Beirão, P S L; Cruz, J S; Rodrigues, A L P

2017-06-05

Breast cancer is the most common cancer among women and its metastatic potential is responsible for numerous deaths. Thus, the need to find new targets for improving treatment, and even finding the cure, becomes increasingly greater. Ion channels are known to participate in several physiological functions, such as muscle contraction, cell volume regulation, immune response and cell proliferation. In breast cancer, different types of ion channels have been associated with tumorigenesis. Recently, voltage-gated Na+ channels (VGSC) have been implicated in the processes that lead to increased tumor aggressiveness. To explain this relationship, different theories, associated with pH changes, gene expression and intracellular Ca2+, have been proposed in an attempt to better understand the role of these ion channels in breast cancer. However, these theories are having difficulty being accepted because most of the findings are contrary to the present scientific knowledge. Several studies have shown that VGSC are related to different types of cancer, making them a promising pharmacological target against this debilitating disease. Molecular biology and cell electrophysiology have been used to look for new forms of treatment aiming to reduce aggressiveness and the disease progress.

Is there a role for voltage-gated Na+ channels in the aggressiveness of breast cancer?

PubMed Central

Rhana, P.; Trivelato, R.R.; Beirão, P.S.L.; Cruz, J.S.; Rodrigues, A.L.P.

2017-01-01

Breast cancer is the most common cancer among women and its metastatic potential is responsible for numerous deaths. Thus, the need to find new targets for improving treatment, and even finding the cure, becomes increasingly greater. Ion channels are known to participate in several physiological functions, such as muscle contraction, cell volume regulation, immune response and cell proliferation. In breast cancer, different types of ion channels have been associated with tumorigenesis. Recently, voltage-gated Na+ channels (VGSC) have been implicated in the processes that lead to increased tumor aggressiveness. To explain this relationship, different theories, associated with pH changes, gene expression and intracellular Ca2+, have been proposed in an attempt to better understand the role of these ion channels in breast cancer. However, these theories are having difficulty being accepted because most of the findings are contrary to the present scientific knowledge. Several studies have shown that VGSC are related to different types of cancer, making them a promising pharmacological target against this debilitating disease. Molecular biology and cell electrophysiology have been used to look for new forms of treatment aiming to reduce aggressiveness and the disease progress. PMID:28591378

Mechanism of voltage-gated channel formation in lipid membranes.

PubMed

Guidelli, Rolando; Becucci, Lucia

2016-04-01

Although several molecular models for voltage-gated ion channels in lipid membranes have been proposed, a detailed mechanism accounting for the salient features of experimental data is lacking. A general treatment accounting for peptide dipole orientation in the electric field and their nucleation and growth kinetics with ion channel formation is provided. This is the first treatment that explains all the main features of the experimental current-voltage curves of peptides forming voltage-gated channels available in the literature. It predicts a regime of weakly voltage-dependent conductance, followed by one of strong voltage-dependent conductance at higher voltages. It also predicts values of the parameters expressing the exponential dependence of conductance upon voltage and peptide bulk concentration for both regimes, in good agreement with those reported in the literature. Most importantly, the only two adjustable parameters involved in the kinetics of nucleation and growth of ion channels can be varied over broad ranges without affecting the above predictions to a significant extent. Thus, the fitting of experimental current-voltage curves stems naturally from the treatment and depends only slightly upon the choice of the kinetic parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

An interview with Arthur M. "Buzz" Brown, M.D., Ph.D. by Vicki Glaser.

PubMed

Brown, Arthur M

2008-12-01

Dr. Arthur M. "Buzz" Brown is the founder and CEO of ChanTest Corporation, an ion channel company specializing in drug discovery and safety services. He is Adjunct Professor of Physiology and Biophysics, School of Medicine, Case Western Reserve University. Dr. Brown has more than 30 years of experience in ion channel structure-function relationships and their associations with human health. He established world-leading ion channel departments at University of Texas Medical Branch, Baylor College of Medicine, and Case Western Reserve University. His lab first applied liquid ion exchanger ion-selective microelectrodes to single cells, introduced the concept of membrane delimited action of G proteins on ion channels, identified the ion channel conduction pathway or pore of voltage-gated channels and inwardly rectifying potassium channels, showed that the human ether-à-go-go-related gene potassium channel was the molecular target for lethal arrhythmias associated with noncardiac drugs, and established that noncardiac drugs could also produce lethal arrhythmias by inhibiting ion channel trafficking. Dr. Brown holds eight patents on ion channel methodology and application of ion channel pharmacology to therapeutics.

The probability of quantal secretion near a single calcium channel of an active zone.

PubMed Central

Bennett, M R; Farnell, L; Gibson, W G

2000-01-01

A Monte Carlo analysis has been made of calcium dynamics and quantal secretion at microdomains in which the calcium reaches very high concentrations over distances of <50 nm from a channel and for which calcium dynamics are dominated by diffusion. The kinetics of calcium ions in microdomains due to either the spontaneous or evoked opening of a calcium channel, both of which are stochastic events, are described in the presence of endogenous fixed and mobile buffers. Fluctuations in the number of calcium ions within 50 nm of a channel are considerable, with the standard deviation about half the mean. Within 10 nm of a channel these numbers of ions can give rise to calcium concentrations of the order of 100 microM. The temporal changes in free calcium and calcium bound to different affinity indicators in the volume of an entire varicosity or bouton following the opening of a single channel are also determined. A Monte Carlo analysis is also presented of how the dynamics of calcium ions at active zones, after the arrival of an action potential and the stochastic opening of a calcium channel, determine the probability of exocytosis from docked vesicles near the channel. The synaptic vesicles in active zones are found docked in a complex with their calcium-sensor associated proteins and a voltage-sensitive calcium channel, forming a secretory unit. The probability of quantal secretion from an isolated secretory unit has been determined for different distances of an open calcium channel from the calcium sensor within an individual unit: a threefold decrease in the probability of secretion of a quantum occurs with a doubling of the distance from 25 to 50 nm. The Monte Carlo analysis also shows that the probability of secretion of a quantum is most sensitive to the size of the single-channel current compared with its sensitivity to either the binding rates of the sites on the calcium-sensor protein or to the number of these sites that must bind a calcium ion to trigger exocytosis of a vesicle. PMID:10777721

Ion channel recordings on an injection-molded polymer chip.

PubMed

Tanzi, Simone; Matteucci, Marco; Christiansen, Thomas Lehrmann; Friis, Søren; Christensen, Mette Thylstrup; Garnaes, Joergen; Wilson, Sandra; Kutchinsky, Jonatan; Taboryski, Rafael

2013-12-21

In this paper, we demonstrate recordings of the ion channel activity across the cell membrane in a biological cell by employing the so-called patch clamping technique on an injection-molded polymer microfluidic device. The findings will allow direct recordings of ion channel activity to be made using the cheapest materials and production platform to date and with the potential for very high throughput. The employment of cornered apertures for cell capture allowed the fabrication of devices without through holes and via a scheme comprising master origination by dry etching in a silicon substrate, electroplating in nickel and injection molding of the final part. The most critical device parameters were identified as the length of the patching capillary and the very low surface roughness on the inside of the capillary. The cross-sectional shape of the orifice was found to be less critical, as both rectangular and semicircular profiles seemed to have almost the same ability to form tight seals with cells with negligible leak currents. The devices were functionally tested using human embryonic kidney cells expressing voltage-gated sodium channels (Nav1.7) and benchmarked against a commercial state-of-the-art system for automated ion channel recordings. These experiments considered current-voltage (IV) relationships for activation and inactivation of the Nav1.7 channels and their sensitivity to a local anesthetic, lidocaine. Both IVs and lidocaine dose-response curves obtained from the injection-molded polymer device were in good agreement with data obtained from the commercial system.

Molecular cloning of ion channels in Felis catus that are related to periodic paralyses in man: a contribution to the understanding of the genetic susceptibility to feline neck ventroflexion and paralysis

PubMed Central

Zapata, Marlyn; Kunii, Ilda S.; Paninka, Rolf M.; Simões, Denise M. N.; Castillo, Víctor A.; Reche, Archivaldo; Maciel, Rui M. B.; Dias da Silva, Magnus R.

2014-01-01

ABSTRACT Neck ventroflexion in cats has different causes; however, the most common is the hypokalemia associated with flaccid paralysis secondary to chronic renal failure. In humans, the most common causes of acute flaccid paralysis are hypokalemia precipitated by thyrotoxicosis and familial forms linked to mutations in sodium, potassium, and calcium channel genes. Here, we describe the sequencing and analysis of skeletal muscle ion channels in Felis catus that could be related to periodic paralyses in humans, contributing to the understanding of the genetic susceptibility to feline neck ventroflexion and paralysis. We studied genomic DNA from eleven cats, including five animals that were hyperthyroid with hypokalemia, although only one presented with muscle weakness, and six healthy control domestic cats. We identified the ion channel ortholog genes KCNJ2, KCNJ12, KCNJ14, CACNA1S and SCN4A in the Felis catus genome, together with several polymorphic variants. Upon comparative alignment with other genomes, we found that Felis catus provides evidence for a high genomic conservation of ion channel sequences. Although we hypothesized that neck ventroflexion in cats could be associated with a thyrotoxic or familial periodic paralysis channel mutation, we did not identify any previously detected human channel mutation in the hyperthyroid cat presenting hypokalemia. However, based on the small number of affected cats in this study, we cannot yet rule out this molecular mechanism. Notwithstanding, hyperthyroidism should still be considered as a differential diagnosis in hypokalemic feline paralysis. PMID:25063199

Phosphate barrier on pore-filled cation-exchange membrane for blocking complexing ions in presence of non-complexing ions

NASA Astrophysics Data System (ADS)

Chavan, Vivek; Agarwal, Chhavi; Shinde, Rakesh N.

2018-06-01

In present work, an approach has been used to form a phosphate groups bearing surface barrier on a cation-exchange membrane (CEM). Using optimized conditions, the phosphate bearing monomer bis[2-(methacryloyloxy)ethyl] phosphate has been grafted on the surface of the host poly(ethersulfone) membranes using UV light induced polymerization. The detailed characterizations have shown that less than a micron layer of phosphate barrier is formed without disturbing the original microporous structure of the host membrane. The pores of thus formed membrane have been blocked by cationic-gel formed by in situ UV-initiator induced polymerization of 2-acrylamido-2-methyl-1-propane sulphonic acid along with crosslinker ethylene glycol dimethacrylate in the pores of the membrane. UV-initiator is required for pore-filling as UV light would not penetrate the interior matrix of the membrane. The phosphate functionalized barrier membrane has been examined for permselectivity using a mixture of representative complexing Am3+ ions and non-complexing Cs+ ions. This experiment has demonstrated that complex forming Am3+ ions are blocked by phosphate barrier layer while non-complexing Cs+ ions are allowed to pass through the channels formed by the crosslinked cationic gel.

Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution

PubMed Central

Wu, Jianping; Li, Zhangqiang; Xie, Tian; Peng, Wei; Yin, Changcheng; Li, Xueming; Scheres, Sjors H.W.; Shi, Yigong; Yan, Nieng

2014-01-01

The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryo-microscopy. Three previously uncharacterized domains, named Central, Handle, and Helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the Central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities. PMID:25517095

Plant Ion Channels: Gene Families, Physiology, and Functional Genomics Analyses

PubMed Central

Ward, John M.; Mäser, Pascal; Schroeder, Julian I.

2016-01-01

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization-and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide–gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport. PMID:18842100

Plant ion channels: gene families, physiology, and functional genomics analyses.

PubMed

Ward, John M; Mäser, Pascal; Schroeder, Julian I

2009-01-01

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

BIOLOGICAL AND BIOPHYSICAL PROPERTIES OF VASCULAR CONNEXIN CHANNELS

PubMed Central

Johnstone, Scott; Isakson, Brant; Locke, Darren

2010-01-01

Intercellular channels formed by connexin proteins play a pivotal role in the direct movement of ions and larger cytoplasmic solutes between vascular endothelial cells, between vascular smooth muscle cells, and between endothelial and smooth muscle cells. Multiple genetic and epigenetic factors modulate connexin expression levels and/or channel function, including cell type-independent and cell type-specific transcription factors, posttranslational modification and localized membrane targeting. Additionally, differences in protein-protein interactions, including those between connexins, significantly contribute to both vascular homeostasis and disease progression. The biophysical properties of the connexin channels identified in the vasculature, those formed by Cx37, Cx40, Cx43 and/or Cx45 proteins, are discussed in this review in the physiological and pathophysiological context of vessel function. PMID:19815177

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

PubMed

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

2001-08-14

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

Investigation of MeV-Cu implantation and channeling effects into porous silicon formation

NASA Astrophysics Data System (ADS)

Ahmad, M.; Naddaf, M.

2011-11-01

P-type (1 1 1) silicon wafers were implanted by copper ions (2.5 MeV) in channeling and random directions using ion beam accelerator of the Atomic Energy Commission of Syria (AECS). The effect of implantation direction on formation process of porous silicon (PS) using electrochemical etching method has been investigated using scanning electron microscope (SEM) and photoluminescence (PL) techniques. SEM observations revealed that the size, shape and density of the formed pores are highly affected by the direction of beam implantation. This in turn is seen to influence the PL behavior of the PS.

Modeling and simulation of Li-ion conduction in poly(ethylene oxide)

NASA Astrophysics Data System (ADS)

Gitelman, L.; Israeli, M.; Averbuch, A.; Nathan, M.; Schuss, Z.; Golodnitsky, D.

2007-12-01

Polyethylene oxide (PEO) containing a lithium salt (e.g., LiI) serves as a solid polymer electrolyte (SPE) in thin-film batteries and its ionic conductivity is a key parameter of their performance. We model and simulate Li + ion conduction in a single PEO molecule. Our simplified stochastic model of ionic motion is based on an analogy between protein channels of biological membranes that conduct Na +, K +, and other ions, and the PEO helical chain that conducts Li + ions. In contrast with protein channels and salt solutions, the PEO is both the channel and the solvent for the lithium salt (e.g., LiI). The mobile ions are treated as charged spherical Brownian particles. We simulate Smoluchowski dynamics in channels with a radius of ca. 0.1 nm and study the effect of stretching and temperature on ion conductivity. We assume that each helix (molecule) forms a random angle with the axis between these electrodes and the polymeric film is composed of many uniformly distributed oriented boxes that include molecules with the same direction. We further assume that mechanical stretching aligns the molecular structures in each box along the axis of stretching (intra-box alignment). Our model thus predicts the PEO conductivity as a function of the stretching, the salt concentration and the temperature. The computed enhancement of the ionic conductivity in the stretch direction is in good agreement with experimental results. The simulation results are also in qualitative agreement with recent theoretical and experimental results.

Molecular basis of proton block of L-type Ca2+ channels.

PubMed

Chen, X H; Bezprozvanny, I; Tsien, R W

1996-11-01

Hydrogen ions are important regulators of ion flux through voltage-gated Ca2+ channels but their site of action has been controversial. To identify molecular determinants of proton block of L-type Ca2+ channels, we combined site-directed mutagenesis and unitary current recordings from wild-type (WT) and mutant L-type Ca2+ channels expressed in Xenopus oocytes. WT channels in 150 mM K+ displayed two conductance states, deprotonated (140 pS) and protonated (45 pS), as found previously in native L-type Ca2+ channels. Proton block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-EIV) that form the locus of high affinity Ca2+ interaction. Glu(E)-->Gln(Q) substitution in either repeats I or III abolished the high-conductance state, as if the titration site had become permanently protonated. While the EIQ mutant displayed only an approximately 40 pS conductance, the EIIIQ mutant showed the approximately 40 pS conductance plus additional pH-sensitive transitions to an even lower conductance level. The EIVQ mutant exhibited the same deprotonated and protonated conductance states as WT, but with an accelerated rate of deprotonation. The EIIQ mutant was unusual in exhibiting three conductance states (approximately 145, 102, 50 pS, respectively). Occupancy of the low conductance state increased with external acidification, albeit much higher proton concentration was required than for WT. In contrast, the equilibrium between medium and high conductance levels was apparently pH-insensitive. We concluded that the protonation site in L-type Ca2+ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats I, II, and III, acting in concert. EIV lies to the cytoplasmic side of the site but exerts an additional stabilizing influence on protonation, most likely via electrostatic interaction. These findings are likely to hold for all voltage-gated Ca2+ channels and provide a simple molecular explanation for the modulatory effect of H+ ions on open channel flux and the competition between H+ ions and permeant divalent cations. The characteristics of H+ interactions advanced our picture of the functional interplay between P-region glutamates, with important implications for the mechanism of Ca2+ selectivity and permeation.

Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

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

Suzuki, Nobuhiro; Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602; Yamazaki, Yasuo

2008-10-01

The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinitiesmore » on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.« less

Voltage Gated Ion Channel Function: Gating, Conduction, and the Role of Water and Protons

PubMed Central

Kariev, Alisher M.; Green, Michael E.

2012-01-01

Ion channels, which are found in every biological cell, regulate the concentration of electrolytes, and are responsible for multiple biological functions, including in particular the propagation of nerve impulses. The channels with the latter function are gated (opened) by a voltage signal, which allows Na+ into the cell and K+ out. These channels have several positively charged amino acids on a transmembrane domain of their voltage sensor, and it is generally considered, based primarily on two lines of experimental evidence, that these charges move with respect to the membrane to open the channel. At least three forms of motion, with greatly differing extents and mechanisms of motion, have been proposed. There is a “gating current”, a capacitative current preceding the channel opening, that corresponds to several charges (for one class of channel typically 12–13) crossing the membrane field, which may not require protein physically crossing a large fraction of the membrane. The coupling to the opening of the channel would in these models depend on the motion. The conduction itself is usually assumed to require the “gate” of the channel to be pulled apart to allow ions to enter as a section of the protein partially crosses the membrane, and a selectivity filter at the opposite end of the channel determines the ion which is allowed to pass through. We will here primarily consider K+ channels, although Na+ channels are similar. We propose that the mechanism of gating differs from that which is generally accepted, in that the positively charged residues need not move (there may be some motion, but not as gating current). Instead, protons may constitute the gating current, causing the gate to open; opening consists of only increasing the diameter at the gate from approximately 6 Å to approximately 12 Å. We propose in addition that the gate oscillates rather than simply opens, and the ion experiences a barrier to its motion across the channel that is tuned by the water present within the channel. Our own quantum calculations as well as numerous experiments of others are interpreted in terms of this hypothesis. It is also shown that the evidence that supports the motion of the sensor as the gating current can also be consistent with the hypothesis we present. PMID:22408417

Homology Model of the GABAA Receptor Examined Using Brownian Dynamics

PubMed Central

O'Mara, Megan; Cromer, Brett; Parker, Michael; Chung, Shin-Ho

2005-01-01

We have developed a homology model of the GABAA receptor, using the subunit combination of α1β2γ2, the most prevalent type in the mammalian brain. The model is produced in two parts: the membrane-embedded channel domain and the extracellular N-terminal domain. The pentameric transmembrane domain model is built by modeling each subunit by homology with the equivalent subunit of the heteropentameric acetylcholine receptor transmembrane domain. This segment is then joined with the extracellular domain built by homology with the acetylcholine binding protein. The all-atom model forms a wide extracellular vestibule that is connected to an oval chamber near the external surface of the membrane. A narrow, cylindrical transmembrane channel links the outer segment of the pore to a shallow intracellular vestibule. The physiological properties of the model so constructed are examined using electrostatic calculations and Brownian dynamics simulations. A deep energy well of ∼80 kT accommodates three Cl− ions in the narrow transmembrane channel and seven Cl− ions in the external vestibule. Inward permeation takes place when one of the ions queued in the external vestibule enters the narrow segment and ejects the innermost ion. The model, when incorporated into Brownian dynamics, reproduces key experimental features, such as the single-channel current-voltage-concentration profiles. Finally, we simulate the γ2 K289M epilepsy inducing mutation and examine Cl− ion permeation through the mutant receptor. PMID:15749776

Compounds having aromatic rings and side-chain amide-functionality and a method for transporting monovalent anions across biological membranes using the same

DOEpatents

Davis, Jeffery T [College Park, MD; Sidorov, Vladimir [Richmond, VA; Kotch, Frank W [New Phila., PA

2008-04-08

A compound containing at least two aromatic rings covalently bonded together, with each aromatic ring containing at least one oxyacetamide-based side chain, the compound being capable of forming a chloride ion channel across a lipid bilayer, and transporting chloride ion across the lipid bilayer.

Free RCK arrangement in Kch, a putative escherichia coli potassium channel, as suggested by electron crystallography.

PubMed

Kuang, Qie; Purhonen, Pasi; Jegerschöld, Caroline; Koeck, Philip J B; Hebert, Hans

2015-01-06

The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bilayer lipid composition modulates the activity of dermaseptins, polycationic antimicrobial peptides.

PubMed

Duclohier, Hervé

2006-05-01

The primary targets of defense peptides are plasma membranes, and the induced irreversible depolarization is sufficient to exert antimicrobial activity although secondary modes of action might be at work. Channels or pores underlying membrane permeabilization are usually quite large with single-channel conductances two orders of magnitude higher than those exhibited by physiological channels involved, e.g., in excitability. Accordingly, the ion specificity and selectivity are quite low. Whereas, e.g., peptaibols favor cation transport, polycationic or basic peptides tend to form anion-specific pores. With dermaseptin B2, a 33 residue long and mostly alpha-helical peptide isolated from the skin of the South American frog Phyllomedusa bicolor, we found that the ion specificity of its pores induced in bilayers is modulated by phospholipid-charged headgroups. This suggests mixed lipid-peptide pore lining instead of the more classical barrel-stave model. Macroscopic conductance is nearly voltage independent, and concentration dependence suggests that the pores are mainly formed by dermaseptin tetramers. The two most probable single-channel events are well resolved at 200 and 500 pS (in 150 mM NaCl) with occasional other equally spaced higher or lower levels. In contrast to previous molecular dynamics previsions, this study demonstrates that dermaseptins are able to form pores, although a related analog (B6) failed to induce any significant conductance. Finally, the model of the pore we present accounts for phospholipid headgroups intercalated between peptide helices lining the pore and for one of the most probable single-channel conductance.

Gradient isolator for flow field of fuel cell assembly

DOEpatents

Ernst, W.D.

1999-06-15

Isolator(s) include isolating material and optionally gasketing material strategically positioned within a fuel cell assembly. The isolating material is disposed between a solid electrolyte and a metal flow field plate. Reactant fluid carried by flow field plate channel(s) forms a generally transverse electrochemical gradient. The isolator(s) serve to isolate electrochemically a portion of the flow field plate, for example, transversely outward from the channel(s), from the electrochemical gradient. Further, the isolator(s) serve to protect a portion of the solid electrolyte from metallic ions. 4 figs.

Gradient isolator for flow field of fuel cell assembly

DOEpatents

Ernst, William D.

1999-01-01

Isolator(s) include isolating material and optionally gasketing material strategically positioned within a fuel cell assembly. The isolating material is disposed between a solid electrolyte and a metal flow field plate. Reactant fluid carried by flow field plate channel(s) forms a generally transverse electrochemical gradient. The isolator(s) serve to isolate electrochemically a portion of the flow field plate, for example, transversely outward from the channel(s), from the electrochemical gradient. Further, the isolator(s) serve to protect a portion of the solid electrolyte from metallic ions.

Properties of a Novel Ion-Exchange Film

NASA Technical Reports Server (NTRS)

Street, Kenneth W.; Hill, Carol M.; Philipp, Warren H.; Tanner, Stephen P.; Gorse, Joseph; Lusk, Amy; Taylor, Jason; Dickens, Jason

2002-01-01

A new ion-exchange material (based on polyacrylic acid) and some of its analytical applications have been reported. This paper contains data on the ion-exchange properties of the film form of the material and its potential application to the decontamination of waste water and drinking water. The film has a high exchange capacity of 5 to 6 meq/g and a pK(sub a) of 5.7. The calcium form is the most effective for removing metal ions from solution, and the optimum pH range is between 5 and 7. The exchange rates are slower for the film than for bead and powder forms of the ion-exchange material; otherwise, the properties are similar. The film is effective when hard water solutions are employed and also when metal ions are in the complex matrix of waste water from electroplating. The film can be used in flow systems having a flow channel large enough to allow passage of turbid solutions.

Properties of a Novel Ion-Exchange Film

NASA Technical Reports Server (NTRS)

Street, Kenneth W.; Hill, Carol M.; Philipp, Warren H.; Tanner, Stephen P.; Gorse, Joseph; Lusk, Amy; Taylor, Jason; Dickens, Jason

2004-01-01

A new ion-exchange material (based on polyacrylic acid) and some of its analytical applications have been reported. This paper contains data on the ion-exchange properties of the film form of the material and its potential application to the decontamination of waste water and drinking water. The film has a high exchange capacity of 5 to 6 meq/g and a pK(sub a) of 5.7. The calcium form is the most effective for removing metal ions from solution, and the optimum pH range is between 5 and 7. The exchange rates are slower for the film than for bead and powder forms of the ion-exchange material; otherwise, the properties are similar. The film is effective when hard water solutions are employed and also when metal ions are in the complex matrix of waste water from electroplating. The film can be used in flow systems having a flow channel large enough to allow passage of turbid solutions.

Nonlinear electronic stopping power of channeled slow light ions in ZnSe: Evidence of energy loss caused by formation and breaking of chemical bond

NASA Astrophysics Data System (ADS)

Li, Chang-kai; Wang, Feng; Gao, Cong-Zhang; Liao, Bin; Ouyang, Xiao-ping; Zhang, Feng-Shou

2018-07-01

Electronic stopping power of helium ions in a semiconductor material ZnSe has been investigated through non-adiabatic dynamics simulations at energies of a few keV under channeling condition. The stopping power is predicted to be proportional to velocity for the trajectory along middle axis of a 〈 1 1 0 〉 channel, as expected for the linear response theory accounts for election-hole pair creation. While for the off-center channeling trajectory, a counterintuitive of electronic stopping power versus velocity is observed. Our study, presented herein, finds a non-trivial connection between charge transfer and the force experienced by the projectile. Charge transfer can produce, throughout the collision process, additional force by continuously forming and breaking instantaneous chemical bonds between the projectile and the neighboring host atoms.

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

PubMed Central

Siebert, Adam P.; Ma, Zhongming; Grevet, Jeremy D.; Demuro, Angelo; Parker, Ian; Foskett, J. Kevin

2013-01-01

CALHM1 (calcium homeostasis modulator 1) forms a plasma membrane ion channel that mediates neuronal excitability in response to changes in extracellular Ca2+ concentration. Six human CALHM homologs exist with no homology to other proteins, although CALHM1 is conserved across >20 species. Here we demonstrate that CALHM1 shares functional and quaternary and secondary structural similarities with connexins and evolutionarily distinct innexins and their vertebrate pannexin homologs. A CALHM1 channel is a hexamer, comprised of six monomers, each of which possesses four transmembrane domains, cytoplasmic amino and carboxyl termini, an amino-terminal helix, and conserved extracellular cysteines. The estimated pore diameter of the CALHM1 channel is ∼14 Å, enabling permeation of large charged molecules. Thus, CALHMs, connexins, and pannexins and innexins are structurally related protein families with shared and distinct functional properties. PMID:23300080

Ion mobility spectrometer using frequency-domain separation

DOEpatents

Martin, Stephen J.; Butler, Michael A.; Frye, Gregory C.; Schubert, W. Kent

1998-01-01

An apparatus and method is provided for separating and analyzing chemical species in an ion mobility spectrometer using a frequency-domain technique wherein the ions generated from the chemical species are selectively transported through an ion flow channel having a moving electrical potential therein. The moving electrical potential allows the ions to be selected according to ion mobility, with certain of the ions being transported to an ion detector and other of the ions being effectively discriminated against. The apparatus and method have applications for sensitive chemical detection and analysis for monitoring of exhaust gases, hazardous waste sites, industrial processes, aerospace systems, non-proliferation, and treaty verification. The apparatus can be formed as a microelectromechanical device (i.e. a micromachine).

Quartz antenna with hollow conductor

DOEpatents

Leung, Ka-Ngo; Benabou, Elie

2002-01-01

A radio frequency (RF) antenna for plasma ion sources is formed of a hollow metal conductor tube disposed within a glass tube. The hollow metal tubular conductor has an internal flow channel so that there will be no coolant leakage if the outer glass tube of the antenna breaks. A portion of the RF antenna is formed into a coil; the antenna is used for inductively coupling RF power to a plasma in an ion source chamber. The antenna is made by first inserting the metal tube inside the glass tube, and then forming the glass/metal composite tube into the desired coil shape.

An ion channel library for drug discovery and safety screening on automated platforms.

PubMed

Wible, Barbara A; Kuryshev, Yuri A; Smith, Stephen S; Liu, Zhiqi; Brown, Arthur M

2008-12-01

Ion channels represent the third largest class of targets in drug discovery after G-protein coupled receptors and kinases. In spite of this ranking, ion channels continue to be under exploited as drug targets compared with the other two groups for several reasons. First, with 400 ion channel genes and an even greater number of functional channels due to mixing and matching of individual subunits, a systematic collection of ion channel-expressing cell lines for drug discovery and safety screening has not been available. Second, the lack of high-throughput functional assays for ion channels has limited their use as drug targets. Now that automated electrophysiology has come of age and provided the technology to assay ion channels at medium to high throughput, we have addressed the need for a library of ion channel cell lines by constructing the Ion Channel Panel (ChanTest Corp., Cleveland, OH). From 400 ion channel genes, a collection of 82 of the most relevant human ion channels for drug discovery, safety, and human disease has been assembled.Each channel has been stably overexpressed in human embryonic kidney 293 or Chinese hamster ovary cells. Cell lines have been selected and validated on automated electrophysiology systems to facilitate cost-effective screening for safe and selective compounds at earlier stages in the drug development process. The screening and validation processes as well as the relative advantages of different screening platforms are discussed.

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

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

Derebe, Mehabaw G.; Sauer, David B.; Zeng, Weizhong

2015-11-30

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K{sup +} channel selectivity filters. Combined with single channel electrophysiology, we show that only themore » channel with four ion binding sites is K{sup +} selective, whereas those with two or three are nonselective and permeate Na{sup +} and K{sup +} equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K{sup +} channels is essential for highly selective and efficient permeation of K{sup +} ions.« less

An embryo of protocells: The capsule of graphene with selective ion channels

PubMed Central

Li, Zhan; Wang, Chunmei; Tian, Longlong; Bai, Jing; Yao, Huijun; Zhao, Yang; Zhang, Xin; Cao, Shiwei; Qi, Wei; Wang, Suomin; Shi, Keliang; Xu, Youwen; Mingliang, Zhang; Liu, Bo; Qiu, Hongdeng; Liu, Jie; Wu, Wangsuo; Wang, Xiaoli; Wenzhen, An

2015-01-01

The synthesis of artificial cell is a route for searching the origin of protocell. Here, we create a novel cell model of graphene capsules with selective ion channels, indicating that graphene might be an embryo of protocell membrane. Firstly, we found that the highly oxidized graphene and phospholipid-graphene oxide composite would curl into capsules under a strongly acidic saturated solution of heavy metallic salt solution at low temperature. Secondly, L-amino acids exhibited higher reactivity than D-amino acids on graphene oxides to form peptides, and the formed peptides in the influence of graphene would be transformed into a secondary structure, promoting the formation of left-handed proteins. Lastly, monolayer nanoporous graphene, prepared by unfocused 84Kr25+, has a high selectivity for permeation of the monovalent metal ions ( Rb+ > K+ > Cs+ > Na+ > Li+, based on permeation concentration), but does not allow Cl- go through. It is similar to K+ channels, which would cause an influx of K+ into capsule of graphene with the increase of pH in the primitive ocean, creating a suitable inner condition for the origin of life. Therefore, we built a model cell of graphene, which would provide a route for reproducing the origin of life. PMID:25989440

An embryo of protocells: The capsule of graphene with selective ion channels

DOE PAGES

Li, Zhan; Wang, Chunmei; Tian, Longlong; ...

2015-05-19

In this study, the synthesis of artificial cell is a route for searching the origin of protocell. Here, we create a novel cell model of graphene capsules with selective ion channels, indicating that graphene might be an embryo of protocell membrane. Firstly, we found that the highly oxidized graphene and phospholipid-graphene oxide composite would curl into capsules under a strongly acidic saturated solution of heavy metallic salt solution at low temperature. Secondly, L-amino acids exhibited higher reactivity than D-amino acids on graphene oxides to form peptides, and the formed peptides in the influence of graphene would be transformed into amore » secondary structure, promoting the formation of left-handed proteins. Lastly, monolayer nanoporous graphene, prepared by unfocused 84Kr 25+, has a high selectivity for permeation of the monovalent metal ions ( Rb + > K + > Cs + > Na + > Li +, based on permeation concentration), but does not allow Cl- go through. It is similar to K+ channels, which would cause an influx of K + into capsule of graphene with the increase of pH in the primitive ocean, creating a suitable inner condition for the origin of life. Therefore, we built a model cell of graphene, which would provide a route for reproducing the origin of life.« less

Chemotherapy drugs form ion pores in membranes due to physical interactions with lipids.

PubMed

Ashrafuzzaman, Mohammad; Tseng, Chih-Yuan; Duszyk, Marek; Tuszynski, Jack A

2012-12-01

We demonstrate the effects on membrane of the tubulin-binding chemotherapy drugs: thiocolchicoside and taxol. Electrophysiology recordings across lipid membranes in aqueous phases containing drugs were used to investigate the drug effects on membrane conductance. Molecular dynamics simulation of the chemotherapy drug-lipid complexes was used to elucidate the mechanism at an atomistic level. Both drugs are observed to induce stable ion-flowing pores across membranes. Discrete pore current-time plots exhibit triangular conductance events in contrast to rectangular ones found for ion channels. Molecular dynamics simulations indicate that drugs and lipids experience electrostatic and van der Waals interactions for short periods of time when found within each other's proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides and lipids due to mainly their charge properties while forming peptide-induced ion channels in lipid bilayers. Experimental and in silico studies together suggest that the chemotherapy drugs induce ion pores inside lipid membranes due to drug-lipid physical interactions. The findings reveal cytotoxic effects of drugs on the cell membrane, which may aid in novel drug development for treatment of cancer and other diseases. © 2012 John Wiley & Sons A/S.

Poisson-Nernst-Planck-Fermi theory for modeling biological ion channels

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

Liu, Jinn-Liang, E-mail: jinnliu@mail.nhcue.edu.tw; Eisenberg, Bob, E-mail: beisenbe@rush.edu

2014-12-14

A Poisson-Nernst-Planck-Fermi (PNPF) theory is developed for studying ionic transport through biological ion channels. Our goal is to deal with the finite size of particle using a Fermi like distribution without calculating the forces between the particles, because they are both expensive and tricky to compute. We include the steric effect of ions and water molecules with nonuniform sizes and interstitial voids, the correlation effect of crowded ions with different valences, and the screening effect of water molecules in an inhomogeneous aqueous electrolyte. Including the finite volume of water and the voids between particles is an important new part ofmore » the theory presented here. Fermi like distributions of all particle species are derived from the volume exclusion of classical particles. Volume exclusion and the resulting saturation phenomena are especially important to describe the binding and permeation mechanisms of ions in a narrow channel pore. The Gibbs free energy of the Fermi distribution reduces to that of a Boltzmann distribution when these effects are not considered. The classical Gibbs entropy is extended to a new entropy form — called Gibbs-Fermi entropy — that describes mixing configurations of all finite size particles and voids in a thermodynamic system where microstates do not have equal probabilities. The PNPF model describes the dynamic flow of ions, water molecules, as well as voids with electric fields and protein charges. The model also provides a quantitative mean-field description of the charge/space competition mechanism of particles within the highly charged and crowded channel pore. The PNPF results are in good accord with experimental currents recorded in a 10{sup 8}-fold range of Ca{sup 2+} concentrations. The results illustrate the anomalous mole fraction effect, a signature of L-type calcium channels. Moreover, numerical results concerning water density, dielectric permittivity, void volume, and steric energy provide useful details to study a variety of physical mechanisms ranging from binding, to permeation, blocking, flexibility, and charge/space competition of the channel.« less

Novel styrylbenzothiazolium dye-based sensor for mercury, cyanide and hydroxide ions

NASA Astrophysics Data System (ADS)

Gwon, Seon-Young; Rao, Boddu Ananda; Kim, Hak-Soo; Son, Young-A.; Kim, Sung-Hoon

2015-06-01

We report the design and synthesis of a novel styrylbenzothiazolium (3) derivative developed as a fluorescent and colorimetric chemodosimeter with high selectivity toward Hg2+, CN- and OH- ions. An obvious loss of pink color in the presence of Hg2+ and CN- ions could make it a suitable "naked eye" indicator. We propose a sensing mechanism whereby the benzenoid form is changed to a quinoid form upon Hg2+ binding in a 1:1 stoichiometric ratio. More significantly, the styrylbenzothiazolium-Hg2+ and styrylbenzothiazolium-CN- complexes exhibited a dual-channel chromo-fluorogenic response. The sensors exhibit remarkable Hg2+-, CN--, and OH--selective red fluorescence but remain dark-green in the presence of a wide range of tested metal ions and anions.

Ion channeling studies on mixed phases formed in metalorganic chemical vapor deposition grown Mg-doped GaN on Al2O3(0001)

NASA Astrophysics Data System (ADS)

Sundaravel, B.; Luo, E. Z.; Xu, J. B.; Wilson, I. H.; Fong, W. K.; Wang, L. S.; Surya, C.

2000-01-01

Rutherford backscattering spectrometry and ion channeling were used to determine the relative quantities of wurtzite and zinc-blende phases in metalorganic chemical vapor deposition grown Mg-doped GaN(0001) on an Al2O3(0001) substrate with a GaN buffer layer. Offnormal axial channeling scans were used. High-resolution x-ray diffraction measurements also confirmed the presence of mixed phases. The in-plane orientation was found to be GaN[11¯0]‖GaN[112¯0]‖Al2O3[112¯0]. The effects of rapid thermal annealing on the relative phase content, thickness and crystalline quality of the GaN epilayer were also studied.

Aspects of the homeostaic plasticity of GABAA receptor-mediated inhibition

PubMed Central

Mody, Istvan

2005-01-01

Plasticity of ligand-gated ion channels plays a critical role in nervous system development, circuit formation and refinement, and pathological processes. Recent advances have mainly focused on the plasticity of channels gated by excitatory amino acids, including their acclaimed role in learning and memory. These receptors, together with voltage-gated ion channels, have also been known to be subjected to a homeostatic form of plasticity that prevents destabilization of the neurone's function and that of the network during various physiological processes. To date, the plasticity of GABAA receptors has been examined mainly from a developmental and a pathological point of view. Little is known about homeostatic mechanisms governing their plasticity. This review summarizes some of the findings on the homeostatic plasticity of tonic and phasic inhibitory activity. PMID:15528237

Membranes with well-defined ions transport channels fabricated via solvent-responsive layer-by-layer assembly method for vanadium flow battery.

PubMed

Xu, Wanxing; Li, Xianfeng; Cao, Jingyu; Zhang, Hongzhang; Zhang, Huamin

2014-02-06

In this work we presented a general strategy for the fabrication of membranes with well-defined ions transport channels through solvent-responsive layer-by-layer assembly (SR-LBL). Multilayered poly (diallyldimethylammonium chloride) (PDDA) and poly (acrylic acid) (PAA) complexes were first introduced on the inner pore wall and the surface of sulfonated poly (ether ether ketone)/poly (ether sulfone) (PES/SPEEK) nanofiltration membranes to form ions transport channels with tuned radius. This type of membranes are highly efficient for the separators of batteries especially vanadium flow batteries (VFBs): the VFBs assembled with prepared membranes exhibit an outstanding performance in a wide current density range, which is much higher than that assembled with commercial Nafion 115 membranes. This idea could inspire the development of membranes for other flow battery systems, as well as create further progress in similar areas such as fuel cells, electro-dialysis, chlor-alkali cells, water electrolysis and so on.

Membranes with well-defined ions transport channels fabricated via solvent-responsive layer-by-layer assembly method for vanadium flow battery

PubMed Central

Xu, Wanxing; Li, Xianfeng; Cao, Jingyu; Zhang, Hongzhang; Zhang, Huamin

2014-01-01

In this work we presented a general strategy for the fabrication of membranes with well-defined ions transport channels through solvent-responsive layer-by-layer assembly (SR-LBL). Multilayered poly (diallyldimethylammonium chloride) (PDDA) and poly (acrylic acid) (PAA) complexes were first introduced on the inner pore wall and the surface of sulfonated poly (ether ether ketone)/poly (ether sulfone) (PES/SPEEK) nanofiltration membranes to form ions transport channels with tuned radius. This type of membranes are highly efficient for the separators of batteries especially vanadium flow batteries (VFBs): the VFBs assembled with prepared membranes exhibit an outstanding performance in a wide current density range, which is much higher than that assembled with commercial Nafion 115 membranes. This idea could inspire the development of membranes for other flow battery systems, as well as create further progress in similar areas such as fuel cells, electro-dialysis, chlor-alkali cells, water electrolysis and so on. PMID:24500376

Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles.

PubMed

Ding, Qiang; Heller, Brigitte; Capuccino, Juan M V; Song, Bokai; Nimgaonkar, Ila; Hrebikova, Gabriela; Contreras, Jorge E; Ploss, Alexander

2017-01-31

Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis globally. Of HEV's three ORFs, the function of ORF3 has remained elusive. Here, we demonstrate that via homophilic interactions ORF3 forms multimeric complexes associated with intracellular endoplasmic reticulum (ER)-derived membranes. HEV ORF3 shares several structural features with class I viroporins, and the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein. ORF3's ion channel function is further evidenced by its ability to mediate ionic currents when expressed in Xenopus laevis oocytes. Furthermore, we identified several positions in ORF3 critical for its formation of multimeric complexes, ion channel activity, and, ultimately, release of infectious particles. Collectively, our data demonstrate a previously undescribed function of HEV ORF3 as a viroporin, which may serve as an attractive target in developing direct-acting antivirals.

Sensing muscle ischemia: coincident detection of acid and ATP via interplay of two ion channels.

PubMed

Birdsong, William T; Fierro, Leonardo; Williams, Frank G; Spelta, Valeria; Naves, Ligia A; Knowles, Michelle; Marsh-Haffner, Josephine; Adelman, John P; Almers, Wolfhard; Elde, Robert P; McCleskey, Edwin W

2010-11-18

Ischemic pain--examples include the chest pain of a heart attack and the leg pain of a 30 s sprint--occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis. Copyright © 2010 Elsevier Inc. All rights reserved.

Iodine Plasma Species Measurements in a Hall Effect Thruster Plume

DTIC Science & Technology

2013-04-01

direction f = species fraction 0g = gravitational constant at Earth’s surface, 9.81 m/s 2 I = current, subscripts b for beam, c for cathode, d for...Hall effect thruster uses crossed electric and magnetic fields to generate and accelerate ions. The gas in the discharge is partially ionized, although...early 1960s.10 Ions are weakly magnetized and most are accelerated directly out of the channel, forming the ion beam. The bulk of the cathode

Comparative Study of the Energetics of Ion Permeation in Kv1.2 and KcsA Potassium Channels

PubMed Central

Baştuğ, Turgut; Kuyucak, Serdar

2011-01-01

Biological ion channels rely on a multi-ion transport mechanism for fast yet selective permeation of ions. The crystal structure of the KcsA potassium channel provided the first microscopic picture of this process. A similar mechanism is assumed to operate in all potassium channels, but the validity of this assumption has not been well investigated. Here, we examine the energetics of ion permeation in Shaker Kv1.2 and KcsA channels, which exemplify the six-transmembrane voltage-gated and two-transmembrane inward-rectifier channels. We study the feasibility of binding a third ion to the filter and the concerted motion of ions in the channel by constructing the potential of mean force for K+ ions in various configurations. For both channels, we find that a pair of K+ ions can move almost freely within the filter, but a relatively large free-energy barrier hinders the K+ ion from stepping outside the filter. We discuss the effect of the CMAP dihedral energy correction that was recently incorporated into the CHARMM force field on ion permeation dynamics. PMID:21281577

Signaling complexes of voltage-gated calcium channels

PubMed Central

Turner, Ray W; Anderson, Dustin

2011-01-01

Voltage-gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage-gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead form complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily. PMID:21832880

Structure of the TRPV1 ion channel determined by electron cryo-microscopy.

PubMed

Liao, Maofu; Cao, Erhu; Julius, David; Cheng, Yifan

2013-12-05

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5-6 (S5-S6) and the intervening pore loop, which is flanked by S1-S4 voltage-sensor-like domains. TRPV1 has a wide extracellular 'mouth' with a short selectivity filter. The conserved 'TRP domain' interacts with the S4-S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

Structure of the TRPV1 ion channel determined by electron cryo-microscopy

PubMed Central

Liao, Maofu; Cao, Erhu; Julius, David; Cheng, Yifan

2014-01-01

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here, we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane helices S5–S6 and the intervening pore loop, which is flanked by S1–S4 voltage sensor-like domains. TRPV1 has a wide extracellular ‘mouth’ with short selectivity filter. The conserved ‘TRP domain’ interacts with the S4–S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including N-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function. PMID:24305160

Energy variational analysis of ions in water and channels: Field theory for primitive models of complex ionic fluids

PubMed Central

Eisenberg, Bob; Hyon, YunKyong; Liu, Chun

2010-01-01

Ionic solutions are mixtures of interacting anions and cations. They hardly resemble dilute gases of uncharged noninteracting point particles described in elementary textbooks. Biological and electrochemical solutions have many components that interact strongly as they flow in concentrated environments near electrodes, ion channels, or active sites of enzymes. Interactions in concentrated environments help determine the characteristic properties of electrodes, enzymes, and ion channels. Flows are driven by a combination of electrical and chemical potentials that depend on the charges, concentrations, and sizes of all ions, not just the same type of ion. We use a variational method EnVarA (energy variational analysis) that combines Hamilton’s least action and Rayleigh’s dissipation principles to create a variational field theory that includes flow, friction, and complex structure with physical boundary conditions. EnVarA optimizes both the action integral functional of classical mechanics and the dissipation functional. These functionals can include entropy and dissipation as well as potential energy. The stationary point of the action is determined with respect to the trajectory of particles. The stationary point of the dissipation is determined with respect to rate functions (such as velocity). Both variations are written in one Eulerian (laboratory) framework. In variational analysis, an “extra layer” of mathematics is used to derive partial differential equations. Energies and dissipations of different components are combined in EnVarA and Euler–Lagrange equations are then derived. These partial differential equations are the unique consequence of the contributions of individual components. The form and parameters of the partial differential equations are determined by algebra without additional physical content or assumptions. The partial differential equations of mixtures automatically combine physical properties of individual (unmixed) components. If a new component is added to the energy or dissipation, the Euler–Lagrange equations change form and interaction terms appear without additional adjustable parameters. EnVarA has previously been used to compute properties of liquid crystals, polymer fluids, and electrorheological fluids containing solid balls and charged oil droplets that fission and fuse. Here we apply EnVarA to the primitive model of electrolytes in which ions are spheres in a frictional dielectric. The resulting Euler–Lagrange equations include electrostatics and diffusion and friction. They are a time dependent generalization of the Poisson–Nernst–Planck equations of semiconductors, electrochemistry, and molecular biophysics. They include the finite diameter of ions. The EnVarA treatment is applied to ions next to a charged wall, where layering is observed. Applied to an ion channel, EnVarA calculates a quick transient pile-up of electric charge, transient and steady flow through the channel, stationary “binding” in the channel, and the eventual accumulation of salts in “unstirred layers” near channels. EnVarA treats electrolytes in a unified way as complex rather than simple fluids. Ad hoc descriptions of interactions and flow have been used in many areas of science to deal with the nonideal properties of electrolytes. It seems likely that the variational treatment can simplify, unify, and perhaps derive and improve those descriptions. PMID:20849161

Dynamic ion-ion and water-ion interactions in ion channels.

PubMed Central

Wu, J V

1992-01-01

The dynamic interactions among ions and water molecules in ion channels are treated based on an assumption that ions at binding sites can be knocked off by both transient entering ions and local water molecules. The theory, when applied to a single-site model K+ channel, provides solutions for super- and subsaturations, flux-ratio exponent (n') greater than 1, osmotic streaming current, activity-dependent reversal potentials, and anomalous mole-fraction behavior. The analysis predicts that: (a) the saturation may but, in general, does not follow the Michaelis-Menten relation; (b) streaming current results from imbalanced water-ion knock-off interactions; (c) n' greater than 1 even for single-site channels, but it is unlikely to exceed 1.4 unless the pore is occupied by one or more ion(s); (d) in the calculation involving two permeant ion species with similar radii, the heavier ions show higher affinity; the ion-ion knock-off dissociation from the site is more effective when two interacting ions are identical. Therefore, the "multi-ion behaviors" found in most ion channels are the consequences of dynamic ion-ion and water-ion interactions. The presence of these interactions does not require two or more binding sites in channels. PMID:1376158

The Control of Male Fertility by Spermatozoan Ion Channels

PubMed Central

Lishko, Polina V.; Kirichok, Yuriy; Ren, Dejian; Navarro, Betsy; Chung, Jean-Ju

2014-01-01

Ion channels control the sperm ability to fertilize the egg by regulating sperm maturation in the female reproductive tract and by triggering key sperm physiological responses required for successful fertilization such as hyperactivated motility, chemotaxis, and the acrosome reaction. CatSper, a pH-regulated, calcium-selective ion channel, and KSper (Slo3) are core regulators of sperm tail calcium entry and sperm hyperactivated motility. Many other channels had been proposed as regulating sperm activity without direct measurements. With the development of the sperm patch-clamp technique, CatSper and KSper have been confirmed as the primary spermatozoan ion channels. In addition, the voltage-gated proton channel Hv1 has been identified in human sperm tail, and the P2X2 ion channel has been identified in the midpiece of mouse sperm. Mutations and deletions in sperm-specific ion channels affect male fertility in both mice and humans without affecting other physiological functions. The uniqueness of sperm ion channels makes them ideal pharmaceutical targets for contraception. In this review we discuss how ion channels regulate sperm physiology. PMID:22017176

Phosphorylation-Dependent Regulation of Ryanodine Receptors

PubMed Central

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function. PMID:11352932

Immunohistochemical and in situ mRNA hybridisation techniques to determine the distribution of ion channels in human brain: a study of neuronal voltage-dependent calcium channels.

PubMed

McCormack, A L; Day, N C; Craig, P J; Smith, W; Beattie, R E; Volsen, S G

1997-08-01

The molecular, structural and functional characterisation of ion channels in the CNS forms an area of intense investigation in current brain research. For strategic and logistical reasons, rodents have historically been the species of choice for these studies. The examination of human CNS tissues generally presents the investigator with specific challenges that are often less problematic in animal studies, e.g. post-mortem delay/agonal status, and thus both the experimental design and techniques must be manipulated accordingly. Since much pharmaceutical interest is currently focused on neuronal ion channels, the examination of their expression in human brain material is of particular importance. We describe here the details of methods that we have developed and used successfully in the study of the expression of voltage-dependent calcium channels (VDCCs) in human CNS tissues. Presynaptic neuronal VDCCs control neurotransmitter release and are important new drug targets. They are composed of three subunits, alpha 1, beta and alpha 2/delta and multiple gene classes of each protein have been identified. Little is known, however, about the distribution of neuronal VDCCs in the human central nervous system, although initial studies have been performed in rat and rabbit.

Ion channel blockers for the treatment of neuropathic pain.

PubMed

Colombo, Elena; Francisconi, Simona; Faravelli, Laura; Izzo, Emanuela; Pevarello, Paolo

2010-05-01

Neuropathic pain, a severe chronic pain condition characterized by a complex pathophysiology, is a largely unmet medical need. Ion channels, which underlie cell excitability, are heavily implicated in the biological mechanisms that generate and sustain neuropathic pain. This review highlights the biological evidence supporting the involvement of voltage-, proton- and ligand-gated ion channels in the neuropathic pain setting. Ion channel modulators at different research or development stages are reviewed and referenced. Ion channel modulation is one of the main avenues to achieve novel, improved neuropathic pain treatments. Voltage-gated sodium and calcium channel and glutamate receptor modulators are likely to produce new, improved agents in the future. Rationally targeting subtypes of known ion channels, tackling recently discovered ion channel targets or combining drugs with different mechanism of action will be primary sources of new drugs in the longer term.

Channel waveguides in glass via silver-sodium field-assisted ion exchange

NASA Technical Reports Server (NTRS)

Forrest, K.; Pagano, S. J.; Viehmann, W.

1986-01-01

Multimode channel waveguides have been formed in sodium aluminosilicate glass by field-assisted diffusion of Ag(+) ions from vacuum-evaporated Ag films. The two-dimensional refractive index profiles of the waveguides were controlled by varying the diffusion time, the diffusion temperature, and the electric field strength. Estimates of the diffusion rate through a strip aperture were obtained, assuming the electric field was strong 120-240 V/mm. The maximum change in refractive index in the sodium aluminosilicate glasses was estimated near 65 percent of the change in soda-lime silicate glass. The physical properties of the glasses are given in a table.

Murine epithelial cells: isolation and culture.

PubMed

Davidson, Donald J; Gray, Michael A; Kilanowski, Fiona M; Tarran, Robert; Randell, Scott H; Sheppard, David N; Argent, Barry E; Dorin, Julia R

2004-08-01

We describe an air-liquid interface primary culture method for murine tracheal epithelial cells on semi-permeable membranes, forming polarized epithelia with a high transepithelial resistance, differentiation to ciliated and secretory cells, and physiologically appropriate expression of key genes and ion channels. We also describe the isolation of primary murine nasal epithelial cells for patch-clamp analysis, generating polarised cells with physiologically appropriate distribution and ion channel expression. These methods enable more physiologically relevant analysis of murine airway epithelial cells in vitro and ex vivo, better utilisation of transgenic mouse models of human pulmonary diseases, and have been approved by the European Working Group on CFTR expression.

Non-synaptic ion channels in insects--basic properties of currents and their modulation in neurons and skeletal muscles.

PubMed

Wicher, D; Walther, C; Wicher, C

2001-08-01

Insects are favoured objects for studying information processing in restricted neuronal networks, e.g. motor pattern generation or sensory perception. The analysis of the underlying processes requires knowledge of the electrical properties of the cells involved. These properties are determined by the expression pattern of ionic channels and by the regulation of their function, e.g. by neuromodulators. We here review the presently available knowledge on insect non-synaptic ion channels and ionic currents in neurons and skeletal muscles. The first part of this article covers genetic and structural informations, the localization of channels, their electrophysiological and pharmacological properties, and known effects of second messengers and modulators such as neuropeptides or biogenic amines. In a second part we describe in detail modulation of ionic currents in three particularly well investigated preparations, i.e. Drosophila photoreceptor, cockroach DUM (dorsal unpaired median) neuron and locust jumping muscle. Ion channel structures are almost exclusively known for the fruitfly Drosophila, and most of the information on their function has also been obtained in this animal, mainly based on mutational analysis and investigation of heterologously expressed channels. Now the entire genome of Drosophila has been sequenced, it seems almost completely known which types of channel genes--and how many of them--exist in this animal. There is much knowledge of the various types of channels formed by 6-transmembrane--spanning segments (6TM channels) including those where four 6TM domains are joined within one large protein (e.g. classical Na+ channel). In comparison, two TM channels and 4TM (or tandem) channels so far have hardly been explored. There are, however, various well characterized ionic conductances, e.g. for Ca2+, Cl- or K+, in other insect preparations for which the channels are not yet known. In some of the larger insects, i.e. bee, cockroach, locust and moth, rather detailed information has been established on the role of ionic currents in certain physiological or behavioural contexts. On the whole, however, knowledge of non-synaptic ion channels in such insects is still fragmentary. Modulation of ion currents usually involves activation of more or less elaborate signal transduction cascades. The three detailed examples for modulation presented in the second part indicate, amongst other things, that one type of modulator usually leads to concerted changes of several ion currents and that the effects of different modulators in one type of cell may overlap. Modulators participate in the adaptive changes of the various cells responsible for different physiological or behavioural states. Further study of their effects on the single cell level should help to understand how small sets of cells cooperate in order to produce the appropriate output.

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress

PubMed Central

Lee, Chun Pong; Maksaev, Grigory; Jensen, Gregory S.; Murcha, Monika W.; Wilson, Margaret E.; Fricker, Mark; Hell, Ruediger; Haswell, Elizabeth S.; Millar, A. Harvey; Sweetlove, Lee

2016-01-01

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in E. coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing but is complementary and appears to be important under similar conditions. PMID:27505616

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress.

PubMed

Lee, Chun Pong; Maksaev, Grigory; Jensen, Gregory S; Murcha, Monika W; Wilson, Margaret E; Fricker, Mark; Hell, Ruediger; Haswell, Elizabeth S; Millar, A Harvey; Sweetlove, Lee J

2016-12-01

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Probing structural features of Alzheimer's amyloid-β pores in bilayers using site-specific amino acid substitutions.

PubMed

Capone, Ricardo; Jang, Hyunbum; Kotler, Samuel A; Kagan, Bruce L; Nussinov, Ruth; Lal, Ratnesh

2012-01-24

A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes.

Probing Structural Features of Alzheimer’s Amyloid-β Pores in Bilayers Using Site-Specific Amino Acid Substitutions

PubMed Central

2012-01-01

A current hypothesis for the pathology of Alzheimer’s disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ1–42 peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes. PMID:22242635

Monovalent Cation Permeation through the Connexin40 Gap Junction Channel

PubMed Central

Beblo, Dolores A.; Veenstra, Richard D.

1997-01-01

The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γj in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γj are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO3 (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li+ are: Cs+ (1.38), Rb+ (1.32), K+ (1.31), Na+ (1.16), TMA+ (0.53), TEA+ (0.45), TBA+ (0.03), Cl− (0.19), glutamate− (0.04), and NO3− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius. PMID:9101408

Measurement of Single Channel Currents from Cardiac Gap Junctions

NASA Astrophysics Data System (ADS)

Veenstra, Richard D.; Dehaan, Robert L.

1986-08-01

Cardiac gap junctions consist of arrays of integral membrane proteins joined across the intercellular cleft at points of cell-to-cell contact. These junctional proteins are thought to form pores through which ions can diffuse from cytosol to cytosol. By monitoring whole-cell currents in pairs of embryonic heart cells with two independent patch-clamp circuits, the properties of single gap junction channels have been investigated. These channels had a conductance of about 165 picosiemens and underwent spontaneous openings and closings that were independent of voltage. Channel activity and macroscopic junctional conductance were both decreased by the uncoupling agent 1-octanol.

Membraneless seawater desalination

DOEpatents

Crooks, Richard A.; Knust, Kyle N.; Perdue, Robbyn K.

2018-04-03

Disclosed are microfluidic devices and systems for the desalination of water. The devices and systems can include an electrode configured to generate an electric field gradient in proximity to an intersection formed by the divergence of two microfluidic channels from an inlet channel. Under an applied bias and in the presence of a pressure driven flow of saltwater, the electric field gradient can preferentially direct ions in saltwater into one of the diverging microfluidic channels, while desalted water flows into second diverging channel. Also provided are methods of using the devices and systems described herein to decrease the salinity of water.

Inhibition of Voltage-Gated K+ Channel Kv1.5 by Antiarrhythmic Drugs.

PubMed

Chen, Rong; Chung, Shin-Ho

2018-05-08

Molecular dynamics simulations are employed to determine the inhibitory mechanisms of three drugs, 5-(4-phenoxybutoxy)psoralen (PAP-1), vernakalant, and flecainide, on the voltage-gated K + channel Kv1.5, a target for the treatment of cardiac arrhythmia. At neutral pH, PAP-1 is neutral, whereas the other two molecules carry one positive charge. We show that PAP-1 forms stable dimers in water, primarily through hydrophobic interactions between aromatic rings. All three molecules bind to the cavity between the Ile508 and Val512 residues from the four subunits of the channel. Once bound, the drug molecules are flexible, with the average root-mean-square fluctuation being between 2 and 3 Å, which is larger than the radius of gyration of a bulky amino acid. The presence of a monomeric PAP-1 causes the permeating K + ion to dehydrate, thereby creating a significant energy barrier. In contrast, vernakalant blocks the ion permeation primarily via an electrostatic mechanism and, therefore, must be in the protonated and charged form to be effective.

Thermodynamic limitations on the temperature sensitivity of cell-membrane ion channels: Trouble with enthalpy uncertainty

NASA Astrophysics Data System (ADS)

Zheltikov, A. M.

2018-06-01

Energy exchange between a thermodynamic ensemble of heat- and cold-activated cell-membrane ion channels and the surrounding heat reservoir is shown to impose fundamental limitations on the performance of such channels as temperature-controlled gates for thermal cell activation. Analysis of unavoidable thermodynamic internal-energy fluctuations caused by energy exchange between the ion channels and the heat bath suggests that the resulting enthalpy uncertainty is too high for a robust ion-current gating by a single ion channel, implying that large ensembles of ion channels are needed for thermal cell activation. We argue, based on this thermodynamic analysis, that, had thermosensitive cell-membrane ion channels operated individually, rather than as large ensembles, robust thermal cell activation would have been impossible because of thermodynamic fluctuations.

Ion mobility spectrometer using frequency-domain separation

DOEpatents

Martin, S.J.; Butler, M.A.; Frye, G.C.; Schubert, W.K.

1998-08-04

An apparatus and method are provided for separating and analyzing chemical species in an ion mobility spectrometer using a frequency-domain technique wherein the ions generated from the chemical species are selectively transported through an ion flow channel having a moving electrical potential therein. The moving electrical potential allows the ions to be selected according to ion mobility, with certain of the ions being transported to an ion detector and other of the ions being effectively discriminated against. The apparatus and method have applications for sensitive chemical detection and analysis for monitoring of exhaust gases, hazardous waste sites, industrial processes, aerospace systems, non-proliferation, and treaty verification. The apparatus can be formed as a microelectromechanical device (i.e. a micromachine). 6 figs.

Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I.

PubMed

Kruse, Martin; Schulze-Bahr, Eric; Corfield, Valerie; Beckmann, Alf; Stallmeyer, Birgit; Kurtbay, Güven; Ohmert, Iris; Schulze-Bahr, Ellen; Brink, Paul; Pongs, Olaf

2009-09-01

Progressive familial heart block type I (PFHBI) is a progressive cardiac bundle branch disease in the His-Purkinje system that exhibits autosomal-dominant inheritance. In 3 branches of a large South African Afrikaner pedigree with an autosomal-dominant form of PFHBI, we identified the mutation c.19G-->A in the transient receptor potential cation channel, subfamily M, member 4 gene (TRPM4) at chromosomal locus 19q13.3. This mutation predicted the amino acid substitution p.E7K in the TRPM4 amino terminus. TRPM4 encodes a Ca2+-activated nonselective cation (CAN) channel that belongs to the transient receptor potential melastatin ion channel family. Quantitative analysis of TRPM4 mRNA content in human cardiac tissue showed the highest expression level in Purkinje fibers. Cellular expression studies showed that the c.19G-->A missense mutation attenuated deSUMOylation of the TRPM4 channel. The resulting constitutive SUMOylation of the mutant TRPM4 channel impaired endocytosis and led to elevated TRPM4 channel density at the cell surface. Our data therefore revealed a gain-of-function mechanism underlying this type of familial heart block.

Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I

PubMed Central

Kruse, Martin; Schulze-Bahr, Eric; Corfield, Valerie; Beckmann, Alf; Stallmeyer, Birgit; Kurtbay, Güven; Ohmert, Iris; Schulze-Bahr, Ellen; Brink, Paul; Pongs, Olaf

2009-01-01

Progressive familial heart block type I (PFHBI) is a progressive cardiac bundle branch disease in the His-Purkinje system that exhibits autosomal-dominant inheritance. In 3 branches of a large South African Afrikaner pedigree with an autosomal-dominant form of PFHBI, we identified the mutation c.19G→A in the transient receptor potential cation channel, subfamily M, member 4 gene (TRPM4) at chromosomal locus 19q13.3. This mutation predicted the amino acid substitution p.E7K in the TRPM4 amino terminus. TRPM4 encodes a Ca2+-activated nonselective cation (CAN) channel that belongs to the transient receptor potential melastatin ion channel family. Quantitative analysis of TRPM4 mRNA content in human cardiac tissue showed the highest expression level in Purkinje fibers. Cellular expression studies showed that the c.19G→A missense mutation attenuated deSUMOylation of the TRPM4 channel. The resulting constitutive SUMOylation of the mutant TRPM4 channel impaired endocytosis and led to elevated TRPM4 channel density at the cell surface. Our data therefore revealed a gain-of-function mechanism underlying this type of familial heart block. PMID:19726882

Non-Equilibrium Dynamics Contribute to Ion Selectivity in the KcsA Channel

PubMed Central

Haas, Stephan; Farley, Robert A.

2014-01-01

The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski’s Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na+ and K+ ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na+ and K+. These structural rearrangements facilitate entry of K+ ions into the selectivity filter and permeation through the channel, and rejection of Na+ ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K+. Estimates of the K+/Na+ selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na+ ions, the “punch through” relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation. PMID:24465882

Crystal structures of a pentameric ion channel gated by alkaline pH show a widely open pore and identify a cavity for modulation.

PubMed

Hu, Haidai; Nemecz, Ákos; Van Renterghem, Catherine; Fourati, Zaineb; Sauguet, Ludovic; Corringer, Pierre-Jean; Delarue, Marc

2018-04-24

Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.

STIM and Orai proteins and the non-capacitative ARC channels

PubMed Central

Shuttleworth, Trevor J.

2012-01-01

The ARC channel is a small conductance, highly Ca2+-selective ion channel whose activation is specifically dependent on low concentrations of arachidonic acid acting at an intracellular site. They are widely distributed in diverse cell types where they provide an alternative, store-independent pathway for agonist-activated Ca2+ entry. Although biophysically similar to the store-operated CRAC channels, these two conductances function under distinct conditions of agonist stimulation, with the ARC channels providing the predominant route of Ca2+ entry during the oscillatory signals generated at low agonist concentrations. Despite these differences in function, like the CRAC channel, activation of the ARC channels is dependent on STIM1, but it is the pool of STIM1 that constitutively resides in the plasma membrane that is responsible. Similarly, both channels are formed by Orai proteins but, whilst the CRAC channel pore is a tetrameric assembly of Orai1 subunits, the ARC channel pore is formed by a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. There is increasing evidence that the activity of these channels plays a critical role a variety of different cellular activities. PMID:22201777

ASIC3 channels in multimodal sensory perception.

PubMed

Li, Wei-Guang; Xu, Tian-Le

2011-01-19

Acid-sensing ion channels (ASICs), which are members of the sodium-selective cation channels belonging to the epithelial sodium channel/degenerin (ENaC/DEG) family, act as membrane-bound receptors for extracellular protons as well as nonproton ligands. At least five ASIC subunits have been identified in mammalian neurons, which form both homotrimeric and heterotrimeric channels. The highly proton sensitive ASIC3 channels are predominantly distributed in peripheral sensory neurons, correlating with their roles in multimodal sensory perception, including nociception, mechanosensation, and chemosensation. Different from other ASIC subunit composing ion channels, ASIC3 channels can mediate a sustained window current in response to mild extracellular acidosis (pH 7.3-6.7), which often occurs accompanied by many sensory stimuli. Furthermore, recent evidence indicates that the sustained component of ASIC3 currents can be enhanced by nonproton ligands including the endogenous metabolite agmatine. In this review, we first summarize the growing body of evidence for the involvement of ASIC3 channels in multimodal sensory perception and then discuss the potential mechanisms underlying ASIC3 activation and mediation of sensory perception, with a special emphasis on its role in nociception. We conclude that ASIC3 activation and modulation by diverse sensory stimuli represent a new avenue for understanding the role of ASIC3 channels in sensory perception. Furthermore, the emerging implications of ASIC3 channels in multiple sensory dysfunctions including nociception allow the development of new pharmacotherapy.

Physical and functional interactions between a glioma cation channel and integrin-β1 require α-actinin

PubMed Central

Rooj, Arun K.; Liu, Zhiyong; McNicholas, Carmel M.

2015-01-01

Major plasma membrane components of the tumor cell, ion channels, and integrins play crucial roles in metastasis. Glioma cells express an amiloride-sensitive nonselective cation channel composed of acid-sensing ion channel (ASIC)-1 and epithelial Na+ channel (ENaC) α- and γ-subunits. Inhibition of this channel is associated with reduced cell migration and proliferation. Using the ASIC-1 subunit as a reporter for the channel complex, we found a physical and functional interaction between this channel and integrin-β1. Short hairpin RNA knockdown of integrin-β1 attenuated the amiloride-sensitive current, which was due to loss of surface expression of ASIC-1. In contrast, upregulation of membrane expression of integrin-β1 increased the surface expression of ASIC-1. The link between the amiloride-sensitive channel and integrin-β1 was mediated by α-actinin. Downregulation of α-actinin-1 or -4 attenuated the amiloride-sensitive current. Mutation of the putative binding site for α-actinin on the COOH terminus of ASIC-1 reduced the membrane localization of ASIC-1 and also resulted in attenuation of the amiloride-sensitive current. Our data suggest a novel interaction between the amiloride-sensitive glioma cation channel and integrin-β1, mediated by α-actinin. This interaction may form a mechanism by which channel activity can regulate glioma cell proliferation and migration. PMID:26108662

Ion concentrations and velocity profiles in nanochannel electroosmotic flows

NASA Astrophysics Data System (ADS)

Qiao, R.; Aluru, N. R.

2003-03-01

Ion distributions and velocity profiles for electroosmotic flow in nanochannels of different widths are studied in this paper using molecular dynamics and continuum theory. For the various channel widths studied in this paper, the ion distribution near the channel wall is strongly influenced by the finite size of the ions and the discreteness of the solvent molecules. The classical Poisson-Boltzmann equation fails to predict the ion distribution near the channel wall as it does not account for the molecular aspects of the ion-wall and ion-solvent interactions. A modified Poisson-Boltzmann equation based on electrochemical potential correction is introduced to account for ion-wall and ion-solvent interactions. The electrochemical potential correction term is extracted from the ion distribution in a smaller channel using molecular dynamics. Using the electrochemical potential correction term extracted from molecular dynamics (MD) simulation of electroosmotic flow in a 2.22 nm channel, the modified Poisson-Boltzmann equation predicts the ion distribution in larger channel widths (e.g., 3.49 and 10.00 nm) with good accuracy. Detailed studies on the velocity profile in electro-osmotic flow indicate that the continuum flow theory can be used to predict bulk fluid flow in channels as small as 2.22 nm provided that the viscosity variation near the channel wall is taken into account. We propose a technique to embed the velocity near the channel wall obtained from MD simulation of electroosmotic flow in a narrow channel (e.g., 2.22 nm wide channel) into simulation of electroosmotic flow in larger channels. Simulation results indicate that such an approach can predict the velocity profile in larger channels (e.g., 3.49 and 10.00 nm) very well. Finally, simulation of electroosmotic flow in a 0.95 nm channel indicates that viscosity cannot be described by a local, linear constitutive relationship that the continuum flow theory is built upon and thus the continuum flow theory is not applicable for electroosmotic flow in such small channels.

Arrhythmogenic KCNE gene variants: current knowledge and future challenges

PubMed Central

Crump, Shawn M.; Abbott, Geoffrey W.

2014-01-01

There are twenty-five known inherited cardiac arrhythmia susceptibility genes, all of which encode either ion channel pore-forming subunits or proteins that regulate aspects of ion channel biology such as function, trafficking, and localization. The human KCNE gene family comprises five potassium channel regulatory subunits, sequence variants in each of which are associated with cardiac arrhythmias. KCNE gene products exhibit promiscuous partnering and in some cases ubiquitous expression, hampering efforts to unequivocally correlate each gene to specific native potassium currents. Likewise, deducing the molecular etiology of cardiac arrhythmias in individuals harboring rare KCNE gene variants, or more common KCNE polymorphisms, can be challenging. In this review we provide an update on putative arrhythmia-causing KCNE gene variants, and discuss current thinking and future challenges in the study of molecular mechanisms of KCNE-associated cardiac rhythm disturbances. PMID:24478792

The Experimental Study of Novel Pseudospark Hollow Cathode Plasma Electron Gun

NASA Astrophysics Data System (ADS)

Gu, Xiaowei; Meng, Lin; Sun, Yiqin; Yu, Xinhua

2008-11-01

The high-power microwave devices with plasma-filled have unique properties. One of the major problems associated with plasma-filled microwave sources is that ions from the plasma drift toward the gun regions of the tube. This bombardment is particularly dangerous for the gun, where high-energy ion impacts can damage the cathode surface and degrade its electron emission capabilities. One of the techniques investigated to mitigate this issue is to replace the material cathode with plasma cathode. Now, we study the novel electron gun (E-gun) that can be suitable for high power microwave device applications, adopting two forms of discharge channel, 1: a single hole channel, the structure can produce a solid electron beam; 2: porous holes channel, the structure can generate multiple electronic injection which is similar to the annular electron beam.

Microfluidic array platform for simultaneous lipid bilayer membrane formation.

PubMed

Zagnoni, M; Sandison, M E; Morgan, H

2009-01-01

In recent years, protein array technologies have found widespread applications in proteomics. However, new methods for high-throughput analysis of protein-protein and protein-compound interactions are still required. In this paper, an array of lipid bilayer membranes formed within a microfluidic system with integrated electrodes is presented. The system is comprised of three layers that are clamped together, thus rendering the device cleanable and reusable. The device microfluidics enable the simultaneous formation of an array of lipid bilayers using a previously developed air-exposure technique, thereby avoiding the need to manually form individual bilayers. The Ag/AgCl electrodes allow for ion channel measurements, each of the sites being independently addressable. Typically, a 50% yield in simultaneous lipid bilayer formation over 12 sites was obtained and ion channel recordings have been acquired over multiple sites. This system has great potential for the development of an automatable platform of suspended lipid bilayer arrays.

Oxidative Stress and Maxi Calcium-Activated Potassium (BK) Channels

PubMed Central

Hermann, Anton; Sitdikova, Guzel F.; Weiger, Thomas M.

2015-01-01

All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences. PMID:26287261

Fabrication of Amorphous Indium Gallium Zinc Oxide Thin Film Transistor by using Focused Ion Beam

NASA Astrophysics Data System (ADS)

Zhu, Wencong

Compared with other transparent semiconductors, amorphous indium gallium zinc oxide (a-IGZO) has both good uniformity and high electron mobility, which make it as a good candidate for displays or large-scale transparent circuit. The goal of this research is to fabricate alpha-IGZO thin film transistor (TFT) with channel milled by focused ion beam (FIB). TFTs with different channel geometries can be achieved by applying different milling strategies, which facilitate modifying complex circuit. Technology Computer-Aided Design (TCAD) was also introduced to understand the effect of trapped charges on the device performance. The investigation of the trapped charge at IGZO/SiO2 interface was performed on the IGZO TFT on p-Silicon substrate with thermally grown SiO2 as dielectric. The subgap density-of-state model was used for the simulation, which includes conduction band-tail trap states and donor-like state in the subgap. The result shows that the de-trapping and donor-state ionization determine the interface trapped charge density at various gate biases. Simulation of IGZO TFT with FIB defined channel on the same substrate was also applied. The drain and source were connected intentionally during metal deposition and separated by FIB milling. Based on the simulation, the Ga ions in SiO2 introduced by the ion beam was drifted by gate bias and affects the saturation drain current. Both side channel and direct channel transparent IGZO TFTs were fabricated on the glass substrate with coated ITO. Higher ion energy (30 keV) was used to etch through the substrate between drain and source and form side channels at the corner of milled trench. Lower ion energy (16 keV) was applied to stop the milling inside IGZO thin film and direct channel between drain and source was created. Annealing after FIB milling removed the residual Ga ions and the devices show switch feature. Direct channel shows higher saturation drain current (~10-6 A) compared with side channel (~10-7 A) because of its shorter channel length and wider width, however, it also exhibit higher gate leakage current (>10-7 A) than side channel (<10-7 A) due to larger Ga ion implantation and diffusion region in SiO2 after annealing. Hysteresis window increase and positive VON shift were also observed due to the interface trap density increase and carrier density suppression both by Ga ions. Laser interference lithography was applied to define the IGZO active region, which gives more flexibility on TFT channel dimension and circuit modification. He-Cd laser with 325 nm wavelength was used to define 2D array of IGZO islands with period of 2.5 im. Logic gate array was designed and fabricated by combining this 2D array of IGZO islands and FIB direct channel milling. After annealing, device shows on-off feature, but high temperature (400 °C) release more free carrier and results in negative shift of VON. The row selection voltage was also introduced in the design of logic gate array to act as switch of input signals to each row separately. However, due to the long input signal sweeping time, the leakage current cannot be overlooked. The idea can be verified by AC or short pulse input signal.

Ion Channel Gene Expression in Lung Adenocarcinoma: Potential Role in Prognosis and Diagnosis

PubMed Central

Ko, Jae-Hong; Gu, Wanjun; Lim, Inja; Bang, Hyoweon; Ko, Eun A.; Zhou, Tong

2014-01-01

Ion channels are known to regulate cancer processes at all stages. The roles of ion channels in cancer pathology are extremely diverse. We systematically analyzed the expression patterns of ion channel genes in lung adenocarcinoma. First, we compared the expression of ion channel genes between normal and tumor tissues in patients with lung adenocarcinoma. Thirty-seven ion channel genes were identified as being differentially expressed between the two groups. Next, we investigated the prognostic power of ion channel genes in lung adenocarcinoma. We assigned a risk score to each lung adenocarcinoma patient based on the expression of the differentially expressed ion channel genes. We demonstrated that the risk score effectively predicted overall survival and recurrence-free survival in lung adenocarcinoma. We also found that the risk scores for ever-smokers were higher than those for never-smokers. Multivariate analysis indicated that the risk score was a significant prognostic factor for survival, which is independent of patient age, gender, stage, smoking history, Myc level, and EGFR/KRAS/ALK gene mutation status. Finally, we investigated the difference in ion channel gene expression between the two major subtypes of non-small cell lung cancer: adenocarcinoma and squamous-cell carcinoma. Thirty ion channel genes were identified as being differentially expressed between the two groups. We suggest that ion channel gene expression can be used to improve the subtype classification in non-small cell lung cancer at the molecular level. The findings in this study have been validated in several independent lung cancer cohorts. PMID:24466154

Drug-induced Inhibition and Trafficking Disruption of ion Channels: Pathogenesis of QT Abnormalities and Drug-induced Fatal Arrhythmias

PubMed Central

Cubeddu, Luigi X.

2016-01-01

Risk of severe and fatal ventricular arrhythmias, presenting as Torsade de Pointes (TdP), is increased in congenital and acquired forms of long QT syndromes (LQTS). Drug-induced inhibition of K+ currents, IKs, IKr, IK1, and/or Ito, delay repolarization, prolong QT, and increase the risk of TdP. Drug-induced interference with IKr is the most common cause of acquired LQTS/TdP. Multiple drugs bind to KNCH2-hERG-K+ channels affecting IKr, including antiarrythmics, antibiotics, antivirals, azole-antifungals, antimalarials, anticancer, antiemetics, prokinetics, antipsychotics, and antidepressants. Azithromycin has been recently added to this list. In addition to direct channel inhibition, some drugs interfere with the traffic of channels from the endoplasmic reticulum to the cell membrane, decreasing mature channel membrane density; e.g., pentamidine, geldalamicin, arsenic trioxide, digoxin, and probucol. Other drugs, such as ketoconazole, fluoxetine, norfluoxetine, citalopram, escitalopram, donepezil, tamoxifen, endoxifen, atazanavir, and roxitromycin, induce both direct channel inhibition and impaired channel trafficking. Although many drugs prolong the QT interval, TdP is a rare event. The following conditions increase the risk of drug-induced TdP: a) Disease states/electrolyte levels (heart failure, structural cardiac disease, bradycardia, hypokalemia); b) Pharmacogenomic variables (presence of congenital LQTS, subclinical ion-channel mutations, history of or having a relative with history of drug-induced long QT/TdP); c) Pharmacodynamic and kinetic factors (high doses, women, elderly, metabolism inhibitors, combining two or more QT prolonging drugs, drugs that prolong the QT and increase QT dispersion, and drugs with multiple actions on ion channels). Because most of these conditions are preventable, careful evaluation of risk factors and increased knowledge of drug use associated with repolarization abnormalities are strongly recommended. PMID:26926294

Effects of free and combined chlorine and exposure duration on rainbow trout, channel catfish, and emerald shiners

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

Brooks, A.S.; Bartos, J.M.

1984-11-01

Laboratory bioassays to determine the acute toxicity of monochloramine, dichloramine, hypochlorous acid, and hypochlorite ion to emerald shiners Notropis atherinoides, channel catfish Ictalurus punctatus, and rainbow trout Salmo gairdneri were conducted. Four exposure regimes typical of chlorination schedules at operating steam electric power plants were used. Fish were exposed for single 15-minute, 30-minute, 120-minute, and 4 x 30-minute periods. Based on median lethal concentrations (LC50s), the nominal solution of hypochlorous acid was the most toxic, followed closely by the solution dominated by dichloramine; nominal monochloramine and hypochlorite ion solutions had a third to a quarter the toxicity of the othermore » two. On the average, emerald shiners were 1.8 times more sensitive than channel catfish and 3.3 times more sensitive than rainbow trout to the four forms of chlorine. The fish were most tolerant of chlorine during short-duration exposures and least tolerant during the continuous 120-minute exposures. The differences in toxicity noted among the chlorine forms suggest that, when chlorination regimes and regulations are established, careful attention should be paid not only to total residual chlorine but to both the chlorine forms and fish species present and the duration of exposure. 24 references, 1 figure, 1 table.« less

Aerospace Power Scholarly Research Program. Delivery Order 0011: Modeling Lithium-Ion Conducting Channel

DTIC Science & Technology

2005-12-01

Brinkmann, D. NMR, DSC, and conductivity study of a poly (ethylene oxide) complex electrolyte: PEO(LiClO4)x, Sol. St. Ionics 1986, 18-19, 295. (27...Electrochemical Characterizations of Dilithium Octacyanophthalocyanine Langmuir - Blodgett films, Langmuir 2002, 18, 2223. 20 ...phthalocyanine (Li2Pc) has been used in this development since it can undergo molecular self-assembly to form the ionic ally conducting channel. The

Ion-dipole interactions and their functions in proteins.

PubMed

Sippel, Katherine H; Quiocho, Florante A

2015-07-01

Ion-dipole interactions in biological macromolecules are formed between atomic or molecular ions and neutral protein dipolar groups through either hydrogen bond or coordination. Since their discovery 30 years ago, these interactions have proven to be a frequent occurrence in protein structures, appearing in everything from transporters and ion channels to enzyme active sites to protein-protein interfaces. However, their significance and roles in protein functions are largely underappreciated. We performed PDB data mining to identify a sampling of proteins that possess these interactions. In this review, we will define the ion-dipole interaction and discuss several prominent examples of their functional roles in nature. © 2015 The Protein Society.

Cardiac ion channels

PubMed Central

Priest, Birgit T; McDermott, Jeff S

2015-01-01

Ion channels are critical for all aspects of cardiac function, including rhythmicity and contractility. Consequently, ion channels are key targets for therapeutics aimed at cardiac pathophysiologies such as atrial fibrillation or angina. At the same time, off-target interactions of drugs with cardiac ion channels can be the cause of unwanted side effects. This manuscript aims to review the physiology and pharmacology of key cardiac ion channels. The intent is to highlight recent developments for therapeutic development, as well as elucidate potential mechanisms for drug-induced cardiac side effects, rather than present an in-depth review of each channel subtype. PMID:26556552

Microfabricated Ion Beam Drivers for Magnetized Target Fusion

NASA Astrophysics Data System (ADS)

Persaud, Arun; Seidl, Peter; Ji, Qing; Ardanuc, Serhan; Miller, Joseph; Lal, Amit; Schenkel, Thomas

2015-11-01

Efficient, low-cost drivers are important for Magnetized Target Fusion (MTF). Ion beams offer a high degree of control to deliver the required mega joules of driver energy for MTF and they can be matched to several types of magnetized fuel targets, including compact toroids and solid targets. We describe an ion beam driver approach based on the MEQALAC concept (Multiple Electrostatic Quadrupole Array Linear Accelerator) with many beamlets in an array of micro-fabricated channels. The channels consist of a lattice of electrostatic quadrupoles (ESQ) for focusing and of radio-frequency (RF) electrodes for ion acceleration. Simulations with particle-in-cell and beam envelope codes predict >10x higher current densities compared to state-of-the-art ion accelerators. This increase results from dividing the total ion beam current up into many beamlets to control space charge forces. Focusing elements can be biased taking advantage of high breakdown electric fields in sub-mm structures formed using MEMS techniques (Micro-Electro-Mechanical Systems). We will present results on ion beam transport and acceleration in MEMS based beamlets. Acknowledgments: This work is supported by the U.S. DOE under Contract No. DE-AC02-05CH11231.

Structure and ion channel activity of the human respiratory syncytial virus (hRSV) small hydrophobic protein transmembrane domain

PubMed Central

Gan, Siok Wan; Ng, Lifang; Lin, Xin; Gong, Xiandi; Torres, Jaume

2008-01-01

The small hydrophobic (SH) protein from the human respiratory syncytial virus (hRSV) is a glycoprotein of ∼64 amino acids with one putative α-helical transmembrane domain. Although SH protein is important for viral infectivity, its exact role during viral infection is not clear. Herein, we have studied the secondary structure, orientation, and oligomerization of the transmembrane domain of SH (SH-TM) in the presence of lipid bilayers. Only one oligomer, a pentamer, was observed in PFO-PAGE. Using polarized attenuated total reflection-Fourier transform infrared (PATR-FTIR) spectroscopy, we show that the SH-TM is α-helical. The rotational orientation of SH-TM was determined by site-specific infrared dichroism (SSID) at two consecutive isotopically labeled residues. This orientation is consistent with that of an evolutionary conserved pentameric model obtained from a global search protocol using 13 homologous sequences of RSV. Conductance studies of SH-TM indicate ion channel activity, which is cation selective, and inactive below the predicted pKa of histidine. Thus, our results provide experimental evidence that the transmembrane domain of SH protein forms pentameric α-helical bundles that form cation-selective ion channels in planar lipid bilayers. We provide a model for this pore, which should be useful in mutagenesis studies to elucidate its role during the virus cycle. PMID:18369195

Study of ND3-enhanced MAR processes in D2-N2 plasmas to induce plasma detachment

NASA Astrophysics Data System (ADS)

Abe, Shota; Chakraborty Thakur, Saikat; Doerner, Russ; Tynan, George

2017-10-01

The Molecular Assisted Recombination (MAR) process is thought to be a main channel of volumetric recombination to induce the plasma detachment operation. Authors have focused on a new plasma recombination process supported by ammonia molecules, which will be formed by impurity seeding of N2 for controlling divertor plasma temperature and heat loads in ITER. This ammonia-enhanced MAR process would occur throughout two steps. In this study, the first step of the new MAR process is investigated in low density plasmas (Ne 1016 m-3, Te 4 eV) fueled by D2 and N2. Ion and neutral densities are measured by a calibrated Electrostatic Quadrupole Plasma (EQP) analyzer, combination of an ion energy analyzer and mass spectrometer. The EQP shows formation of ND3 during discharges. Ion densities calculated by a rate equation model are compared with experimental results. We find that the model can reproduce the observed ion densities in the plasma. The model calculation shows that the dominant neutralization channel of Dx+(x =1-3) ions in the volume is the formation of NDy+(y =3 or 4) throughout charge/D+ exchange reactions with ND3. Furthermore, high density plasmas (Ne 1016 m-3) have been achieved to investigate electron-impact dissociative recombination processes of formed NDy+,which is the second step of this MAR process.

Inert gas rejection device for zinc-halogen battery systems

DOEpatents

Hammond, Michael J.; Arendell, Mark W.

1981-01-01

An electrolytic cell for separating chlorine gas from other (foreign) gases, having an anode, a cathode assembly, an aqueous electrolyte, a housing, and a constant voltage power supply. The cathode assembly is generally comprised of a dense graphite electrode having a winding channel formed in the face opposing the anode, a gas impermeable (but liquid permeable) membrane sealed into the side of the cathode electrode over the channel, and a packing of graphite particles contained in the channel of the cathode electrode. The housing separates and parallelly aligns the anode and cathode assembly, and provides a hermetic seal for the cell. In operation, a stream of chlorine and foreign gases enters the cell at the beginning of the cathode electrode channel. The chlorine gas is dissolved into the electrolyte and electrochemically reduced into chloride ions. The chloride ions disfuse through the gas impermeable membrane, and are electrochemically oxidized at the anode into purified chlorine gas. The foreign gases do not participate in the above electrochemical reactions, and are vented from the cell at the end of the cathode electrode channel.

Mapping the function of neuronal ion channels in model and experiment

PubMed Central

Podlaski, William F; Seeholzer, Alexander; Groschner, Lukas N; Miesenböck, Gero; Ranjan, Rajnish; Vogels, Tim P

2017-01-01

Ion channel models are the building blocks of computational neuron models. Their biological fidelity is therefore crucial for the interpretation of simulations. However, the number of published models, and the lack of standardization, make the comparison of ion channel models with one another and with experimental data difficult. Here, we present a framework for the automated large-scale classification of ion channel models. Using annotated metadata and responses to a set of voltage-clamp protocols, we assigned 2378 models of voltage- and calcium-gated ion channels coded in NEURON to 211 clusters. The IonChannelGenealogy (ICGenealogy) web interface provides an interactive resource for the categorization of new and existing models and experimental recordings. It enables quantitative comparisons of simulated and/or measured ion channel kinetics, and facilitates field-wide standardization of experimentally-constrained modeling. DOI: http://dx.doi.org/10.7554/eLife.22152.001 PMID:28267430

Comparison of Ion Chemistries in Octafluoro-2-butene (2-C4F8) and in Octfluorocyclobutane (c-C4F8)

NASA Astrophysics Data System (ADS)

Jiao, Charles; Dejoseph, Charles; Garscadden, Alan

2007-10-01

2-C4F8 is one of the promising candidates to replace c-C4F8 that has been widely used for dielectric etching but is not environmentally friendly. In this study we have investigated electron impact ionization and ion-molecule reactions of 2-C4F8 using Fourier transform mass spectrometry (FTMS), and compared the results with those of c-C4F8 we have studied previously. Electron impact ionization of 2-C4F8 produces 15 ionic species including C4F7,8^+, C3F3,5,6^+, C2F4^+ and CF1-3^+ as the major ions. The total ionization cross section of 2-C4F8 reaches a maximum of 1.8x10-15 cm^2 at 90 eV. The ionization is dominated by the channel forming the parent ion C4F8^+ from 12 to 18 eV, and by the channel forming C3F5^+ from 18 to 70 eV. After 70 eV, CF3^+ becomes the dominant product ion. Among the major ions generated from the electron impact ionization of 2-C4F8, only CF^+, CF2^+ and CF3^+ are found to react with 2-C4F8, via F^- abstraction or charge transfer mechanism. The charge transfer reaction of Ar^++2-C4F8 produces primarily C4F7^+.

Tight junctions of the proximal tubule and their channel proteins.

PubMed

Fromm, Michael; Piontek, Jörg; Rosenthal, Rita; Günzel, Dorothee; Krug, Susanne M

2017-08-01

The renal proximal tubule achieves the majority of renal water and solute reabsorption with the help of paracellular channels which lead through the tight junction. The proteins forming such channels in the proximal tubule are claudin-2, claudin-10a, and possibly claudin-17. Claudin-2 forms paracellular channels selective for small cations like Na + and K + . Independently of each other, claudin-10a and claudin-17 form anion-selective channels. The claudins form the paracellular "pore pathway" and are integrated, together with purely sealing claudins and other tight junction proteins, in the belt of tight junction strands surrounding the tubular epithelial cells. In most species, the proximal tubular tight junction consists of only 1-2 (pars convoluta) to 3-5 (pars recta) horizontal strands. Even so, they seal the tubule very effectively against leak passage of nutrients and larger molecules. Remarkably, claudin-2 channels are also permeable to water so that 20-25% of proximal water absorption may occur paracellularly. Although the exact structure of the claudin-2 channel is still unknown, it is clear that Na + and water share the same pore. Already solved claudin crystal structures reveal a characteristic β-sheet, comprising β-strands from both extracellular loops, which is anchored to a left-handed four-transmembrane helix bundle. This allowed homology modeling of channel-forming claudins present in the proximal tubule. The surface of cation- and anion-selective claudins differ in electrostatic potentials in the area of the proposed ion channel, resulting in the opposite charge selectivity of these claudins. Presently, while models of the molecular structure of the claudin-based oligomeric channels have been proposed, its full understanding has only started.

Product ion distributions for the reactions of NO+ with some physiologically significant volatile organosulfur and organoselenium compounds obtained using a selective reagent ionization time-of-flight mass spectrometer

PubMed Central

Mochalski, Paweł; Unterkofler, Karl; Španěl, Patrik; Smith, David; Amann, Anton

2014-01-01

RATIONALE The reactions of NO+ with volatile organic compounds (VOCs) in Selective Reagent Ionization Time-of-Flight Mass Spectrometry (SRI-TOF-MS) reactors are relatively poorly known, inhibiting their use for trace gas analysis. The rationale for this product ion distribution study was to identify the major product ions of the reactions of NO+ ions with 13 organosulfur compounds and 2 organoselenium compounds in an SRI-TOF-MS instrument and thus to prepare the way for their analysis in exhaled breath, in skin emanations and in the headspace of urine, blood and cell and bacterial cultures. METHODS Product ion distributions have been investigated by a SRI-TOF-MS instrument at an E/N in the drift tube reactor of 130 Td for both dry air and humid air (4.9% absolute humidity) used as the matrix gas. The investigated species were five monosulfides (dimethyl sulfide, ethyl methyl sulfide, methyl propyl sulfide, allyl methyl sulfide and methyl 5-methyl-2-furyl sulfide), dimethyl disulfide, dimethyl trisulfide, thiophene, 2-methylthiophene, 3-methylthiophene, methanethiol, allyl isothiocyanate, dimethyl sulfoxide, and two selenium compounds – dimethyl selenide and dimethyl diselenide. RESULTS Charge transfer was seen to be the dominant reaction mechanism in all reactions under study forming the M+ cations. For methanethiol and allyl isothiocyanate significant fractions were also observed of the stable adduct ions NO+M, formed by ion-molecule association, and [M–H]+ ions, formed by hydride ion transfer. Several other minor product channels are seen for most reactions indicating that the nascent excited intermediate (NOM)+* adduct ions partially fragment along other channels, most commonly by the elimination of neutral CH3, CH4 and/or C2H4 species that are probably bound to an NO molecule. Humidity had little effect on the product ion distributions. CONCLUSIONS The findings of this study are of particular importance for data interpretation in studies of volatile organosulfur and volatile organoselenium compounds employing SRI-TOF-MS in the NO+ mode. © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd. PMID:24975248

United in Diversity: Mechanosensitive Ion Channels in Plants

PubMed Central

Hamilton, Eric S.; Schlegel, Angela M.; Haswell, Elizabeth S.

2015-01-01

Mechanosensitive (MS) ion channels are a common mechanism for perceiving and responding to mechanical force. This class of mechanoreceptors is capable of transducing membrane tension directly into ion flux. In plant systems, MS ion channels have been proposed to play a wide array of roles, from the perception of touch and gravity to the osmotic homeostasis of intracellular organelles. Three families of plant MS ion channels have been identified: the MscS-like (MSL), Mid1-complementing activity (MCA), and two-pore potassium (TPK) families. Channels from these families vary widely in structure and function, localize to multiple cellular compartments, and conduct chloride, calcium, and/or potassium ions. However, they are still likely to represent only a fraction of the MS ion channel diversity in plant systems. PMID:25494462

Ion channel gene expression predicts survival in glioma patients

PubMed Central

Wang, Rong; Gurguis, Christopher I.; Gu, Wanjun; Ko, Eun A; Lim, Inja; Bang, Hyoweon; Zhou, Tong; Ko, Jae-Hong

2015-01-01

Ion channels are important regulators in cell proliferation, migration, and apoptosis. The malfunction and/or aberrant expression of ion channels may disrupt these important biological processes and influence cancer progression. In this study, we investigate the expression pattern of ion channel genes in glioma. We designate 18 ion channel genes that are differentially expressed in high-grade glioma as a prognostic molecular signature. This ion channel gene expression based signature predicts glioma outcome in three independent validation cohorts. Interestingly, 16 of these 18 genes were down-regulated in high-grade glioma. This signature is independent of traditional clinical, molecular, and histological factors. Resampling tests indicate that the prognostic power of the signature outperforms random gene sets selected from human genome in all the validation cohorts. More importantly, this signature performs better than the random gene signatures selected from glioma-associated genes in two out of three validation datasets. This study implicates ion channels in brain cancer, thus expanding on knowledge of their roles in other cancers. Individualized profiling of ion channel gene expression serves as a superior and independent prognostic tool for glioma patients. PMID:26235283

Recording ion channels across soy-extracted lecithin bilayer generated by water-soluble quantum dots

NASA Astrophysics Data System (ADS)

Sarma, Runjun; Mohanta, Dambarudhar

2014-02-01

We report on the quantum dot (QD)-induced ion channels across a soya-derived lecithin bilayer supported on a laser drilled of ~100 μm aperture of cellulose acetate substrate that separates two electrolytic chambers. Adequate current bursts were observed when the bilayer was subjected to a gating voltage. The voltage-dependent current fluctuation, across the bilayer, was attributed to the insertion of ~20 nm sized water-soluble CdSe QDs, forming nanopores due to their spontaneous aggregation. Apart from a closed state, the first observable conductance levels were found as 6.3 and 11 nS, as for the respective biasing voltages of -10 and -20 mV. The highest observable conductance states, at corresponding voltages were ~14.3 and 21.1 nS. Considering two simplified models, we predict that the non-spherical pores (dnspore) can be a better approximation over spherical nanopores (dspore) for exhibiting a definite conductance level. At times, even dnspore ≤ 4dspore and that the non-spherical nanopores were associated with a smaller No. of QDs than the case for spherical nanopores, for a definite conductance state. It seems like the current events are partly stochastic, possibly due to thermal effects on the aggregated QDs that would form nanopores. The dwell time of the states was predicted in the range of 384-411 μs. The ion channel mechanism in natural phospholipid bilayers over artificial ones will provide a closer account to understand ion transport mechanism in live cells and signaling activity including labelling with fluorescent QDs.

Characterization of Membrane Patch-Ion Channel Probes for Scanning Ion Conductance Microscopy.

PubMed

Shi, Wenqing; Zeng, Yuhan; Zhu, Cheng; Xiao, Yucheng; Cummins, Theodore R; Hou, Jianghui; Baker, Lane A

2018-05-01

Integration of dual-barrel membrane patch-ion channel probes (MP-ICPs) to scanning ion conductance microscopy (SICM) holds promise of providing a revolutionized approach of spatially resolved chemical sensing. A series of experiments are performed to further the understanding of the system and to answer some fundamental questions, in preparation for future developments of this approach. First, MP-ICPs are constructed that contain different types of ion channels including transient receptor potential vanilloid 1 and large conductance Ca2 + -activated K + channels to establish the generalizability of the methods. Next, the capability of the MP-ICP platforms in single ion channel activity measurements is proved. In addition, the interplay between the SICM barrel and the ICP barrel is studied. For ion channels gated by uncharged ligands, channel activity at the ICP barrel is unaffected by the SICM barrel potential; whereas for ion channels that are gated by charged ligands, enhanced channel activity can be obtained by biasing the SICM barrel at potentials with opposite polarity to the charge of the ligand molecules. Finally, a proof-of-principle experiment is performed and site-specific molecular/ionic flux sensing is demonstrated at single-ion-channel level, which show that the MP-ICP platform can be used to quantify local molecular/ionic concentrations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ion channel expression patterns in glioblastoma stem cells with functional and therapeutic implications for malignancy.

PubMed

Pollak, Julia; Rai, Karan G; Funk, Cory C; Arora, Sonali; Lee, Eunjee; Zhu, Jun; Price, Nathan D; Paddison, Patrick J; Ramirez, Jan-Marino; Rostomily, Robert C

2017-01-01

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation, migration, and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme, a highly aggressive brain cancer, suggesting that ion channel expression may be perturbed in this population. However, little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing, we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance, expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally, genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes, gene mutations, survival outcomes, regional tumor expression, and experimental responses to loss-of-function. Together, the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance.

Ion channel expression patterns in glioblastoma stem cells with functional and therapeutic implications for malignancy

PubMed Central

Pollak, Julia; Rai, Karan G.; Funk, Cory C.; Arora, Sonali; Lee, Eunjee; Zhu, Jun; Price, Nathan D.; Paddison, Patrick J.; Ramirez, Jan-Marino; Rostomily, Robert C.

2017-01-01

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation, migration, and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme, a highly aggressive brain cancer, suggesting that ion channel expression may be perturbed in this population. However, little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing, we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance, expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally, genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes, gene mutations, survival outcomes, regional tumor expression, and experimental responses to loss-of-function. Together, the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance. PMID:28264064

Organic toxins as tools to understand ion channel mechanisms and structure.

PubMed

Morales-Lázaro, Sara Luz; Hernández-García, Enrique; Serrano-Flores, Barbara; Rosenbaum, Tamara

2015-01-01

Ion channels constitute a varied class of membrane proteins with pivotal roles in cellular physiology and that are fundamental for neuronal signaling, hormone secretion and muscle contractility. Hence, it is not unanticipated that toxins from diverse organisms have evolved to modulate the activity of ion channels. For instance, animals such as cone snails, scorpions, spiders and snakes use toxins to immobilize and capture their prey by affecting ion channel function. This is a beautiful example of an evolutionary process that has led to the development of an injection apparatus from predators and to the existence of toxins with high affinity and specificity for a given target. Toxins have been used in the field of ion channel biophysics for several decades to gain insight into the gating mechanisms and the structure of ion channels. Through the use of these peptides, much has been learned about the ion conduction pathways, voltage-sensing mechanisms, pore sizes, kinetics, inactivation processes, etc. This review examines an assortment of toxins that have been used to study different ion channels and describes some key findings about the structure-function relationships in these proteins through the details of the toxin-ion channel interactions.

Molecular dynamics investigation of Cl(-) transport through the closed and open states of the 2α12β2γ2 GABA(A) receptor.

PubMed

Xie, Hong-Bo; Wang, Jian; Sha, Yu; Cheng, Mao-Sheng

2013-01-01

The α1β2γ2 gamma-aminobutyric type A receptor (GABA(A)R) is one of the most widely expressed GABA(A)R subtypes in the mammalian brain. GABA(A)Rsbelonging to the Cys-loop superfamily of ligand-gated ion channels have been identified as key targets for many clinical drugs, and the motions that govern the gating mechanism are still not well understood. In this study, an open-state GABA(A)R was constructed using the structure of the glutamate-gated chloride channel (GluCl), which has a high sequence identity to GABA(A)R. A closed-state model was constructed using the structure of the nicotinic acetylcholine receptor (nAChR). Molecular dynamics simulations of the open-state and closed-state GABA(A)R were performed. We calculated the electrostatic potential of the two conformations, the pore radius of the two ion channels and the root-mean-square fluctuation. We observed the presence of two positively charged girdles around the ion channel and found flexible regions in the GABA(A)R. Then, the free-energy of chloride ion permeations through the closed-state and open-state G GABA(A)R has been estimated using adaptive biasing force (ABF) simulation. For the closed-state G GABA(A)R, we observed two major energy barriers for chloride ion translocation in the transmembrane domain (TMD). For the open-state GABA(A)R, there was only one energy barrier formed by two Thr261 (α1), two Thr255 (β2) and one Thr271 (γ2). By using ABF simulation, the overall free-energy profile is obtained for Cl(-) transporting through GABA(A)R, which gives a complete map of the ion channel of Cl(-) permeation. Copyright © 2013 Elsevier B.V. All rights reserved.

Mechanically Activated Ion Channels

PubMed Central

Ranade, Sanjeev S.; Syeda, Ruhma; Patapoutian, Ardem

2015-01-01

Mechanotransduction, the conversion of physical forces into biochemical signals, is an essential component of numerous physiological processes including not only conscious senses of touch and hearing, but also unconscious senses such as blood pressure regulation. Mechanically activated (MA) ion channels have been proposed as sensors of physical force, but the identity of these channels and an understanding of how mechanical force is transduced has remained elusive. A number of recent studies on previously known ion channels along with the identification of novel MA ion channels have greatly transformed our understanding of touch and hearing in both vertebrates and invertebrates. Here, we present an updated review of eukaryotic ion channel families that have been implicated in mechanotransduction processes and evaluate the qualifications of the candidate genes according to specified criteria. We then discuss the proposed gating models for MA ion channels and highlight recent structural studies of mechanosensitive potassium channels. PMID:26402601

Systematic analysis of the contributions of stochastic voltage gated channels to neuronal noise

PubMed Central

O'Donnell, Cian; van Rossum, Mark C. W.

2014-01-01

Electrical signaling in neurons is mediated by the opening and closing of large numbers of individual ion channels. The ion channels' state transitions are stochastic and introduce fluctuations in the macroscopic current through ion channel populations. This creates an unavoidable source of intrinsic electrical noise for the neuron, leading to fluctuations in the membrane potential and spontaneous spikes. While this effect is well known, the impact of channel noise on single neuron dynamics remains poorly understood. Most results are based on numerical simulations. There is no agreement, even in theoretical studies, on which ion channel type is the dominant noise source, nor how inclusion of additional ion channel types affects voltage noise. Here we describe a framework to calculate voltage noise directly from an arbitrary set of ion channel models, and discuss how this can be use to estimate spontaneous spike rates. PMID:25360105

Interaction of elaiophylin with model bilayer membrane

NASA Astrophysics Data System (ADS)

Genova, J.; Dencheva-Zarkova, M.

2017-01-01

Elaiophylin is a new macrodiolide antibiotic, which is produced by the Streptomyces strains [1]. It displays biological activities against Gram-positive bacteria and fungi. The mode of action of this antibiotic has been attributed to an alteration of the membrane permeability. When this antibiotic is inserted into the bilayer membranes destabilization of the membrane and formation of ion-penetrable channels is observed. The macrodiolide antibiotic forms stable cation selective ion channels in synthetic lipid bilayer membranes. The aim of this work was to study the interactions of Elaiophylin with model bilayer membranes and to get information on the mechanical properties of lipid bilayers in presence of this antibiotic. Patch-clamp technique [2] were used in the study

ZnO nanoparticles modulate the ionic transport and voltage regulation of lysenin nanochannels.

PubMed

Bryant, Sheenah L; Eixenberger, Josh E; Rossland, Steven; Apsley, Holly; Hoffmann, Connor; Shrestha, Nisha; McHugh, Michael; Punnoose, Alex; Fologea, Daniel

2017-12-16

The insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate. The macroscopic conductance of lysenin channels greatly diminished in the presence of cationic ZnO NPs. The inhibitory effects were asymmetrical relative to the direction of the electric field and addition site, suggesting electrostatic interactions between ZnO NPs and a binding site. Similar changes in the macroscopic conductance were observed when lysenin channels were reconstituted in neutral lipid membranes, implicating protein-NP interactions as the major contributor to the reduced transport capabilities. In contrast, no inhibitory effects were observed in the presence of anionic SnO 2 NPs. Additionally, we demonstrate that inhibition of ion transport is not due to the dissolution of ZnO NPs and subsequent interactions of zinc ions with lysenin channels. We conclude that electrostatic interactions between positively charged ZnO NPs and negative charges within the lysenin channels are responsible for the inhibitory effects on the transport of ions. These interactions point to a potential mechanism of cytotoxicity, which may not require NP internalization.

Ion Channels in Obesity: Pathophysiology and Potential Therapeutic Targets

PubMed Central

Vasconcelos, Luiz H. C.; Souza, Iara L. L.; Pinheiro, Lílian S.; Silva, Bagnólia A.

2016-01-01

Obesity is a multifactorial disease related to metabolic disorders and associated with genetic determinants. Currently, ion channels activity has been linked to many of these disorders, in addition to the central regulation of food intake, energetic balance, hormone release and response, as well as the adipocyte cell proliferation. Therefore, the objective of this work is to review the current knowledge about the influence of ion channels in obesity development. This review used different sources of literature (Google Scholar, PubMed, Scopus, and Web of Science) to assess the role of ion channels in the pathophysiology of obesity. Ion channels present diverse key functions, such as the maintenance of physiological homeostasis and cell proliferation. Cell biology and pharmacological experimental evidences demonstrate that proliferating cells exhibit ion channel expression, conductance, and electrical properties different from the resting cells. Thereby, a large variety of ion channels has been identified in the pathogenesis of obesity such as potassium, sodium, calcium and chloride channels, nicotinic acetylcholine receptor and transient receptor potential channels. The fundamental involvement of these channels on the generation of obesity leads to the progress in the knowledge about the mechanisms responsible for the obesity pathophysiology, consequently emerging as new targets for pharmacological modulation. PMID:27065858

Pore Helices Play a Dynamic Role as Integrators of Domain Motion during Kv11.1 Channel Inactivation Gating*

PubMed Central

Perry, Matthew D.; Ng, Chai Ann; Vandenberg, Jamie I.

2013-01-01

Proteins that form ion-selective pores in the membrane of cells are integral to many rapid signaling processes, including regulating the rhythm of the heartbeat. In potassium channels, the selectivity filter is critical for both endowing an exquisite selectivity for potassium ions, as well as for controlling the flow of ions through the pore. Subtle rearrangements in the complex hydrogen-bond network that link the selectivity filter to the surrounding pore helices differentiate conducting (open) from nonconducting (inactivated) conformations of the channel. Recent studies suggest that beyond the selectivity filter, inactivation involves widespread rearrangements of the channel protein. Here, we use rate equilibrium free energy relationship analysis to probe the structural changes that occur during selectivity filter gating in Kv11.1 channels, at near atomic resolution. We show that the pore helix plays a crucial dynamic role as a bidirectional interface during selectivity filter gating. We also define the molecular bases of the energetic coupling between the pore helix and outer helix of the pore domain that occurs early in the transition from open to inactivated states, as well as the coupling between the pore helix and inner helix late in the transition. Our data demonstrate that the pore helices are more than just static structural elements supporting the integrity of the selectivity filter; instead they play a crucial dynamic role during selectivity filter gating. PMID:23471968

Acid-Sensing Ion Channel Pharmacology, Past, Present, and Future ….

PubMed

Rash, Lachlan D

2017-01-01

pH is one of the most strictly controlled parameters in mammalian physiology. An extracellular pH of ~7.4 is crucial for normal physiological processes, and perturbations to this have profound effects on cell function. Acidic microenvironments occur in many physiological and pathological conditions, including inflammation, bone remodeling, ischemia, trauma, and intense synaptic activity. Cells exposed to these conditions respond in different ways, from tumor cells that thrive to neurons that are either suppressed or hyperactivated, often fatally. Acid-sensing ion channels (ASICs) are primary pH sensors in mammals and are expressed widely in neuronal and nonneuronal cells. There are six main subtypes of ASICs in rodents that can form homo- or heteromeric channels resulting in many potential combinations. ASICs are present and activated under all of the conditions mentioned earlier, suggesting that they play an important role in how cells respond to acidosis. Compared to many other ion channel families, ASICs were relatively recently discovered-1997-and there is a substantial lack of potent, subtype-selective ligands that can be used to elucidate their structural and functional properties. In this chapter I cover the history of ASIC channel pharmacology, which began before the proteins were even identified, and describe the current arsenal of tools available, their limitations, and take a glance into the future to predict from where new tools are likely to emerge. © 2017 Elsevier Inc. All rights reserved.

Stability of parallel electroosmotic flow subject to an axial modulated electric field

NASA Astrophysics Data System (ADS)

Suresh, Vinod; Homsy, George

2001-11-01

The stability of parallel electroosmotic flow in a micro-channel subjected to an AC electric field is studied. A spatially uniform time harmonic electric field is applied along the length of a two-dimensional micro-channel containing a dilute electrolytic solution, resulting in a time periodic parallel flow. The top and bottom walls of the channel are maintained at constant potential. The base state ion concentrations and double layer potential are determined using the Poisson-Boltzmann equation in the Debye-Hückel approximation. Experiments by other workers (Santiago et. al., unpublished) have shown that such a system can exhibit instabilities that take the form of mixing motion occurring in the bulk flow outside the double layer. It is shown that such instabilities can potentially result from the coupling of disturbances in the ion concentrations or electric potential to the base state velocity or ion concentrations, respectively. The stability boundary of the system is determined using Floquet theory and its dependence on the modulation frequency and amplitude of the axial electric field is studied.

3-D indium(III)-btc channel frameworks and their ion-exchange properties (btc=1,3,5-benzenetricarboxylate)

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

Lin Zhengzhong; Chen Lian; Yue Chengyang

2006-04-15

Assembly of InCl{sub 3} with 1,3,5-benzenetricarboxylic acid (H{sub 3}btc) and pyridine or pyridine derivatives under hydrothermal conditions produces a series of isostructural coordination polymers with the interesting frameworks: {l_brace}(HL)[In{sub 4}(OH){sub 4}(btc){sub 3}].L.3H{sub 2}O{r_brace} {sub n} {sub ,} L=pyridine (1); L=2-picoline (2); L=4-picoline (3) and {l_brace}(Hdpea)[In{sub 4}(OH){sub 4}(btc){sub 3}].3H{sub 2}O{r_brace} {sub n} (4) (dpea=1,2-di(4-pyridyl)ethane). In these four complexes, carboxyl and hydroxyl oxygen atoms bridge indium(III) centers to form octahedral chain-like sinusoidal curves, which are further interlinked by btc{sup 3-} moieties to generate 3-D frameworks with 1-D channels. The protonated guests HL in 1-3 located at the channels can be fully exchangedmore » by K{sup +} ion or partially exchanged by Sr{sup 2+}, and Ba{sup 2+} ions.« less

Fabrication of Buried Nanochannels From Nanowire Patterns

NASA Technical Reports Server (NTRS)

Choi, Daniel; Yang, Eui-Hyeok

2007-01-01

A method of fabricating channels having widths of tens of nanometers in silicon substrates and burying the channels under overlying layers of dielectric materials has been demonstrated. With further refinement, the method might be useful for fabricating nanochannels for manipulation and analysis of large biomolecules at single-molecule resolution. Unlike in prior methods, burying the channels does not involve bonding of flat wafers to the silicon substrates to cover exposed channels in the substrates. Instead, the formation and burying of the channels are accomplished in a more sophisticated process that is less vulnerable to defects in the substrates and less likely to result in clogging of, or leakage from, the channels. In this method, the first step is to establish the channel pattern by forming an array of sacrificial metal nanowires on an SiO2-on-Si substrate. In particular, the wire pattern is made by use of focused-ion-beam (FIB) lithography and a subsequent metallization/lift-off process. The pattern of metal nanowires is then transferred onto the SiO2 layer by reactive-ion etching, which yields sacrificial SiO2 nanowires covered by metal. After removal of the metal covering the SiO2 nanowires, what remains are SiO2 nanowires on an Si substrate. Plasma-enhanced chemical vapor deposition (PECVD) is used to form a layer of a dielectric material over the Si substrate and over the SiO2 wires on the surface of the substrate. FIB milling is then performed to form trenches at both ends of each SiO2 wire. The trenches serve as openings for the entry of chemicals that etch SiO2 much faster than they etch Si. Provided that the nanowires are not so long that the diffusion of the etching chemicals is blocked, the sacrificial SiO2 nanowires become etched out from between the dielectric material and the Si substrate, leaving buried channels. At the time of reporting the information for this article, channels 3 m long, 20 nm deep, and 80 nm wide (see figure) had been fabricated by this method.

Connexins and Pannexins in Vascular Function and Disease.

PubMed

Molica, Filippo; Figueroa, Xavier F; Kwak, Brenda R; Isakson, Brant E; Gibbins, Jonathan M

2018-06-05

Connexins (Cxs) and pannexins (Panxs) are ubiquitous membrane channel forming proteins that are critically involved in many aspects of vascular physiology and pathology. The permeation of ions and small metabolites through Panx channels, Cx hemichannels and gap junction channels confers a crucial role to these proteins in intercellular communication and in maintaining tissue homeostasis. This review provides an overview of current knowledge with respect to the pathophysiological role of these channels in large arteries, the microcirculation, veins, the lymphatic system and platelet function. The essential nature of these membrane proteins in vascular homeostasis is further emphasized by the pathologies that are linked to mutations and polymorphisms in Cx and Panx genes.

The electronic stopping powers and angular energy-loss dependence of helium and lithium ions in the silicon crystal

NASA Astrophysics Data System (ADS)

Mikšová, R.; Macková, A.; Malinský, P.

2017-09-01

We have measured the electronic stopping powers of helium and lithium ions in the channelling direction of the Si〈1 0 0〉 crystal. The energy range used (2.0-8.0 MeV) was changed by 200 and 400-keV steps. The ratio α between the channelling and random stopping powers was determined as a function of the angle for 2, 3 and 4 MeV 4He+ ions and for 3 and 6 MeV 7Li+,2+ ions. The measurements were carried out using the Rutherford backscattering spectrometry in the channelling mode (RBS-C) in a silicon-on-insulator material. The experimental channelling stopping-power values measured in the channelling direction were then discussed in the frame of the random energy stopping predictions calculated using SRIM-2013 code and the theoretical unitary convolution approximation (UCA) model. The experimental channelling stopping-power values decrease with increasing ion energy. The stopping-power difference between channelled and randomly moving ions increases with the enhanced initial ion energy. The ratio between the channelling and random ion stopping powers α as a function of the ion beam incoming angle for 2, 3 and 4 MeV He+ ions and for 3 and 6 MeV Li+,2+ ions was observed in the range 0.5-1.

Hierarchically porous carbon with high-speed ion transport channels for high performance supercapacitors

NASA Astrophysics Data System (ADS)

Lu, Haoyuan; Li, Qingwei; Guo, Jianhui; Song, Aixin; Gong, Chunhong; Zhang, Jiwei; Zhang, Jingwei

2018-01-01

Hierarchically porous carbons (HPC) are considered as promising electrode materials for supercapacitors, due to their outstanding charge/discharge cycling stabilities and high power densities. However, HPC possess a relatively low ion diffusion rate inside the materials, which challenges their application for high performance supercapacitor. Thus tunnel-shaped carbon pores with a size of tens of nanometers were constructed by inducing the self-assembly of lithocholic acid with ammonium chloride, thereby providing high-speed channels for internal ion diffusion. The as-formed one-dimensional pores are beneficial to the activation process by KOH, providing a large specific surface area, and then facilitate rapid transport of electrolyte ions from macropores to the microporous surfaces. Therefore, the HPC achieve an outstanding gravimetric capacitance of 284 F g-1 at a current density of 0.1 A g-1 and a remarkable capacity retention of 64.8% when the current density increases by 1000 times to 100 A g-1.

High throughput screening technologies for ion channels

PubMed Central

Yu, Hai-bo; Li, Min; Wang, Wei-ping; Wang, Xiao-liang

2016-01-01

Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications. PMID:26657056

Incorporating Born solvation energy into the three-dimensional Poisson-Nernst-Planck model to study ion selectivity in KcsA K+ channels

NASA Astrophysics Data System (ADS)

Liu, Xuejiao; Lu, Benzhuo

2017-12-01

Potassium channels are much more permeable to potassium than sodium ions, although potassium ions are larger and both carry the same positive charge. This puzzle cannot be solved based on the traditional Poisson-Nernst-Planck (PNP) theory of electrodiffusion because the PNP model treats all ions as point charges, does not incorporate ion size information, and therefore cannot discriminate potassium from sodium ions. The PNP model can qualitatively capture some macroscopic properties of certain channel systems such as current-voltage characteristics, conductance rectification, and inverse membrane potential. However, the traditional PNP model is a continuum mean-field model and has no or underestimates the discrete ion effects, in particular the ion solvation or self-energy (which can be described by Born model). It is known that the dehydration effect (closely related to ion size) is crucial to selective permeation in potassium channels. Therefore, we incorporated Born solvation energy into the PNP model to account for ion hydration and dehydration effects when passing through inhomogeneous dielectric channel environments. A variational approach was adopted to derive a Born-energy-modified PNP (BPNP) model. The model was applied to study a cylindrical nanopore and a realistic KcsA channel, and three-dimensional finite element simulations were performed. The BPNP model can distinguish different ion species by ion radius and predict selectivity for K+ over Na+ in KcsA channels. Furthermore, ion current rectification in the KcsA channel was observed by both the PNP and BPNP models. The I -V curve of the BPNP model for the KcsA channel indicated an inward rectifier effect for K+ (rectification ratio of ˜3 /2 ) but indicated an outward rectifier effect for Na+ (rectification ratio of ˜1 /6 ) .

Kainate receptor pore‐forming and auxiliary subunits regulate channel block by a novel mechanism

PubMed Central

Brown, Patricia M. G. E.; Aurousseau, Mark R. P.; Musgaard, Maria; Biggin, Philip C.

2016-01-01

Key points Kainate receptor heteromerization and auxiliary subunits, Neto1 and Neto2, attenuate polyamine ion‐channel block by facilitating blocker permeation.Relief of polyamine block in GluK2/GluK5 heteromers results from a key proline residue that produces architectural changes in the channel pore α‐helical region.Auxiliary subunits exert an additive effect to heteromerization, and thus relief of polyamine block is due to a different mechanism.Our findings have broad implications for work on polyamine block of other cation‐selective ion channels. Abstract Channel block and permeation by cytoplasmic polyamines is a common feature of many cation‐selective ion channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α‐helical region of the selectivity filter. The effect of auxiliary proteins is additive, and therefore the structural basis of polyamine permeation and unblock is through a different mechanism. As native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation‐selective ion channels. PMID:26682513

Ion Velocity Distribution in a Low-Power Cylindrical Hall Thruster

DTIC Science & Technology

2010-07-01

profile inside a CHT – the magnetic field lines are believed to form equipotential surfaces , creating an electric field that has a significant axial...centerline of the channel. The resulting equipotential surfaces provide an electric field profile with a significant outward pointing radial component...pole and creating a region of the channel with a low surface -to-volume area (a cylindrical region), the CHT as developed by Princeton University reduces

Structure and selectivity in bestrophin ion channels

DOE PAGES

Yang, Tingting; Liu, Qun; Kloss, Brian; ...

2014-09-25

Human bestrophin 1 (hBest1) is a calcium-activated chloride channel from the retinal pigment epithelium, where it can suffer mutations associated with vitelliform macular degeneration, or Best disease. We describe the structure of a bacterial homolog (KpBest) of hBest1 and functional characterizations of both channels. KpBest is a pentamer that forms a five-helix transmembrane pore, closed by three rings of conserved hydrophobic residues, and has a cytoplasmic cavern with a restricted exit. From electrophysiological analysis of structure-inspired mutations in KpBest and hBest1, we find a subtle control of ion selectivity in the bestrophins, including reversal of anion/cation selectivity, and dramatic activationmore » by mutations at the exit restriction. Lastly, a homology model of hBest1 shows the locations of disease-causing mutations and suggests possible roles in regulation.« less

Giga-ohm seals on intracellular membranes: a technique for studying intracellular ion channels in intact cells.

PubMed

Jonas, E A; Knox, R J; Kaczmarek, L K

1997-07-01

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Photoionization and Photofragmentation of Carbon Fullerene Molecular Ions

NASA Astrophysics Data System (ADS)

Baral, Kiran Kumar

Cross sections are reported for single and double photoionization accompanied by the loss of as many as seven pairs of C atoms of C60 + and C70+ fullerene molecular ions in the photon energy range 18 eV to 150 eV. These measurements were performed at the Advanced Light Source (ALS) by merging a mass-selected ion beam with a beam of monochromatized synchrotron radiation. Threshold energies were determined for the formation of doubly and triply charged fragment ions from parent ions C60+ and C70+. The energy dependences of cross-sections for direct photoionization yielding C60 2+ and C702+ are compared with those for forming different doubly and triply charged fullerene fragment ions. Two-dimensional product ion scans were measured and quantified at four discrete photon energies: 35 eV, 65 eV, 105 eV and 140 eV, in the vacuum ultraviolet region, providing a comprehensive mapping of the product channels involving single ionization of fullerene ions C60+ and C 70+ accompanied by fragmentation. Since fullerenes are composed of even numbers of carbon atoms, the fragmentation occurs by the loss of differing numbers of carbon atom pairs. In addition to pure ionization, fragmentation product channels become relatively more important at higher photon energies.

Ion channels versus ion pumps: the principal difference, in principle.

PubMed

Gadsby, David C

2009-05-01

The incessant traffic of ions across cell membranes is controlled by two kinds of border guards: ion channels and ion pumps. Open channels let selected ions diffuse rapidly down electrical and concentration gradients, whereas ion pumps labour tirelessly to maintain the gradients by consuming energy to slowly move ions thermodynamically uphill. Because of the diametrically opposed tasks and the divergent speeds of channels and pumps, they have traditionally been viewed as completely different entities, as alike as chalk and cheese. But new structural and mechanistic information about both of these classes of molecular machines challenges this comfortable separation and forces its re-evaluation.

Structure, function and translational relevance of aquaporin dual water and ion channels.

PubMed

Yool, Andrea J; Campbell, Ewan M

2012-01-01

Aquaporins have been assumed to be selective for water alone, and aquaglyceroporins are accepted as carrying water and small uncharged solutes including glycerol. This review presents an expanded view of aquaporins as channels with more complex mechanisms of regulation and diverse repertoires of substrate permeabilities than were originally appreciated in the early establishment of the field. The role of aquaporins as dual water and gated ion channels is likely to have physiological and potentially translational relevance, and can be evaluated with newly developed molecular and pharmacological tools. Ion channel activity has been shown for Aquaporins -0, -1, and -6, Drosphila Big Brain, and plant Nodulin-26. Although the concept of ion channel function in aquaporins remains controversial, research advances are beginning to define not only the ion channel function but also the detailed molecular mechanisms that govern and mediate the multifunctional capabilities. With regard to physiological relevance, the adaptive benefit of expression of ion channel activity in aquaporins, implied by amino acid sequence conservation of the ion channel gating domains, suggests they provide more than water or glycerol and solute transport. Dual ion and water channels are of interest for understanding the modulation of transmembrane fluid gradients, volume regulation, and possible signal transduction in tissues expressing classes of aquaporins that have the dual function capability. Other aquaporin classes might be found in future work to have ion channel activities, pending identification of the possible signaling pathways that could govern activation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Influence of 1α, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] on the expression of Sox 9 and the transient receptor potential vanilloid 5/6 ion channels in equine articular chondrocytes.

PubMed

Hdud, Ismail M; Loughna, Paul T

2014-01-01

Sox 9 is a major marker of chondrocyte differentiation. When chondrocytes are cultured in vitro they progressively de-differentiate and this is associated with a decline in Sox 9 expression. The active form of vitamin D, 1, 25 (OH)2D3 has been shown to be protective of cartilage in both humans and animals. In this study equine articular chondrocytes were grown in culture and the effects of 1, 25 (OH)2D3 upon Sox 9 expression examined. The expression of the transient receptor potential vanilloid (TRPV) ion channels 5 and 6 in equine chondrocytes in vitro, we have previously shown, is inversely correlated with de-differentiation. The expression of these channels in response to 1, 25 (OH)2D3 administration was therefore also examined. The active form of vitamin D (1, 25 (OH)2D3) when administered to cultured equine chondrocytes at two different concentrations significantly increased the expression of Sox 9 at both. In contrast 1, 25 (OH)2D3 had no significant effect upon the expression of either TRPV 5 or 6 at either the protein or the mRNA level. The increased expression of Sox 9, in equine articular chondrocytes in vitro, in response to the active form of vitamin D suggests that this compound could be utilized to inhibit the progressive de-differentiation that is normally observed in these cells. It is also supportive of previous studies indicating that 1α, 25-dihydroxyvitamin D3 can have a protective effect upon cartilage in animals in vivo. The previously observed correlation between the degree of differentiation and the expression levels of TRPV 5/6 had suggested that these ion channels may have a direct involvement in, or be modulated by, the differentiation process in vitro. The data in the present study do not support this.

Marine Toxins Targeting Ion Channels

PubMed Central

Arias, Hugo R.

2006-01-01

This introductory minireview points out the importance of ion channels for cell communication. The basic concepts on the structure and function of ion channels triggered by membrane voltage changes, the so-called voltage-gated ion channels (VGICs), as well as those activated by neurotransmitters, the so-called ligand-gated ion channel (LGICs), are introduced. Among the most important VGIC superfamiles, we can name the voltage-gated Na+ (NaV), Ca2+ (CaV), and K+ (KV) channels. Among the most important LGIC super families, we can include the Cys-loop or nicotinicoid, the glutamate-activated (GluR), and the ATP-activated (P2XnR) receptor superfamilies. Ion channels are transmembrane proteins that allow the passage of different ions in a specific or unspecific manner. For instance, the activation of NaV, CaV, or KV channels opens a pore that is specific for Na+, Ca2+, or K+, respectively. On the other hand, the activation of certain LGICs such as nicotinic acetylcholine receptors, GluRs, and P2XnRs allows the passage of cations (e.g., Na+, K+, and/or Ca2+), whereas the activation of other LGICs such as type A γ-butyric acid and glycine receptors allows the passage of anions (e.g., Cl− and/or HCO3−). In this regard, the activation of NaV and CaV as well as ligand-gated cation channels produce membrane depolarization, which finally leads to stimulatory effects in the cell, whereas the activation of KV as well as ligand-gated anion channels induce membrane hyperpolarization that finally leads to inhibitory effects in the cell. The importance of these ion channel superfamilies is emphasized by considering their physiological functions throughout the body as well as their pathophysiological implicance in several neuronal diseases. In this regard, natural molecules, and especially marine toxins, can be potentially used as modulators (e.g., inhibitors or prolongers) of ion channel functions to treat or to alleviate a specific ion channel-linked disease (e.g., channelopaties).

Acid stress mediated adaptive divergence in ion channel function during embryogenesis in Rana arvalis

PubMed Central

Shu, Longfei; Laurila, Anssi; Räsänen, Katja

2015-01-01

Ion channels and pumps are responsible for ion flux in cells, and are key mechanisms mediating cellular function. Many environmental stressors, such as salinity and acidification, are known to severely disrupt ionic balance of organisms thereby challenging fitness of natural populations. Although ion channels can have several vital functions during early life-stages (e.g. embryogenesis), it is currently not known i) how developing embryos maintain proper intracellular conditions when exposed to environmental stress and ii) to what extent environmental stress can drive intra-specific divergence in ion channels. Here we studied the moor frog, Rana arvalis, from three divergent populations to investigate the role of different ion channels and pumps for embryonic survival under acid stress (pH 4 vs 7.5) and whether populations adapted to contrasting acidities differ in the relative role of different ion channel/pumps. We found that ion channels that mediate Ca2+ influx are essential for embryonic survival under acidic pH, and, intriguingly, that populations differ in calcium channel function. Our results suggest that adaptive divergence in embryonic acid stress tolerance of amphibians may in part be mediated by Ca2+ balance. We suggest that ion flux may mediate adaptive divergence of natural populations at early life-stages in the face of environmental stress. PMID:26381453

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

PubMed

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

2009-01-01

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

TRPP2 ion channels: Critical regulators of organ morphogenesis in health and disease.

PubMed

Busch, Tilman; Köttgen, Michael; Hofherr, Alexis

2017-09-01

Ion channels control the membrane potential and mediate transport of ions across membranes. Archetypical physiological functions of ion channels include processes such as regulation of neuronal excitability, muscle contraction, or transepithelial ion transport. In that regard, transient receptor potential ion channel polycystin 2 (TRPP2) is remarkable, because it controls complex morphogenetic processes such as the establishment of properly shaped epithelial tubules and left-right-asymmetry of organs. The fascinating question of how an ion channel regulates morphogenesis has since captivated the attention of scientists in different disciplines. Four loosely connected key insights on different levels of biological complexity ranging from protein to whole organism have framed our understanding of TRPP2 physiology: 1) TRPP2 is a non-selective cation channel; 2) TRPP2 is part of a receptor-ion channel complex; 3) TRPP2 localizes to primary cilia; and 4) TRPP2 is required for organ morphogenesis. In this review, we will discuss the current knowledge in these key areas and highlight some of the challenges ahead. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Ion and Electron Heating Characteristics of Magnetic Re- Connection in Mast Tokamak Merging Experiment

NASA Astrophysics Data System (ADS)

Tanabe, Hiroshi; Inomoto, Michiaki; Ono, Yasushi; Yamada, Takuma; Imazawa, Ryota; Cheng, Chio-Zong

2016-07-01

We present results of recent studies of high power heating of magnetic reconnection, the fundamental process of several astrophysical events such as solar flare, in the Mega Amp Spherical Tokamak (MAST) - the world largest merging experiment. In addition to the previously reported significant reconnection heating up to ˜1keV [1], detailed local profiles of electron and ion temperature have been measured using a ultra-fine 300 channel Ruby- and a 130 channel YAG-Thomson scattering and a new 32 channel ion Doppler tomography diagnostics [2]. 2D profile measurement of electron temperature revealed highly localized heating structure at the X point with the characteristic scale length of 0.02-0.05m0.3T), a thick layer of closed flux surface surrounding the current sheet sustains the temperature profile for longer time than the electron and ion energy relaxation time ˜4-10ms, finally forming triple peak structures of ion and electron temperatures at the X point and in the downstream. While the peak electron temperature at the X point increases with toroidal field, the bulk electron temperature and the ion temperature in the downstream are unaffected. [1] Y. Ono et.al., Plasma Phys. Control. Fusion 54, 124039 (2012) [2] H. Tanabe et. al., Nucl. Fusion 53, 093027 (2013). [3] H. Tanabe et.al., Phys. Rev. Lett. 115, 215004 (2015)

Lunar Ion Transport Near Magnetic Anomalies: Possible Implications for Swirl Formation

NASA Technical Reports Server (NTRS)

Keller, J. W.; Killen, R. M.; Stubbs, T. J.; Farrell, W. M.; Halekas, J. S.

2011-01-01

The bright swirling features on the lunar surface in areas around the Moon but most prominently at Reiner Gamma, have intrigued scientists for many years. After Apollo and later Lunar Prospector (LP} mapped the Lunar magnetic fields from orbit, it was observed that these features are generally associated with crustal magnetic anomalies. This led researchers to propose a number of explanations for the swirls that invoke these fields. Prominent among these include magnetic shielding in the form of a mini-magnetosphere which impedes space weathering by the solar wind, magnetically controlled dust transport, and cometary or asteroidal impacts that would result in shock magnetization with concomitant formation ofthe swirls. In this presentation, we will consider another possibility, that the ambient magnetic and electric fields can transport and channel secondary ions produced by micrometeorite or solar wind ion impacts. In this scenario, ions that are created in these impacts are under the influence of these fields and can drift for significant distances before encountering the magnetic anomalies when their trajectories are disrupted and concentrated onto nearby areas. These ions may then be responsible for chemical alteration of the surface leading either to a brightening effect or a disruption of space weathering processes. To test this hypothesis we have run ion trajectory simulations that show ions from regions about the magnetic anomalies can be channeled into very small areas near the anomalies and although questions remain as to nature of the mechanisms that could lead to brightening of the surface it appears that the channeling effect is consistent with the existence of the swirls.

Differential subcellular distribution of ion channels and the diversity of neuronal function.

PubMed

Nusser, Zoltan

2012-06-01

Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

A single Markov-type kinetic model accounting for the macroscopic currents of all human voltage-gated sodium channel isoforms.

PubMed

Balbi, Pietro; Massobrio, Paolo; Hellgren Kotaleski, Jeanette

2017-09-01

Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH). However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs). The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms) and computationally efficient (i.e. with a minimal set of states and transitions). The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9). By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane potential). The kinetic model, developed in the NEURON simulation environment, appears to be the simplest and most parsimonious way for a detailed phenomenological description of the human VGSCs electrophysiological behaviour.

Functional nicotinic acetylcholine receptor reconstitution in Au(111)-supported thiolipid monolayers

NASA Astrophysics Data System (ADS)

Pissinis, Diego E.; Diaz, Carolina; Maza, Eliana; Bonini, Ida C.; Barrantes, Francisco J.; Salvarezza, Roberto C.; Schilardi, Patricia L.

2015-09-01

The insertion and function of the muscle-type nicotinic acetylcholine receptor (nAChR) in Au(111)-supported thiolipid self-assembled monolayers have been studied by atomic force microscopy (AFM), surface plasmon resonance (SPR), and electrochemical techniques. It was possible for the first time to resolve the supramolecular arrangement of the protein spontaneously inserted in a thiolipid monolayer in an aqueous solution. Geometric supramolecular arrays of nAChRs were observed, most commonly in a triangular form compatible with three nAChR dimers of ~20 nm each. Addition of the full agonist carbamoylcholine activated and opened the nAChR ion channel, as revealed by the increase in capacitance relative to that of the nAChR-thiolipid system under basal conditions. Thus, the self-assembled system appears to be a viable biomimetic model to measure ionic conductance mediated by ion-gated ion channels under different experimental conditions, with potential applications in biotechnology and pharmacology.

Polarized targeting of a shaker-like (A-type) K(+)-channel in the polarized epithelial cell line MDCK.

PubMed

Le Maout, S; Sewing, S; Coudrier, E; Elalouf, J M; Pongs, O; Merot, J

1996-01-01

Functional Kv 1-4 channels were stably expressed in filter-grown MDCK cells which form a polarized epithelium with two distinct plasma membrane domains: a basolateral and an apical cell surface. The Shaker-related Kv 1-4 channels mediated in MDCK cells fast transient (A-type) voltage-activated outward currents having similar properties to the ones reported for Kv 1-4 in the Xenopus oocytes expression system. Immunoblot analysis with specific anti-Kv 1-4 antibodies showed that two Kv 1-4 protein forms are expressed in MDCK cells which most likely represent the glycosylated and non-glycosylated Kv 1-4 protein, respectively. Using immunocytochemistry and confocal microscopy we showed that the Kv 1-4 channels are specifically localized in the basolateral membranes of MDCK cells. Thus, the MDCK cells may provide an important model system to analyse the polarized transport of ion channels such as Kv 1-4, which are distinctly expressed in the mammalian central nervous system.

Ion Channels in Innate and Adaptive Immunity

PubMed Central

Feske, Stefan; Wulff, Heike; Skolnik, Edward Y.

2016-01-01

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy. PMID:25861976

Laser characterization of the unsteady 2-D ion flow field in a Hall thruster with breathing mode oscillations

NASA Astrophysics Data System (ADS)

Lucca Fabris, Andrea; Young, Christopher; MacDonald-Tenenbaum, Natalia; Hargus, William, Jr.; Cappelli, Mark

2016-10-01

Hall thrusters are a mature form of electric propulsion for spacecraft. One commonly observed low frequency (10-50 kHz) discharge current oscillation in these E × B devices is the breathing mode, linked to a propagating ionization front traversing the channel. The complex time histories of ion production and acceleration in the discharge channel and near-field plume lead to interesting dynamics and interactions in the central plasma jet and downstream plume regions. A time-resolved laser-induced fluorescence (LIF) diagnostic non-intrusively measures 2-D ion velocity and relative ion density throughout the plume of a commercial BHT-600 Hall thruster manufactured by Busek Co. Low velocity classes of ions observed in addition to the main accelerated population are linked to propellant ionization outside of the device. Effects of breathing mode dynamics are shown to persist far downstream where modulations in ion velocity and LIF intensity are correlated with discharge current oscillations. This work is sponsored by the U.S. Air Force Office of Scientific Research with Dr. M. Birkan as program manager. C.Y. acknowledges support from the DOE NSSA Stewardship Science Graduate Fellowship under contract DE-FC52-08NA28752.

Ion Trapping with Fast-Response Ion-Selective Microelectrodes Enhances Detection of Extracellular Ion Channel Gradients

PubMed Central

Messerli, Mark A.; Collis, Leon P.; Smith, Peter J.S.

2009-01-01

Previously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 μm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10–55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane. PMID:19217875

Calculating tracer currents through narrow ion channels: Beyond the independent particle model.

PubMed

Coalson, Rob D; Jasnow, David

2018-06-01

Discrete state models of single-file ion permeation through a narrow ion channel pore are employed to analyze the ratio of forward to backward tracer current. Conditions under which the well-known Ussing formula for this ratio hold are explored in systems where ions do not move independently through the channel. Building detailed balance into the rate constants for the model in such a way that under equilibrium conditions (equal rate of forward vs. backward permeation events) the Nernst Equation is satisfied, it is found that in a model where only one ion can occupy the channel at a time, the Ussing formula is always obeyed for any number of binding sites, reservoir concentrations of the ions and electric potential difference across the membrane which the ion channel spans, independent of the internal details of the permeation pathway. However, numerical analysis demonstrates that when multiple ions can occupy the channel at once, the nonequilibrium forward/backward tracer flux ratio deviates from the prediction of the Ussing model. Assuming an appropriate effective potential experienced by ions in the channel, we provide explicit formulae for the rate constants in these models. © 2018 IOP Publishing Ltd.

Endogenous Voltage Potentials and the Microenvironment: Bioelectric Signals that Reveal, Induce and Normalize Cancer

PubMed Central

Chernet, Brook; Levin, Michael

2014-01-01

Cancer may be a disease of geometry: a misregulation of the field of information that orchestrates individual cells’ activities towards normal anatomy. Recent work identified molecular mechanisms underlying a novel system of developmental control: bioelectric gradients. Endogenous spatio-temporal differences in resting potential of non-neural cells provide instructive cues for cell regulation and complex patterning during embryogenesis and regeneration. It is now appreciated that these cues are an important layer of the dysregulation of cell: cell interactions that leads to cancer. Abnormal depolarization of resting potential (Vmem) is a convenient marker for neoplasia and activates a metastatic phenotype in genetically-normal cells in vivo. Moreover, oncogene expression depolarizes cells that form tumor-like structures, but is unable to form tumors if this depolarization is artificially prevented by misexpression of hyperpolarizing ion channels. Vmem triggers metastatic behaviors at considerable distance, mediated by transcriptional and epigenetic effects of electrically-modulated flows of serotonin and butyrate. While in vivo data on voltages in carcinogenesis comes mainly from the amphibian model, unbiased genetic screens and network profiling in rodents and human tissues reveal several ion channel proteins as bona fide oncogene and promising targets for cancer drug development. However, we propose that a focus on specific channel genes is just the tip of the iceberg. Bioelectric state is determined by post-translational gating of ion channels, not only from genetically-specified complements of ion translocators. A better model is a statistical dynamics view of spatial Vmem gradients. Cancer may not originate at the single cell level, since gap junctional coupling results in multi-cellular physiological networks with multiple stable attractors in bioelectrical state space. New medical applications await a detailed understanding of the mechanisms by which organ target morphology stored in real-time patterns of ion flows is perceived or mis-perceived by cells. Mastery of somatic voltage gradients will lead to cancer normalization or rebooting strategies, such as those that occur in regenerating and embryonic organs, resulting in transformative advances in basic biology and oncology. PMID:25525610

ModFossa: A library for modeling ion channels using Python.

PubMed

Ferneyhough, Gareth B; Thibealut, Corey M; Dascalu, Sergiu M; Harris, Frederick C

2016-06-01

The creation and simulation of ion channel models using continuous-time Markov processes is a powerful and well-used tool in the field of electrophysiology and ion channel research. While several software packages exist for the purpose of ion channel modeling, most are GUI based, and none are available as a Python library. In an attempt to provide an easy-to-use, yet powerful Markov model-based ion channel simulator, we have developed ModFossa, a Python library supporting easy model creation and stimulus definition, complete with a fast numerical solver, and attractive vector graphics plotting.

Voltage-dependent ion channels in the mouse RPE: comparison with Norrie disease mice.

PubMed

Wollmann, Guido; Lenzner, Steffen; Berger, Wolfgang; Rosenthal, Rita; Karl, Mike O; Strauss, Olaf

2006-03-01

We studied electrophysiological properties of cultured retinal pigment epithelial (RPE) cells from mouse and a mouse model for Norrie disease. Wild-type RPE cells revealed the expression of ion channels known from other species: delayed-rectifier K(+) channels composed of Kv1.3 subunits, inward rectifier K(+) channels, Ca(V)1.3 L-type Ca(2+) channels and outwardly rectifying Cl(-) channels. Expression pattern and the ion channel characteristics current density, blocker sensitivity, kinetics and voltage-dependence were compared in cells from wild-type and Norrie mice. Although no significant differences were observed, our study provides a base for future studies on ion channel function and dysfunction in transgenic mouse models.

CLC Chloride Channels and Transporters: Structure, Function, Physiology, and Disease.

PubMed

Jentsch, Thomas J; Pusch, Michael

2018-07-01

CLC anion transporters are found in all phyla and form a gene family of eight members in mammals. Two CLC proteins, each of which completely contains an ion translocation parthway, assemble to homo- or heteromeric dimers that sometimes require accessory β-subunits for function. CLC proteins come in two flavors: anion channels and anion/proton exchangers. Structures of these two CLC protein classes are surprisingly similar. Extensive structure-function analysis identified residues involved in ion permeation, anion-proton coupling and gating and led to attractive biophysical models. In mammals, ClC-1, -2, -Ka/-Kb are plasma membrane Cl - channels, whereas ClC-3 through ClC-7 are 2Cl - /H + -exchangers in endolysosomal membranes. Biological roles of CLCs were mostly studied in mammals, but also in plants and model organisms like yeast and Caenorhabditis elegans. CLC Cl - channels have roles in the control of electrical excitability, extra- and intracellular ion homeostasis, and transepithelial transport, whereas anion/proton exchangers influence vesicular ion composition and impinge on endocytosis and lysosomal function. The surprisingly diverse roles of CLCs are highlighted by human and mouse disorders elicited by mutations in their genes. These pathologies include neurodegeneration, leukodystrophy, mental retardation, deafness, blindness, myotonia, hyperaldosteronism, renal salt loss, proteinuria, kidney stones, male infertility, and osteopetrosis. In this review, emphasis is laid on biophysical structure-function analysis and on the cell biological and organismal roles of mammalian CLCs and their role in disease.

Ion-binding properties of the ClC chloride selectivity filter

PubMed Central

Lobet, Séverine; Dutzler, Raimund

2006-01-01

The ClC channels are members of a large protein family of chloride (Cl−) channels and secondary active Cl− transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl− ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl− channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction. PMID:16341087

Simulation of the effect of a magnetically insulated anode on a low-power cylindrical Hall thruster

NASA Astrophysics Data System (ADS)

Yongjie, DING; Hong, LI; Boyang, JIA; Peng, LI; Liqiu, WEI; Yu, XU; Wuji, PENG; Hezhi, SUN; Yong, CAO; Daren, YU

2018-03-01

The intersection point of the characteristic magnetic field line (CMFL) crossing the anode boundary with the discharge channel wall, and its influence on thruster performance and the energy and flux of ions bombarding the channel wall, have been studied numerically. The simulation results demonstrate that with the increase in distance from the crossover point of the CMFL with the channel wall to the bottom of the thruster channel, the ionization rate in the discharge channel gradually increases; meanwhile, the ion energy and ion current density bombarding the channel wall decreases. When the point of the CMFL with the channel wall is at the channel outlet, the thrust, specific impulse, and efficiency are at a maximum, while the ion energy and ion current density bombarding the channel wall are at a minimum. Therefore, to improve the performance and lifetime of the thruster, it is important to control the point of intersection of the CMFL with the channel wall.

Lipid bilayers suspended on microfabricated supports

NASA Astrophysics Data System (ADS)

Ogier, Simon D.; Bushby, Richard J.; Cheng, Yaling; Cox, Tim I.; Evans, Stephen D.; Knowles, Peter F.; Miles, Robert E.; Pattison, Ian

2001-03-01

The plasma membrane, that exists as part of many animal and plant cells, is a regulator for the transport of ions and small molecules across cell boundaries. Two main components involved are the phospholipid bilayer and the transport proteins. This paper details the construction of a micromachined support for bilayers (MSB) as a first step towards the development of highly selective and highly sensitive ion-channel based biosensors. The device consists of a ~100 micrometer hole in a polymeric support above a cavity that can hold ~25 nL of electrolyte. Electrodes attached to the structure allow the resistance of the membranes to be measured using d.c. conductivity. The MSB is made in two halves, using SU8 ultra-thick resist, which are subsequently bonded together to make the final structure. A layer of gold, surrounding the aperture, enables self-assembled monolayers of alkanethiols to be used to make the polymeric structure biocompatible. Lipid membranes have been formed over these holes with resistances comparable with those of natural membranes >10 MOhmcm^2. The ion-channel gramicidin has successfully been incorporated into the bilayer and its activity monitored. It is proposed that this type of device could be used not only for studying membrane transport phenomena but also as part of an ion-channel based biosensor.

KCNE Regulation of K+ Channel Trafficking – a Sisyphean Task?

PubMed Central

Kanda, Vikram A.; Abbott, Geoffrey W.

2012-01-01

Voltage-gated potassium (Kv) channels shape the action potentials of excitable cells and regulate membrane potential and ion homeostasis in excitable and non-excitable cells. With 40 known members in the human genome and a variety of homomeric and heteromeric pore-forming α subunit interactions, post-translational modifications, cellular locations, and expression patterns, the functional repertoire of the Kv α subunit family is monumental. This versatility is amplified by a host of interacting proteins, including the single membrane-spanning KCNE ancillary subunits. Here, examining both the secretory and the endocytic pathways, we review recent findings illustrating the surprising virtuosity of the KCNE proteins in orchestrating not just the function, but also the composition, diaspora and retrieval of channels formed by their Kv α subunit partners. PMID:22754540

Nervous System Complexity Baffles Scientists.

ERIC Educational Resources Information Center

Fox, Jeffrey L.

1982-01-01

New research findings about how nerve cells transmit signals are forcing researchers to overhaul their simplistic ideas about the nervous system. Topics highlighted include the multiple role of peptides in the nervous system, receptor molecules, and molecules that form ion channels within membranes. (Author/JN)

Effects of channel noise on firing coherence of small-world Hodgkin-Huxley neuronal networks

NASA Astrophysics Data System (ADS)

Sun, X. J.; Lei, J. Z.; Perc, M.; Lu, Q. S.; Lv, S. J.

2011-01-01

We investigate the effects of channel noise on firing coherence of Watts-Strogatz small-world networks consisting of biophysically realistic HH neurons having a fraction of blocked voltage-gated sodium and potassium ion channels embedded in their neuronal membranes. The intensity of channel noise is determined by the number of non-blocked ion channels, which depends on the fraction of working ion channels and the membrane patch size with the assumption of homogeneous ion channel density. We find that firing coherence of the neuronal network can be either enhanced or reduced depending on the source of channel noise. As shown in this paper, sodium channel noise reduces firing coherence of neuronal networks; in contrast, potassium channel noise enhances it. Furthermore, compared with potassium channel noise, sodium channel noise plays a dominant role in affecting firing coherence of the neuronal network. Moreover, we declare that the observed phenomena are independent of the rewiring probability.

Brownian dynamics study of ion transport in the vestibule of membrane channels.

PubMed

Li, S C; Hoyles, M; Kuyucak, S; Chung, S H

1998-01-01

Brownian dynamics simulations have been carried out to study the transport of ions in a vestibular geometry, which offers a more realistic shape for membrane channels than cylindrical tubes. Specifically, we consider a torus-shaped channel, for which the analytical solution of Poisson's equation is possible. The system is composed of the toroidal channel, with length and radius of the constricted region of 80 A and 4 A, respectively, and two reservoirs containing 50 sodium ions and 50 chloride ions. The positions of each of these ions executing Brownian motion under the influence of a stochastic force and a systematic electric force are determined at discrete time steps of 50 fs for up to 2.5 ns. All of the systematic forces acting on an ion due to the other ions, an external electric field, fixed charges in the channel protein, and the image charges induced at the water-protein boundary are explicitly included in the calculations. We find that the repulsive dielectric force arising from the induced surface charges plays a dominant role in channel dynamics. It expels an ion from the vestibule when it is deliberately put in it. Even in the presence of an applied electric potential of 100 mV, an ion cannot overcome this repulsive force and permeate the channel. Only when dipoles of a favorable orientation are placed along the sides of the transmembrane segment can an ion traverse the channel under the influence of a membrane potential. When the strength of the dipoles is further increased, an ion becomes detained in a potential well, and the driving force provided by the applied field is not sufficient to drive the ion out of the well. The trajectory of an ion navigating across the channel mostly remains close to the central axis of the pore lumen. Finally, we discuss the implications of these findings for the transport of ions across the membrane.

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions

PubMed Central

Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A.; Bukauskas, Feliksas F.

2018-01-01

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pHi) and cytosolic magnesium ion concentration ([Mg2+]i), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pHi and [Mg2+]i affect junctional conductance (gj) in an interdependent manner; in other words, intracellular acidification cause increase or decay in gj depending on whether [Mg2+]i is high or low, respectively, and intracellular alkalization cause reduction in gj independently of [Mg2+]i. Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pHi and [Mg2+]i. Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pHi and [Mg2+]i. Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg2+]i, while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36*E8Q lost the initial increase of gj at low [Mg2+]i and double mutation lost the sensitivity to high [Mg2+]i. These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg2+ and H+ ions. PMID:29706896

Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6).

PubMed

Azumaya, Caleigh M; Sierra-Valdez, Francisco; Cordero-Morales, Julio F; Nakagawa, Terunaga

2018-05-11

The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here we report a cryoEM structure of the cytoplasmic domain of murine TRPC6 at 3.8Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike in other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Saturation of conductance in single ion channels: the blocking effect of the near reaction field.

PubMed

Nadler, Boaz; Schuss, Zeev; Hollerbach, Uwe; Eisenberg, R S

2004-11-01

The ionic current flowing through a protein channel in the membrane of a biological cell depends on the concentration of the permeant ion, as well as on many other variables. As the concentration increases, the rate of arrival of bath ions to the channel's entrance increases, and typically so does the net current. This concentration dependence is part of traditional diffusion and rate models that predict Michaelis-Menten current-concentration relations for a single ion channel. Such models, however, neglect other effects of bath concentrations on the net current. The net current depends not only on the entrance rate of ions into the channel, but also on forces acting on ions inside the channel. These forces, in turn, depend not only on the applied potential and charge distribution of the channel, but also on the long-range Coulombic interactions with the surrounding bath ions. In this paper, we study the effects of bath concentrations on the average force on an ion in a single ion channel. We show that the force of the reaction field on a discrete ion inside a channel embedded in an uncharged lipid membrane contains a blocking (shielding) term that is proportional to the square root of the ionic bath concentration. We then show that different blocking strengths yield different behavior of the current-concentration and conductance-concentration curves. Our theory shows that at low concentrations, when the blocking force is weak, conductance grows linearly with concentration, as in traditional models, e.g., Michaelis-Menten formulations. As the concentration increases to a range of moderate shielding, conductance grows as the square root of concentration, whereas at high concentrations, with high shielding, conductance may actually decrease with increasing concentrations: the conductance-concentration curve can invert. Therefore, electrostatic interactions between bath ions and the single ion inside the channel can explain the different regimes of conductance-concentration relations observed in experiments.

Comparison of Nernst-Planck and reaction rate models for multiply occupied channels.

PubMed Central

Levitt, D G

1982-01-01

The Nernst-Planck continuum equation for a channel that can be occupied by at most two ions is solved for two different physical cases. The first case is for the assumption that the water and ion cannot get around each other anywhere in the channel, so that if there are two ions in the channel the distance between them is fixed by the number of water molecules between them. The second case is for the assumption that there are regions at he ends of the channel where the ions and water can get around each other. For these two cases, the validity of the simple two-site reaction-rate approximation when there is a continuously varying central energy barrier was evaluated by comparing it with the exact Nernst-Planck solution. For the first continuum case, the kinetics for the continuum and reaction-rate models are nearly identical. For the second case, the agreement depends on the strength of the ion-ion interaction energy. For a low interaction energy (large channel diameter) a high ion concentrations, there is a large difference in the flux as a function of voltage for the two models-with the continuum flux becoming more than four times larger at 250 mV. Simple analytical expressions are derived for the two-ion continuum channel for the case where the ends are in equilibrium with the bulk solution and for the case where ion mobility becomes zero when there are two ions in the channel. The implications of these results for biological channels are discussed. PMID:6280783

Simple Ion Channels: From Structure to Electrophysiology and Back

NASA Technical Reports Server (NTRS)

Pohorille, Andrzej

2018-01-01

A reliable way to establish whether our understanding of a channel is satisfactory is to reproduce its measured ionic conductance over a broad range of applied voltages in computer simulations. In molecular dynamics (MD), this can be done by way of applying an external electric field to the system and counting the number of ions that traverse the channel per unit time. Since this approach is computationally very expensive, we have developed a markedly more efficient alternative in which MD is combined with the electrodiffusion (ED) equation. In this approach, the assumptions of the ED equation can be rigorously tested, and the precision and consistency of the calculated conductance can be determined. We have demonstrated that the full current/voltage dependence and the underlying free energy profile for a simple channel can be reliably calculated from equilibrium or non-equilibrium MD simulations at a single voltage. To carry out MD simulations, a structural model of a channel has to be assumed, which is an important constraint, considering that high-resolution structures are available for only very few simple channels. If the comparison of calculated ionic conductance with electrophysiological data is satisfactory, it greatly increases our confidence that the structure and the function are described sufficiently accurately. We examined the validity of the ED for several channels embedded in phospholipid membranes - four naturally occurring channels: trichotoxin, alamethicin, p7 from hepatitis C virus (HCV) and Vpu from the HIV-1 virus, and a synthetic, hexameric channel, formed by a 21-residue peptide that contains only leucine and serine. All these channels mediate transport of potassium and chloride ions. It was found that the ED equation is satisfactory for these systems. In some of them experimental and calculated electrophysiological properties are in good agreement, whereas in others there are strong indications that the structural models are incorrect.

Trivalent ions modification for high-silica mordenite: A first principles study

NASA Astrophysics Data System (ADS)

Chen, Fayun; Zhang, Laijun; Feng, Gang; Wang, Xuewen; Zhang, Rongbin; Liu, Jianwen

2018-03-01

Using periodic DFT-D3-U methods, the present work give a mechanistic insight into the high silica B-, Al-, Ga- and Fe-MOR with H, Li, Na, and K as charge balance ions. The acid properties of the zeolite were probed via NH3 and pyridine adsorption. It is found that the charge balance ions influence the location of the trivalent ions, the cell volumes, as well as the synthesis difficulty of the zeolites. The energy differences for B, Al, Ga and Fe in different T sites are small for the H-form zeolites, while large for the Na- and K-form zeolites. For H-form MOR, the proton of the sbnd OH group prefers to bond to O(7) and O(3) and pointing to the 12MR for trivalent ions in T1 sites. The proton bonds to O(3), O(2), O(2) and O(5), respectively, for B, Al, Ga and Fe in T2 site of MOR, with the sbnd OH group pointing to intersection of 12MR and the side-pocket, except for the B-MOR that sbnd OH group pointing to the 12MR. For trivalent ions located in T3 and T4 sites, the protons prefers to bond to O(1) and O(2), respectively, with the sbnd OH group pointing to the intersection of 8MR and side-pocket as well as the intersection of 12MR and side-pocket. All incorporated B, Al, Ga, and Fe framework ions are tetra-coordinated, except the B atoms are tri-coordinated. The NH4-form MOR has smaller cell volume than the other form MOR. Na and K are energetically more favored charge balance ions than Li and NH3 for MOR zeolites synthesis, and the H-form zeolite is the most difficult to be synthesized directly. The strength of the Brønsted acidity follows the order: HBMOR < HFeMOR ≈ HGaMOR < HAlMOR, vs. the Lewis acidity order: HBMOR < HAlMOR < HFeMOR ≈ HGaMOR. NH3 could be adsorbed inside all kinds of channels, and especially favors in the small 8MR vs. pyridine could only be adsorbed in the main channel of MOR due to the steric effect. It indicates that the acid sites in the side pocket and the small 8-membered ring and the side pocket could not be effectively determined just by the pyridine adsorption experiments. In comparison, the NH3 adsorption experiments could detect all kinds of Brønsted sites of the MOR zeolites.

Investigating ion channel conformational changes using voltage clamp fluorometry.

PubMed

Talwar, Sahil; Lynch, Joseph W

2015-11-01

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

Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of CaV Function.

PubMed

Findeisen, Felix; Campiglio, Marta; Jo, Hyunil; Abderemane-Ali, Fayal; Rumpf, Christine H; Pope, Lianne; Rossen, Nathan D; Flucher, Bernhard E; DeGrado, William F; Minor, Daniel L

2017-06-21

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (Ca V ) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca V β). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca V β interactions and reduce the entropic penalty associated with AID binding to Ca V β. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca V α 1 :Ca V β interaction that modulate Ca V function in an Ca V β isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca V modulator design.

Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein–Protein Interaction Inhibitors of CaV Function

PubMed Central

2017-01-01

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein–protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein–protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein–protein interaction, the interaction between the voltage-gated calcium channel (CaV) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (CaVβ). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:CaVβ interactions and reduce the entropic penalty associated with AID binding to CaVβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the CaVα1:CaVβ interaction that modulate CaV function in an CaVβ isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein–protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based CaV modulator design. PMID:28278376

Synthetic chloride-selective carbon nanotubes examined by using molecular and stochastic dynamics.

PubMed

Hilder, Tamsyn A; Gordon, Dan; Chung, Shin-Ho

2010-09-22

Synthetic channels, such as nanotubes, offer the possibility of ion-selective nanoscale pores which can broadly mimic the functions of various biological ion channels, and may one day be used as antimicrobial agents, or for treatment of cystic fibrosis. We have designed a carbon nanotube that is selectively permeable to anions. The virtual nanotubes are constructed from a hexagonal array of carbon atoms (graphene) rolled up to form a tubular structure, with an effective radius of 4.53 Å and length of 34 Å. The pore ends are terminated with polar carbonyl groups. The nanotube thus formed is embedded in a lipid bilayer and a reservoir containing ionic solutions is added at each end of the pore. The conductance properties of these synthetic channels are then examined with molecular and stochastic dynamics simulations. Profiles of the potential of mean force at 0 mM reveal that a cation moving across the pore encounters an insurmountable free energy barrier of ∼25 kT in height. In contrast, for anions, there are two energy wells of ∼12 kT near each end of the tube, separated by a central free energy barrier of 4 kT. The conductance of the pore, with symmetrical 500 mM solutions in the reservoirs, is 72 pS at 100 mV. The current saturates with an increasing ionic concentration, obeying a Michaelis-Menten relationship. The pore is normally occupied by two ions, and the rate-limiting step in conduction is the time taken for the resident ion near the exit gate to move out of the energy well. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Simulation of charge transport in ion channels and nanopores with anisotropic permittivity

PubMed Central

Mashl, R. Jay; Lee, Kyu Il; Jakobsson, Eric; Ravaioli, Umberto

2010-01-01

Ion channels are part of nature's solution for regulating biological environments. Every ion channel consists of a chain of amino acids carrying a strong and sharply varying permanent charge, folded in such a way that it creates a nanoscopic aqueous pore spanning the otherwise mostly impermeable membranes of biological cells. These naturally occurring proteins are particularly interesting to device engineers seeking to understand how such nanoscale systems realize device-like functions. Availability of high-resolution structural information from X-ray crystallography, as well as large-scale computational resources, makes it possible to conduct realistic ion channel simulations. In general, a hierarchy of simulation methodologies is needed to study different aspects of a biological system like ion channels. Biology Monte Carlo (BioMOCA), a three-dimensional coarse-grained particle ion channel simulator, offers a powerful and general approach to study ion channel permeation. BioMOCA is based on the Boltzmann Transport Monte Carlo (BTMC) and Particle-Particle-Particle-Mesh (P3M) methodologies developed at the University of Illinois at Urbana-Champaign. In this paper we briefly discuss the various approaches to simulating ion flow in channel systems that are currently being pursued by the biophysics and engineering communities, and present the effect of having anisotropic dielectric constants on ion flow through a number of nanopores with different effective diameters. PMID:20445807

Novel screening techniques for ion channel targeting drugs

PubMed Central

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation. PMID:26556400

Novel screening techniques for ion channel targeting drugs.

PubMed

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

Highly Sensitive and Patchable Pressure Sensors Mimicking Ion-Channel-Engaged Sensory Organs.

PubMed

Chun, Kyoung-Yong; Son, Young Jun; Han, Chang-Soo

2016-04-26

Biological ion channels have led to much inspiration because of their unique and exquisite operational functions in living cells. Specifically, their extreme and dynamic sensing abilities can be realized by the combination of receptors and nanopores coupled together to construct an ion channel system. In the current study, we demonstrated that artificial ion channel pressure sensors inspired by nature for detecting pressure are highly sensitive and patchable. Our ion channel pressure sensors basically consisted of receptors and nanopore membranes, enabling dynamic current responses to external forces for multiple applications. The ion channel pressure sensors had a sensitivity of ∼5.6 kPa(-1) and a response time of ∼12 ms at a frequency of 1 Hz. The power consumption was recorded as less than a few μW. Moreover, a reliability test showed stability over 10 000 loading-unloading cycles. Additionally, linear regression was performed in terms of temperature, which showed no significant variations, and there were no significant current variations with humidity. The patchable ion channel pressure sensors were then used to detect blood pressure/pulse in humans, and different signals were clearly observed for each person. Additionally, modified ion channel pressure sensors detected complex motions including pressing and folding in a high-pressure range (10-20 kPa).

Orientation independence of single-vacancy and single-ion permeability ratios.

PubMed Central

McGill, P; Schumaker, M F

1995-01-01

Single-vacancy models have been proposed as open channel permeation mechanisms for K+ channels. Single-ion models have been used to describe permeation through Na+ channels. This paper demonstrates that these models have a distinctive symmetry property. Their permeability ratios, measured under biionic conditions, are independent of channel orientation when the reversal potential is zero. This symmetry is a property of general m-site single-vacancy channels, m-site shaking-stack channels, as well as m-site single-ion channels. An experimental finding that the permeability ratios of a channel did not have this symmetry would provide evidence that a single-vacancy or single-ion model is an incorrect or incomplete description of permeation. Images FIGURE 1 PMID:7669913

Complementary junction heterostructure field-effect transistor

DOEpatents

Baca, Albert G.; Drummond, Timothy J.; Robertson, Perry J.; Zipperian, Thomas E.

1995-01-01

A complimentary pair of compound semiconductor junction heterostructure field-effect transistors and a method for their manufacture are disclosed. The p-channel junction heterostructure field-effect transistor uses a strained layer to split the degeneracy of the valence band for a greatly improved hole mobility and speed. The n-channel device is formed by a compatible process after removing the strained layer. In this manner, both types of transistors may be independently optimized. Ion implantation is used to form the transistor active and isolation regions for both types of complimentary devices. The invention has uses for the development of low power, high-speed digital integrated circuits.

Complementary junction heterostructure field-effect transistor

DOEpatents

Baca, A.G.; Drummond, T.J.; Robertson, P.J.; Zipperian, T.E.

1995-12-26

A complimentary pair of compound semiconductor junction heterostructure field-effect transistors and a method for their manufacture are disclosed. The p-channel junction heterostructure field-effect transistor uses a strained layer to split the degeneracy of the valence band for a greatly improved hole mobility and speed. The n-channel device is formed by a compatible process after removing the strained layer. In this manner, both types of transistors may be independently optimized. Ion implantation is used to form the transistor active and isolation regions for both types of complimentary devices. The invention has uses for the development of low power, high-speed digital integrated circuits. 10 figs.

The Kinetics and the Permeation Properties of Piezo Channels.

PubMed

Gnanasambandam, R; Gottlieb, P A; Sachs, F

2017-01-01

Piezo channels are eukaryotic, cation-selective mechanosensitive channels (MSCs), which show rapid activation and voltage-dependent inactivation. The kinetics of these channels are largely consistent across multiple cell types and different stimulation paradigms with some minor variability. No accessory subunits that associate with Piezo channels have been reported. They are homotrimers and each ∼300kD monomer has an N-terminal propeller blade-like mechanosensing module, which can confer mechanosensing capabilities on ASIC-1 (the trimeric non-MSC, acid-sensing ion channel-1) and a C-terminal pore module, which influences conductance, selectivity, and channel inactivation. Repeated stimulation can cause domain fracture and diffusion of these channels leading to synchronous loss of inactivation. The reconstituted channels spontaneously open only in asymmetric bilayers but lack inactivation. Mutations that cause hereditary xerocytosis alter PIEZO1 kinetics. The kinetics of the wild-type PIEZO1 and alterations thereof in mutants (M2225R, R2456K, and DhPIEZO1) are summarized in the form of a quantitative model and hosted online. The pore is permeable to alkali ions although Li + permeates poorly. Divalent cations, notably Ca 2+ , traverse the channel and inhibit the flux of monovalents. The large monovalent organic cations such as tetramethyl ammonium and tetraethyl ammonium can traverse the channel, but slowly, suggesting a pore diameter of ∼8Å, and the estimated in-plane area change upon opening is around 6-20nm 2 . Ruthenium red can enter the channel only from the extracellular side and seems to bind in a pocket close to residue 2496. Copyright © 2017 Elsevier Inc. All rights reserved.

In Touch With the Mechanosensitive Piezo Channels: Structure, Ion Permeation, and Mechanotransduction.

PubMed

Geng, J; Zhao, Q; Zhang, T; Xiao, B

2017-01-01

Mechanotransduction, the conversion of mechanical forces into biological signals, plays critical roles in various physiological and pathophysiological processes in mammals, such as conscious sensing of touch, pain, and sound, as well as unconscious sensing of blood flow-associated shear stress, urine flow, and bladder distention. Among the various molecules involved in mechanotransduction, mechanosensitive (MS) cation channels have long been postulated to represent one critical class of mechanotransducers that directly and rapidly converts mechanical force into electrochemical signals. Despite the awareness of their functional significance, the molecular identities of MS cation channels in mammals had remained elusive for decades till the groundbreaking finding that the Piezo family of genes, including Piezo1 and Piezo2, constitutes their essential components. Since their identification about 6years ago, tremendous progress has been made in understanding their physiological and pathophysiological importance in mechanotransduction and their structure-function relationships of being the prototypic class of mammalian MS cation channels. On the one hand, Piezo proteins have been demonstrated to serve as physiologically and pathophysiologically important mechanotransducers for most, if not all, mechanotransduction processes. On the other hand, they have been shown to form a remarkable three-bladed, propeller-shaped homotrimeric channel complex comprising a separable ion-conducting pore module and mechanotransduction modules. In this chapter, we review the major advancements, with a particular focus on the structural and biophysical features that enable Piezo proteins to serve as sophisticated MS cation channels for force sensing, transduction, and ion conduction. Copyright © 2017 Elsevier Inc. All rights reserved.

Voltage-gated sodium channel β subunits: The power outside the pore in brain development and disease.

PubMed

Hull, Jacob M; Isom, Lori L

2018-04-01

Voltage gated sodium channels (VGSCs) were first identified in terms of their role in the upstroke of the action potential. The underlying proteins were later identified as saxitoxin and scorpion toxin receptors consisting of α and β subunits. We now know that VGSCs are heterotrimeric complexes consisting of a single pore forming α subunit joined by two β subunits; a noncovalently linked β1 or β3 and a covalently linked β2 or β4 subunit. VGSC α subunits contain all the machinery necessary for channel cell surface expression, ion conduction, voltage sensing, gating, and inactivation, in one central, polytopic, transmembrane protein. VGSC β subunits are more than simple accessories to α subunits. In the more than two decades since the original cloning of β1, our knowledge of their roles in physiology and pathophysiology has expanded immensely. VGSC β subunits are multifunctional. They confer unique gating mechanisms, regulate cellular excitability, affect brain development, confer distinct channel pharmacology, and have functions that are independent of the α subunits. The vast array of functions of these proteins stems from their special station in the channelome: being the only known constituents that are cell adhesion and intra/extracellular signaling molecules in addition to being part of channel complexes. This functional trifecta and how it goes awry demonstrates the power outside the pore in ion channel signaling complexes, broadening the term channelopathy beyond defects in ion conduction. This article is part of the Special Issue entitled 'Channelopathies.' Copyright © 2017 Elsevier Ltd. All rights reserved.

Ion-imprinted nanoparticles for the concurrent estimation of Pb(II) and Cu(II) ions over a two channel surface plasmon resonance-based fiber optic platform

NASA Astrophysics Data System (ADS)

Shrivastav, Anand Mohan; Gupta, Banshi D.

2018-01-01

We report the design, fabrication, and characterization of an optical fiber sensor based on the surface plasmon resonance (SPR) technique for the simultaneous determination of lead (Pb) and copper (Cu) metal ions in aqueous samples. Two cascade channels over a single optical fiber are fabricated by removing cladding from two well-separated regions of the fiber. SPR working as a transducing mechanism for the sensor is realized by coating thin films of copper and silver over unclad cores of channel I and channel II, respectively. Ion-imprinted nanoparticles for both ions are separately synthesized and coated over the metal-coated unclad cores of the fiber as the recognition layers for sensor fabrication. A first channel having layer of Pb(II) ion-imprinted nanoparticles detects Pb(II) ions and a second channel having layer of Cu(II) ion-imprinted nanoparticles are used for the detection of Cu(II) ions. Both channels are characterized using the wavelength interrogation method. The sensor operates in the range between 0 to 1000 μg/L and 0 to 1000 mg/L for Pb(II) and Cu(II) ions, respectively. These ranges cover water resources and the human body for these ions. The sensitivities of channel I and channel II are found to be 8.19×104 nm/(μg/L) and 4.07×105 nm/(mg/L) near the lowest concentration of Pb(II) and Cu(II) ions, respectively. The sensor can detect concentrations of Pb(II) and Cu(II) ions as low as 4.06 × 10-12 g/L and 8.18 × 10-10 g/L, respectively, which are the least among the reported values in the literature. Further, the probe is simple, cost effective, highly selective, and applicable for online monitoring and remote sensing.

Selectivity and permeation of alkali metal ions in K+-channels.

PubMed

Furini, Simone; Domene, Carmen

2011-06-24

Ion conduction in K(+)-channels is usually described in terms of concerted movements of K(+) progressing in a single file through a narrow pore. Permeation is driven by an incoming ion knocking on those ions already inside the protein. A fine-tuned balance between high-affinity binding and electrostatic repulsive forces between permeant ions is needed to achieve efficient conduction. While K(+)-channels are known to be highly selective for K(+) over Na(+), some K(+) channels conduct Na(+) in the absence of K(+). Other ions are known to permeate K(+)-channels with a more moderate preference and unusual conduction features. We describe an extensive computational study on ion conduction in K(+)-channels rendering free energy profiles for the translocation of three different alkali ions and some of their mixtures. The free energy maps for Rb(+) translocation show at atomic level why experimental Rb(+) conductance is slightly lower than that of K(+). In contrast to K(+) or Rb(+), external Na(+) block K(+) currents, and the sites where Na(+) transport is hindered are characterized. Translocation of K(+)/Na(+) mixtures is energetically unfavorable owing to the absence of equally spaced ion-binding sites for Na(+), excluding Na(+) from a channel already loaded with K(+). Copyright © 2011 Elsevier Ltd. All rights reserved.

Of channels and pumps: different ways to boost the aldosterone?

PubMed

Bandulik, S

2017-07-01

The mineralocorticoid aldosterone is a major factor controlling the salt and water balance and thereby also the arterial blood pressure. Accordingly, primary aldosteronism (PA) characterized by an inappropriately high aldosterone secretion is the most common form of secondary hypertension. The physiological stimulation of aldosterone synthesis in adrenocortical glomerulosa cells by angiotensin II and an increased plasma K + concentration depends on a membrane depolarization and an increase in the cytosolic Ca 2+ activity. Recurrent gain-of-function mutations of ion channels and transporters have been identified in a majority of cases of aldosterone-producing adenomas and in familial forms of PA. In this review, the physiological role of these genes in the regulation of aldosterone synthesis and the altered function of the mutant proteins as well are described. The specific changes of the membrane potential and the cellular ion homoeostasis in adrenal cells expressing the different mutants are compared, and their impact on autonomous aldosterone production and proliferation is discussed. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Automated Patch-Clamp Methods for the hERG Cardiac Potassium Channel.

PubMed

Houtmann, Sylvie; Schombert, Brigitte; Sanson, Camille; Partiseti, Michel; Bohme, G Andrees

2017-01-01

The human Ether-a-go-go Related Gene (hERG) product has been identified as a central ion channel underlying both familial forms of elongated QT interval on the electrocardiogram and drug-induced elongation of the same QT segment. Indeed, reduced function of this potassium channel involved in the repolarization of the cardiac action potential can produce a type of life-threatening cardiac ventricular arrhythmias called Torsades de Pointes (TdP). Therefore, hERG inhibitory activity of newly synthetized molecules is a relevant structure-activity metric for compound prioritization and optimization in medicinal chemistry phases of drug discovery. Electrophysiology remains the gold standard for the functional assessment of ion channel pharmacology. The recent years have witnessed automatization and parallelization of the manual patch-clamp technique, allowing higher throughput screening on recombinant hERG channels. However, the multi-well plate format of automatized patch-clamp does not allow visual detection of potential micro-precipitation of poorly soluble compounds. In this chapter we describe bench procedures for the culture and preparation of hERG-expressing CHO cells for recording on an automated patch-clamp workstation. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.

A New Poisson-Nernst-Planck Model with Ion-Water Interactions for Charge Transport in Ion Channels.

PubMed

Chen, Duan

2016-08-01

In this work, we propose a new Poisson-Nernst-Planck (PNP) model with ion-water interactions for biological charge transport in ion channels. Due to narrow geometries of these membrane proteins, ion-water interaction is critical for both dielectric property of water molecules in channel pore and transport dynamics of mobile ions. We model the ion-water interaction energy based on realistic experimental observations in an efficient mean-field approach. Variation of a total energy functional of the biological system yields a new PNP-type continuum model. Numerical simulations show that the proposed model with ion-water interaction energy has the new features that quantitatively describe dielectric properties of water molecules in narrow pores and are possible to model the selectivity of some ion channels.

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery

PubMed Central

Imbrici, Paola; Liantonio, Antonella; Camerino, Giulia M.; De Bellis, Michela; Camerino, Claudia; Mele, Antonietta; Giustino, Arcangela; Pierno, Sabata; De Luca, Annamaria; Tricarico, Domenico; Desaphy, Jean-Francois; Conte, Diana

2016-01-01

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets. PMID:27242528

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery.

PubMed

Imbrici, Paola; Liantonio, Antonella; Camerino, Giulia M; De Bellis, Michela; Camerino, Claudia; Mele, Antonietta; Giustino, Arcangela; Pierno, Sabata; De Luca, Annamaria; Tricarico, Domenico; Desaphy, Jean-Francois; Conte, Diana

2016-01-01

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets.

Roderick MacKinnon and Ion Channels - Potassium Channels and Sodium

Science.gov Websites

very first potassium channel structure, which revealed the way that positively charged potassium ions explain how a fundamental feedback loop worked. Now, with the structure of the voltage-dependent ion channel, based on research carried out at The Rockefeller University, the National Synchrotron Light

Optical control of trimeric P2X receptors and acid-sensing ion channels.

PubMed

Browne, Liam E; Nunes, João P M; Sim, Joan A; Chudasama, Vijay; Bragg, Laricia; Caddick, Stephen; North, R Alan

2014-01-07

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Optical control of trimeric P2X receptors and acid-sensing ion channels

PubMed Central

Browne, Liam E.; Nunes, João P. M.; Sim, Joan A.; Chudasama, Vijay; Bragg, Laricia; Caddick, Stephen; Alan North, R.

2014-01-01

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis–trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues. PMID:24367083

Disposable, Autonomic, Energy-Converting Ion Channel Sensor Materials

DTIC Science & Technology

2018-07-02

variant forms well-defined pores _____________________________ 26 4.2. Another pore-forming peptide, Ceratotoxin A, displays alamethicin-like activity ...bilayer recordings to examine the activity of these compounds on the single-pore level. We plan to use modified CtxA for targeted cell killing...strongly dependent on entropy of activation . Tethering is one strategy towards achieving this goal. A manuscript regarding this work is currently in

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

PubMed Central

Kalsi, Sumit; Powl, Andrew M.; Wallace, B.A.; Morgan, Hywel; de Planque, Maurits R.R.

2014-01-01

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms. PMID:24739164

Molecular dynamics of water and monovalent-ions transportation mechanisms of pentameric sarcolipin.

PubMed

Cao, Yipeng; Wu, Xue; Lee, Imshik; Wang, Xinyu

2016-01-01

The Sarcolipin (SLN) is a transmembrane protein that can form a self-assembled pentamer. In this work, the homology modeling and all-atom molecular dynamic (MD) simulation was performed to study the model of SLN pentamer in POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane. The potential of mean force (PMF) was calculated for transmembrane transportation of Na(+), Cl(-) and water molecule along the pore channel of penta-SLN complex. The root mean square deviation (RMSD) of the SLN pentamer in POPC membrane showed that the stabilized SLN protein complex could exist in the membrane and that the Na(+) and Cl(-) could not permeate through the channel when the pore was under the vacuum state, but the water could permeate through from cytoplasm to lumen. Under the aqueous state, our simulation demonstrated that hydrated state of Na(+) and Cl(-) could pass through the channel. The PMF and radii of the pore showed that the channel had a gate at Leu(21) that is a key hydrophobicity residue in the channel. Our simulations help to clarify and to understand better the SLN pentamer channel that had a hydrophobic gate and could switch Na(+) and Cl(-) ion permeability by hydrated and vacuum states. © 2015 Wiley Periodicals, Inc.

Receptor for protons: First observations on Acid Sensing Ion Channels.

PubMed

Krishtal, Oleg

2015-07-01

The history of ASICs began in 1980 with unexpected observation. The concept of highly selective Na(+) current gated by specific receptors for protons was not easily accepted. It took 16 years to get these receptor/channels cloned and start a new stage in their investigation. "The receptor for protons" became ASIC comprising under this name a family of receptor/channels ubiquitous for mammalian nervous system, both peripheral and central. The role of ASICs as putative nociceptors was suggested almost immediately after their discovery. This role subsequently was proven in many forms of pain-related phenomena. Many other functions of ASICs have been also found or primed for speculations both in physiology and in disease. Despite the width of field and strength of efforts, numerous basic questions are to be answered before we understand how the local changes in pH in the nervous tissue transform into electric and messenger signaling via ASICs as transducers. This article is part of the Special Issue entitled 'Acid-Sensing Ion Channels in the Nervous System'. Copyright © 2015. Published by Elsevier Ltd.

Moving Fe2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates.

PubMed

Behera, Rabindra K; Theil, Elizabeth C

2014-06-03

Ferritin biominerals are protein-caged metabolic iron concentrates used for iron-protein cofactors and oxidant protection (Fe(2+) and O2 sequestration). Fe(2+) passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe(2+) substrate movement to ferritin enzyme (Fox) sites. Fe(2+) and O2 substrates are coupled via a diferric peroxo (DFP) intermediate, λmax 650 nm, which decays to [Fe(3+)-O-Fe(3+)] precursors of caged ferritin biominerals. Structural studies show multiple conformations for conserved, carboxylate residues E136 and E57, which are between ferritin ion channel exits and enzymatic sites, suggesting functional connections. Here we show that E136 and E57 are required for ferritin enzyme activity and thus are functional links between ferritin ion channels and enzymatic sites. DFP formation (Kcat and kcat/Km), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe(2+) concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe(2+) access), Mn(2+) < Co(2+) < Cu(2+) < Zn(2+), reflecting metal ion-protein binding stabilities. Fe(2+)-Cys126 binding in ferritin ion channels, observed as Cu(2+)-S-Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu(2+) inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe(2+) movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade that moves external Fe(2+) into ferritin enzymatic sites. The results clarify Fe(2+) transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations.

Global versus local mechanisms of temperature sensing in ion channels.

PubMed

Arrigoni, Cristina; Minor, Daniel L

2018-05-01

Ion channels turn diverse types of inputs, ranging from neurotransmitters to physical forces, into electrical signals. Channel responses to ligands generally rely on binding to discrete sensor domains that are coupled to the portion of the channel responsible for ion permeation. By contrast, sensing physical cues such as voltage, pressure, and temperature arises from more varied mechanisms. Voltage is commonly sensed by a local, domain-based strategy, whereas the predominant paradigm for pressure sensing employs a global response in channel structure to membrane tension changes. Temperature sensing has been the most challenging response to understand and whether discrete sensor domains exist for pressure and temperature has been the subject of much investigation and debate. Recent exciting advances have uncovered discrete sensor modules for pressure and temperature in force-sensitive and thermal-sensitive ion channels, respectively. In particular, characterization of bacterial voltage-gated sodium channel (BacNa V ) thermal responses has identified a coiled-coil thermosensor that controls channel function through a temperature-dependent unfolding event. This coiled-coil thermosensor blueprint recurs in other temperature sensitive ion channels and thermosensitive proteins. Together with the identification of ion channel pressure sensing domains, these examples demonstrate that "local" domain-based solutions for sensing force and temperature exist and highlight the diversity of both global and local strategies that channels use to sense physical inputs. The modular nature of these newly discovered physical signal sensors provides opportunities to engineer novel pressure-sensitive and thermosensitive proteins and raises new questions about how such modular sensors may have evolved and empowered ion channel pores with new sensibilities.

A disruptive sequencer meets disruptive publishing.

PubMed

Loman, Nick; Goodwin, Sarah; Jansen, Hans; Loose, Matt

2015-01-01

Nanopore sequencing was recently made available to users in the form of the Oxford Nanopore MinION. Released to users through an early access programme, the MinION is made unique by its tiny form factor and ability to generate very long sequences from single DNA molecules. The platform is undergoing rapid evolution with three distinct nanopore types and five updates to library preparation chemistry in the last 18 months. To keep pace with the rapid evolution of this sequencing platform, and to provide a space where new analysis methods can be openly discussed, we present a new F1000Research channel devoted to updates to and analysis of nanopore sequence data.

Ion-binding properties of a K+ channel selectivity filter in different conformations.

PubMed

Liu, Shian; Focke, Paul J; Matulef, Kimberly; Bian, Xuelin; Moënne-Loccoz, Pierre; Valiyaveetil, Francis I; Lockless, Steve W

2015-12-08

K(+) channels are membrane proteins that selectively conduct K(+) ions across lipid bilayers. Many voltage-gated K(+) (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K(+) channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na(+) or low [K(+)]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K(+) ion binding to channels with an inactivated or conductive selectivity filter is different from K(+) ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.

Large fraction of crystal directions leads to ion channeling

NASA Astrophysics Data System (ADS)

Nordlund, K.; Djurabekova, F.; Hobler, G.

2016-12-01

It is well established that when energetic ions are moving in crystals, they may penetrate much deeper if they happen to be directed in some specific crystal directions. This `channeling' effect is utilized for instance in certain ion beam analysis methods and has been described by analytical theories and atomistic computer simulations. However, there have been very few systematic studies of channeling in directions other than the principal low-index ones. We present here a molecular dynamics-based approach to calculate ion channeling systematically over all crystal directions, providing ion `channeling maps' that easily show in which directions channeling is expected. The results show that channeling effects can be quite significant even at energies below 1 keV, and that in many cases, significant planar channeling occurs also in a wide range of crystal directions between the low-index principal ones. In all of the cases studied, a large fraction (˜20 -60 % ) of all crystal directions show channeling. A practical implication of this is that modern experiments on randomly oriented nanostructures will have a large probability of channeling. It also means that when ion irradiations are carried out on polycrystalline samples, channeling effects on the results cannot a priori be assumed to be negligible. The maps allow for easy selection of good `nonchanneling' directions in experiments or alternatively finding wide channels for beneficial uses of channeling. We implement channeling theory to also give the fraction of channeling directions in a manner directly comparable to the simulations. The comparison shows good qualitative agreement. In particular, channeling theory is very good at predicting which channels are active at a given energy. This is true down to sub-keV energies, provided the penetration depth is not too small.

Molecular mechanism of ATP binding and ion channel activation in P2X receptors

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

Hattori, Motoyuki; Gouaux, Eric

P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure ofmore » the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.« less

Alkyl ether lipids, ion channels and lipid raft reorganization in cancer therapy.

PubMed

Jaffrès, Paul-Alain; Gajate, Consuelo; Bouchet, Ana Maria; Couthon-Gourvès, Hélène; Chantôme, Aurélie; Potier-Cartereau, Marie; Besson, Pierre; Bougnoux, Philippe; Mollinedo, Faustino; Vandier, Christophe

2016-09-01

Synthetic alkyl lipids, such as the ether lipids edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) and ohmline (1-O-hexadecyl-2-O-methyl-rac-glycero-3-β-lactose), are forming a class of antitumor agents that target cell membranes to induce apoptosis and to decrease cell migration/invasion, leading to the inhibition of tumor and metastasis development. In this review, we present the structure-activity relationship of edelfosine and ohmline, and we point out differences and similarities between these two amphiphilic compounds. We also discuss the mechanisms of action of these synthetic alkyl ether lipids (involving, among other structures and molecules, membrane domains, Fas/CD95 death receptor signaling, and ion channels), and highlight a key role for lipid rafts in the underlying process. The reorganization of lipid raft membrane domains induced by these alkyl lipids affects the function of death receptors and ion channels, thus leading to apoptosis and/or inhibition of cancer cell migration. The possible therapeutic use of these alkyl lipids and the clinical perspectives for these lipids in prevention or/and treatment of tumor development and metastasis are also discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Ion Channels in Brain Metastasis

PubMed Central

Klumpp, Lukas; Sezgin, Efe C.; Eckert, Franziska; Huber, Stephan M.

2016-01-01

Breast cancer, lung cancer and melanoma exhibit a high metastatic tropism to the brain. Development of brain metastases severely worsens the prognosis of cancer patients and constrains curative treatment options. Metastasizing to the brain by cancer cells can be dissected in consecutive processes including epithelial–mesenchymal transition, evasion from the primary tumor, intravasation and circulation in the blood, extravasation across the blood–brain barrier, formation of metastatic niches, and colonization in the brain. Ion channels have been demonstrated to be aberrantly expressed in tumor cells where they regulate neoplastic transformation, malignant progression or therapy resistance. Moreover, many ion channel modulators are FDA-approved drugs and in clinical use proposing ion channels as druggable targets for future anti-cancer therapy. The present review article aims to summarize the current knowledge on the function of ion channels in the different processes of brain metastasis. The data suggest that certain channel types involving voltage-gated sodium channels, ATP-release channels, ionotropic neurotransmitter receptors and gap junction-generating connexins interfere with distinct processes of brain metastazation. PMID:27618016

Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide

NASA Astrophysics Data System (ADS)

Wang, Yukun; Chen, Charles H.; Hu, Dan; Ulmschneider, Martin B.; Ulmschneider, Jakob P.

2016-11-01

Many antimicrobial peptides (AMPs) selectively target and form pores in microbial membranes. However, the mechanisms of membrane targeting, pore formation and function remain elusive. Here we report an experimentally guided unbiased simulation methodology that yields the mechanism of spontaneous pore assembly for the AMP maculatin at atomic resolution. Rather than a single pore, maculatin forms an ensemble of structurally diverse temporarily functional low-oligomeric pores, which mimic integral membrane protein channels in structure. These pores continuously form and dissociate in the membrane. Membrane permeabilization is dominated by hexa-, hepta- and octamers, which conduct water, ions and small dyes. Pores form by consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models. The diversity of the pore architectures--formed by a single sequence--may be a key feature in preventing bacterial resistance and could explain why sequence-function relationships in AMPs remain elusive.

Modulatory mechanisms and multiple functions of somatodendritic A-type K+ channel auxiliary subunits

PubMed Central

Jerng, Henry H.; Pfaffinger, Paul J.

2014-01-01

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders. PMID:24723849

Modulatory mechanisms and multiple functions of somatodendritic A-type K (+) channel auxiliary subunits.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2014-01-01

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.

WIMP detection and slow ion dynamics in carbon nanotube arrays.

PubMed

Cavoto, G; Cirillo, E N M; Cocina, F; Ferretti, J; Polosa, A D

2016-01-01

Large arrays of aligned carbon nanotubes (CNTs), open at one end, could be used as target material for the directional detection of weakly interacting dark matter particles (WIMPs). As a result of a WIMP elastic scattering on a CNT, a carbon ion might be injected in the body of the array and propagate through multiple collisions within the lattice. The ion may eventually emerge from the surface with open end CNTs, provided that its longitudinal momentum is large enough to compensate energy losses and its transverse momentum approaches the channeling conditions in a single CNT. Therefore, the angle formed between the WIMP wind apparent orientation and the direction of parallel carbon nanotube axes must be properly chosen. We focus on very low ion recoil kinetic energies, related to low mass WIMPs ([Formula: see text] GeV) where most of the existing experiments have low sensitivity. Relying on some exact results on two-dimensional lattices of circular obstacles, we study the low energy ion motion in the transverse plane with respect to CNT directions. New constraints are obtained on how to devise the CNT arrays to maximize the target channeling efficiency.

New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.

PubMed

Yamamura, Hisao; Suzuki, Yoshiaki; Imaizumi, Yuji

2015-05-01

Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Streaming potentials in gramicidin channels measured with ion-selective microelectrodes.

PubMed Central

Tripathi, S; Hladky, S B

1998-01-01

Streaming potentials have been measured for gramicidin channels with a new method employing ion-selective microelectrodes. It is shown that ideally ion-selective electrodes placed at the membrane surface record the true streaming potential. Using this method for ion concentrations below 100 mM, approximately seven water molecules are transported whenever a sodium, potassium, or cesium ion, passes through the channel. This new method confirms earlier measurements (Rosenberg, P.A., and A. Finkelstein. 1978. Interaction of ions and water in gramicidin A channels. J. Gen. Physiol. 72:327-340) in which the streaming potentials were calculated as the difference between electrical potentials measured in the presence of gramicidin and in the presence of the ion carriers valinomycin and nonactin. PMID:9635745

Ion Selectivity Mechanism in a Bacterial Pentameric Ligand-Gated Ion Channel

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

Fritsch, Sebastian; Ivanov, Ivaylo; Wang, Hailong

2010-01-01

The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel ismore » open for a sodium ion to transport, but presents a 11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2 ) at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl in the middle of the pore for both GLIC and the E-2 A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.« less

Big Potassium (BK) ion channels in biology, disease and possible targets for cancer immunotherapy

PubMed Central

Ge, Lisheng; Hoa, Neil T.; Wilson, Zechariah; Arismendi-Morillo, Gabriel; Kong, Xia-Tang; Tajhya, Rajeev B.; Beeton, Christine; Jadus, Martin R.

2017-01-01

The Big Potassium (BK) ion channel is commonly known by a variety of names (Maxi-K, KCNMA1, slo, Stretch-activated potassium channels, KCa1.1). Each name reflects a different physical property displayed by this single ion channel. This transmembrane channel is found on nearly every cell type of the body and has its own distinctive roles for that tissue type. The BKα channel contains the pore that releases potassium ions from intracellular stores. This ion channel is found on the cell membrane, endoplasmic reticulum, Golgi and mitochondria. Complex splicing pathways produce different isoforms. The BKα channels can be phosphorylated, palmitoylated and myristylated. BK is composed of a homo-tetramer that interacts with β and γ chains. These accessory proteins provide a further modulating effect on the functions of BKα channels. BK channels play important roles in cell division and migration. In this review, we will focus on the biology of BK channels, especially its role, and that it has in the immune response towards cancer. Recent proteomic studies have linked BK channels with various proteins. Some of these interactions offer further insight into the role that BK channels have with cancers, especially with brain tumors. This review shows that BK channels have a complex interplay with intracellular components of cancer cells and still have plenty of secrets to be discovered. PMID:25027630

Never at rest: insights into the conformational dynamics of ion channels from cryo-electron microscopy.

PubMed

Lau, Carus; Hunter, Mark J; Stewart, Alastair; Perozo, Eduardo; Vandenberg, Jamie I

2018-04-01

The tightly regulated opening and closure of ion channels underlies the electrical signals that are vital for a wide range of physiological processes. Two decades ago the first atomic level view of ion channel structures led to a detailed understanding of ion selectivity and conduction. In recent years, spectacular developments in the field of cryo-electron microscopy have resulted in cryo-EM superseding crystallography as the technique of choice for determining near-atomic resolution structures of ion channels. Here, we will review the recent developments in cryo-EM and its specific application to the study of ion channel gating. We will highlight the advantages and disadvantages of the current technology and where the field is likely to head in the next few years. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Channels Active in the Excitability of Nerves and Skeletal Muscles across the Neuromuscular Junction: Basic Function and Pathophysiology

ERIC Educational Resources Information Center

Goodman, Barbara E.

2008-01-01

Ion channels are essential for the basic physiological function of excitable cells such as nerve, skeletal, cardiac, and smooth muscle cells. Mutations in genes that encode ion channels have been identified to cause various diseases and disorders known as channelopathies. An understanding of how individual ion channels are involved in the…

The Origin and Early Evolution of Membrane Proteins

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

2006-01-01

The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

LOW CONDUCTANCE HCN1 ION CHANNELS AUGMENT THE FREQUENCY RESPONSE OF ROD AND CONE PHOTORECEPTORS

PubMed Central

Barrow, Andrew J.; Wu, Samuel M.

2009-01-01

Hyperpolarization-activated cyclic nucleotide gated (HCN) ion channels are expressed in several tissues throughout the body, including the heart, the CNS, and the retina. HCN channels are found in many neurons in the retina, but their most established role is in generating the hyperpolarization-activated current, Ih, in photoreceptors. This current makes the light response of rod and cone photoreceptors more transient, an effect similar to that of a high-pass filter. A unique property of HCN channels is their small single channel current, which is below the thermal noise threshold of measuring electronics. We use nonstationary fluctuation analysis (NSFA) in the intact retina to estimate the conductance of single HCN channels, revealing a conductance of approximately 650 fS in both rod and cone photoreceptors. We also analyze the properties of HCN channels in salamander rods and cones, from the biophysical to the functional level, showing that HCN1 is the predominant isoform in both cells, and demonstrate how HCN1 channels speed up the light response of both rods and cones under distinct adaptational conditions. We show that in rods and cones, HCN channels increase the natural frequency response of single cells by modifying the photocurrent input, which is limited in its frequency response by the speed of a molecular signaling cascade. In doing so, HCN channels form the first of several systems in the retina that augment the speed of the visual response, allowing an animal to perceive visual stimuli that change more quickly than the underlying photocurrent. PMID:19420251

Study of shallow junction formation by boron-containing cluster ion implantation of silicon and two-stage annealing

NASA Astrophysics Data System (ADS)

Lu, Xin-Ming

Shallow junction formation made by low energy ion implantation and rapid thermal annealing is facing a major challenge for ULSI (ultra large scale integration) as the line width decreases down to the sub micrometer region. The issues include low beam current, the channeling effect in low energy ion implantation and TED (transient enhanced diffusion) during annealing after ion implantation. In this work, boron containing small cluster ions, such as GeB, SiB and SiB2, was generated by using the SNICS (source of negative ion by cesium sputtering) ion source to implant into Si substrates to form shallow junctions. The use of boron containing cluster ions effectively reduces the boron energy while keeping the energy of the cluster ion beam at a high level. At the same time, it reduces the channeling effect due to amorphization by co-implanted heavy atoms like Ge and Si. Cluster ions have been used to produce 0.65--2keV boron for low energy ion implantation. Two stage annealing, which is a combination of low temperature (550°C) preannealing and high temperature annealing (1000°C), was carried out to anneal the Si sample implanted by GeB, SiBn clusters. The key concept of two-step annealing, that is, the separation of crystal regrowth, point defects removal with dopant activation from dopant diffusion, is discussed in detail. The advantages of the two stage annealing include better lattice structure, better dopant activation and retarded boron diffusion. The junction depth of the two stage annealed GeB sample was only half that of the one-step annealed sample, indicating that TED was suppressed by two stage annealing. Junction depths as small as 30 nm have been achieved by two stage annealing of sample implanted with 5 x 10-4/cm2 of 5 keV GeB at 1000°C for 1 second. The samples were evaluated by SIMS (secondary ion mass spectrometry) profiling, TEM (transmission electron microscopy) and RBS (Rutherford Backscattering Spectrometry)/channeling. Cluster ion implantation in combination with two-step annealing is effective in fabricating ultra-shallow junctions.

Dominant-negative suppression of big brain ion channel activity by mutation of a conserved glutamate in the first transmembrane domain.

PubMed

Yool, Andrea J

2007-01-01

The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila. The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+.

PubMed

Li, Mufeng; Silberberg, Shai D; Swartz, Kenton J

2013-09-03

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg(2+). Here we investigated the active forms of ATP and found that the action of MgATP(2-) and ATP(4-) differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP(2-) promotes opening with very low efficacy. In contrast, both free ATP and MgATP(2-) robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg(2+) to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP(2-) and weak regulation by Mg(2+). These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP(2-) and regulation by Mg(2+), and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

PubMed Central

Li, Mufeng; Silberberg, Shai D.; Swartz, Kenton J.

2013-01-01

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons. PMID:23959888

Dielectric Self-Energy in Poisson-Boltzmann and Poisson-Nernst-Planck Models of Ion Channels

PubMed Central

Corry, Ben; Kuyucak, Serdar; Chung, Shin-Ho

2003-01-01

We demonstrated previously that the two continuum theories widely used in modeling biological ion channels give unreliable results when the radius of the conduit is less than two Debye lengths. The reason for this failure is the neglect of surface charges on the protein wall induced by permeating ions. Here we attempt to improve the accuracy of the Poisson-Boltzmann and Poisson-Nernst-Planck theories, when applied to channel-like environments, by including a specific dielectric self-energy term to overcome spurious shielding effects inherent in these theories. By comparing results with Brownian dynamics simulations, we show that the inclusion of an additional term in the equations yields significant qualitative improvements. The modified theories perform well in very wide and very narrow channels, but are less successful at intermediate sizes. The situation is worse in multi-ion channels because of the inability of the continuum theories to handle the ion-to-ion interactions correctly. Thus, further work is required if these continuum theories are to be reliably salvaged for quantitative studies of biological ion channels in all situations. PMID:12770869

Crystal structure of the ATP-gated P2X[subscript 4] ion channel in the closed state

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

Kawate, Toshimitsu; Michel, Jennifer Carlisle; Birdsong, William T.

2009-08-13

P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X{sub 4} receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit-subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in {beta}-strands, have largemore » acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an {approx}8 {angstrom} slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.« less

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

PubMed Central

Buchanan, Susan K; Lukacik, Petra; Grizot, Sylvestre; Ghirlando, Rodolfo; Ali, Maruf M U; Barnard, Travis J; Jakes, Karen S; Kienker, Paul K; Esser, Lothar

2007-01-01

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane. PMID:17464289

An (e, 2e+ ion) study of electron-impact ionization and fragmentation of tetrafluoromethane at low energies

NASA Astrophysics Data System (ADS)

Hossen, Khokon; Ren, Xueguang; Wang, Enliang; Kumar, S. V. K.; Dorn, Alexander

2018-03-01

We study ionization and fragmentation of tetrafluoromethane (CF4) molecule induced by electron impact at low energies ( E 0 = 38 and 67 eV). We use a reaction microscope combined with a pulsed photoemission electron beam for our experimental investigation. The momentum vectors of the two outgoing electrons (energies E 1, E 2) and one fragment ion are detected in triple coincidence (e, 2e+ ion). After dissociation, the fragment products observed are CF3 +, CF2 +, CF+, F+ and C+. For CF3 + and CF2 + channels, we measure the ionized orbitals binding energies, the kinetic energy (KE) of the charged fragments and the two-dimensional (2D) correlation map between binding energy (BE) and KE of the fragments. From the BE and KE spectra, we conclude which molecular orbitals contribute to particular fragmentation channels of CF4. We also measure the total ionization cross section for the formation of CF3 + and CF2 + ions as function of projectile energy. We compare our results with earlier experiments and calculations for electron-impact and photoionization. The major contribution to CF3 + formation originates from ionization of the 4t2 orbital while CF2 + is mainly formed after 3t2 orbital ionization. We also observe a weak contribution of the (4a1)-1 state for the channel CF3 +.

Channel-forming activity in the venom of the cockroach-hunting wasp, Ampulex compressa.

PubMed

Gincel, Dan; Haspel, Gal; Libersat, Frederic

2004-05-01

The parasitoid solitary wasp Ampulex compressa uses the cockroach Periplaneta americana as a food supply for its larvae. To subdue its prey, the wasp injects a venom cocktail into the brain of the cockroach. We investigated channel activity of A. compressa venom by collecting venom and incorporating it into a planar lipid bilayer. The venom, reconstituted into the bilayer, showed ion channel activity, forming a fast-fluctuating channel with a small conductance of 20+/-0.1pS, with no voltage sensitivity. These channels were not observed when the venom was digested with proteases before application to the bilayer, but were not affected by exposure to protease after their incorporation into the bilayer, indicating that the active venom component is a peptide. The channels were found to be cation selective with similar selectivity for the monovalent cations K(+), Li(+) and Na(+), but showed high selectivity against anions (Cl(-)) and divalent cations (Ca(2+) and Mg(2+)). This study is the first demonstration and biophysical characterization of channel activity in the venom of A. compressa. The possible functional significance of this channel activity is discussed in light of the unusual nature of the effects of this wasp venom on the behavior of its prey.

Unraveling the mechanism of selective ion transport in hydrophobic subnanometer channels.

PubMed

Li, Hui; Francisco, Joseph S; Zeng, Xiao Cheng

2015-09-01

Recently reported synthetic organic nanopore (SONP) can mimic a key feature of natural ion channels, i.e., selective ion transport. However, the physical mechanism underlying the K(+)/Na(+) selectivity for the SONPs is dramatically different from that of natural ion channels. To achieve a better understanding of the selective ion transport in hydrophobic subnanometer channels in general and SONPs in particular, we perform a series of ab initio molecular dynamics simulations to investigate the diffusivity of aqua Na(+) and K(+) ions in two prototype hydrophobic nanochannels: (i) an SONP with radius of 3.2 Å, and (ii) single-walled carbon nanotubes (CNTs) with radii of 3-5 Å (these radii are comparable to those of the biological potassium K(+) channels). We find that the hydration shell of aqua Na(+) ion is smaller than that of aqua K(+) ion but notably more structured and less yielding. The aqua ions do not lower the diffusivity of water molecules in CNTs, but in SONP the diffusivity of aqua ions (Na(+) in particular) is strongly suppressed due to the rugged inner surface. Moreover, the aqua Na(+) ion requires higher formation energy than aqua K(+) ion in the hydrophobic nanochannels. As such, we find that the ion (K(+) vs. Na(+)) selectivity of the (8, 8) CNT is ∼20× higher than that of SONP. Hence, the (8, 8) CNT is likely the most efficient artificial K(+) channel due in part to its special interior environment in which Na(+) can be fully solvated, whereas K(+) cannot. This work provides deeper insights into the physical chemistry behind selective ion transport in nanochannels.

Conformational plasticity in the selectivity filter of the TRPV2 ion channel.

PubMed

Zubcevic, Lejla; Le, Son; Yang, Huanghe; Lee, Seok-Yong

2018-05-01

Transient receptor potential vanilloid (TRPV) channels are activated by ligands and heat and are involved in various physiological processes. In contrast to the architecturally related voltage-gated cation channels, TRPV1 and TRPV2 subtypes possess another activation gate at the selectivity filter that can open widely enough to permeate large organic cations. Despite recent structural advances, the mechanism of selectivity filter gating and permeation for both metal ions and large molecules by TRPV1 or TRPV2 is not well known. Here, we determined two crystal structures of rabbit TRPV2 in its Ca 2+ -bound and resiniferatoxin (RTx)- and Ca 2+ -bound forms, to 3.9 Å and 3.1 Å, respectively. Notably, our structures show that RTx binding leads to two-fold symmetric opening of the selectivity filter of TRPV2 that is wide enough for large organic cation permeation. Combined with functional characterizations, our studies reveal a structural basis for permeation of Ca 2+ and large organic cations in TRPV2.

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

PubMed

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

2014-04-25

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

Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Baetz, Ulrike; Krügel, Undine; Martinoia, Enrico; De Angeli, Alexis

2013-10-01

Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.

Quantum mechanical calculations of charge effects on gating the KcsA channel.

PubMed

Kariev, Alisher M; Znamenskiy, Vasiliy S; Green, Michael E

2007-05-01

A series of ab initio (density functional) calculations were carried out on side chains of a set of amino acids, plus water, from the (intracellular) gating region of the KcsA K(+) channel. Their atomic coordinates, except hydrogen, are known from X-ray structures [D.A. Doyle, J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, R. MacKinnon, The structure of the potassium channel: molecular basis of K(+) conduction and selectivity, Science 280 (1998) 69-77; R. MacKinnon, S.L. Cohen, A. Kuo, A. Lee, B.T. Chait, Structural conservation in prokaryotic and eukaryotic potassium channels, Science 280 (1998) 106-109; Y. Jiang, A. Lee, J. Chen, M. Cadene, B.T. Chait, R. MacKinnon, The open pore conformation of potassium channels. Nature 417 (2001) 523-526], as are the coordinates of some water oxygen atoms. The 1k4c structure is used for the starting coordinates. Quantum mechanical optimization, in spite of the starting configuration, places the atoms in positions much closer to the 1j95, more tightly closed, configuration. This state shows four water molecules forming a "basket" under the Q119 side chains, blocking the channel. When a hydrated K(+) approaches this "basket", the optimized system shows a strong set of hydrogen bonds with the K(+) at defined positions, preventing further approach of the K(+) to the basket. This optimized structure with hydrated K(+) added shows an ice-like 12 molecule nanocrystal of water. If the water molecules exchange, unless they do it as a group, the channel will remain blocked. The "basket" itself appears to be very stable, although it is possible that the K(+) with its hydrating water molecules may be more mobile, capable of withdrawing from the gate. It is also not surprising that water essentially freezes, or forms a kind of glue, in a nanometer space; this agrees with experimental results on a rather different, but similarly sized (nm dimensions) system [K.B. Jinesh, J.W.M. Frenken, Capillary condensation in atomic scale friction: how water acts like a glue, Phys. Rev. Lett. 96 (2006) 166103/1-4]. It also agrees qualitatively with simulations on channels [A. Anishkin, S. Sukharev, Water dynamics and dewetting transitions in the small mechanosensitive channel MscS, Biophys. J. 86 (2004) 2883-2895; O. Beckstein, M.S.P. Sansom, Liquid-vapor oscillations of water in hydrophobic nanopores, Proc. Natl Acad. Sci. U. S. A. 100 (2003) 7063-7068] and on featureless channel-like systems [J. Lu, M.E. Green, Simulation of water in a pore with charges: application to a gating mechanism for ion channels, Prog. Colloid Polym. Sci. 103 (1997) 121-129], in that it forms a boundary on water that is not obvious from the liquid state. The idea that a structure is stable, even if individual molecules exchange, is well known, for example from the hydration shell of ions. We show that when charges are added in the form of protons to the domains (one proton per domain), the optimized structure is open. No stable water hydrogen bonds hold it together; an opening of 11.0 A appears, measured diagonally between non-neighboring domains as glutamine 119 carbonyl O-O distance. This is comparable to the opening in the MthK potassium channel structure that is generally agreed to be open. The appearance of the opening is in rather good agreement with that found by Perozo and coworkers. In contrast, in the uncharged structure this diagonal distance is 6.5 A, and the water "basket" constricts the uncharged opening still further, with the ice-like structure that couples the K(+) ion to the gating region freezing the entrance to the channel. Comparison with our earlier model for voltage gated channels suggests that a similar mechanism may apply in those channels.

Selectivity Mechanism of the Voltage-gated Proton Channel, HV1

NASA Astrophysics Data System (ADS)

Dudev, Todor; Musset, Boris; Morgan, Deri; Cherny, Vladimir V.; Smith, Susan M. E.; Mazmanian, Karine; Decoursey, Thomas E.; Lim, Carmay

2015-05-01

Voltage-gated proton channels, HV1, trigger bioluminescence in dinoflagellates, enable calcification in coccolithophores, and play multifarious roles in human health. Because the proton concentration is minuscule, exquisite selectivity for protons over other ions is critical to HV1 function. The selectivity of the open HV1 channel requires an aspartate near an arginine in the selectivity filter (SF), a narrow region that dictates proton selectivity, but the mechanism of proton selectivity is unknown. Here we use a reduced quantum model to elucidate how the Asp-Arg SF selects protons but excludes other ions. Attached to a ring scaffold, the Asp and Arg side chains formed bidentate hydrogen bonds that occlude the pore. Introducing H3O+ protonated the SF, breaking the Asp-Arg linkage and opening the conduction pathway, whereas Na+ or Cl- was trapped by the SF residue of opposite charge, leaving the linkage intact, thus preventing permeation. An Asp-Lys SF behaved like the Asp-Arg one and was experimentally verified to be proton-selective, as predicted. Hence, interacting acidic and basic residues form favorable AspH0-H2O0-Arg+ interactions with hydronium but unfavorable Asp--X-/X+-Arg+ interactions with anions/cations. This proposed mechanism may apply to other proton-selective molecules engaged in bioenergetics, homeostasis, and signaling.

Promotion of Water Channels for Enhanced Ion Transport in 14 nm Diameter Carbon Nanotubes.

PubMed

Sheng, Jiadong; Zhu, Qi; Zeng, Xian; Yang, Zhaohui; Zhang, Xiaohua

2017-03-29

Ion transport plays an important role in solar-to-electricity conversion, drug delivery, and a variety of biological processes. Carbon nanotube (CNT) is a promising material as an ion transporter in the applications of the mimicking of natural ion channels, desalination, and energy harvesting. Here, we demonstrate a unique, enhanced ion transport through a vertically aligned multiwall CNT membrane after the application of an electric potential across CNT membranes. Interestingly, electrowetting arising from the application of an electric potential is critical for the enhancement of overall ion transport rate through CNT membranes. The wettability of a liquid with high surface tension on the interior channel walls of CNTs increases during an electric potential treatment and promotes the formation of water channels in CNTs. The formation of water channels in CNTs induces an increase in overall ion diffusion through CNT membranes. This phenomenon is also related to a decrease in the charge transfer resistance of CNTs (R ct ) after an electric potential is applied. Correspondingly, the enhanced ion flow rate gives rise to an enhancement in the capacitive performance of CNT based membranes. Our observations might have profound impact on the development of CNT based energy storage devices as well as artificial ion channels.

Properties of the intracellular transient receptor potential (TRP) channel in yeast, Yvc1.

PubMed

Chang, Yiming; Schlenstedt, Gabriel; Flockerzi, Veit; Beck, Andreas

2010-05-17

Transient receptor potential (TRP) channels are found among mammals, flies, worms, ciliates, Chlamydomonas, and yeast but are absent in plants. These channels are believed to be tetramers of proteins containing six transmembrane domains (TMs). Their primary structures are diverse with sequence similarities only in some short amino acid sequence motifs mainly within sequences covering TM5, TM6, and adjacent domains. In the yeast genome, there is one gene encoding a TRP-like sequence. This protein forms an ion channel in the vacuolar membrane and is therefore called Yvc1 for yeast vacuolar conductance 1. In the following we summarize its prominent features. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

PubMed

Catarsi, S; Drapeau, P

1997-08-01

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

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

Structures of closed and open states of a voltage-gated sodium channel

PubMed Central

Lenaeus, Michael J.; Gamal El-Din, Tamer M.; Ramanadane, Karthik; Pomès, Régis; Zheng, Ning; Catterall, William A.

2017-01-01

Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltage-sensing and pore-forming domains, but they are homotetramers of four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NaVAb mutants that capture tightly closed and open states at a resolution of 2.8–3.2 Å. Introduction of two humanizing mutations in the S6 segment (NaVAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NavAb/1–226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ∼10 Å. Molecular dynamics and free-energy simulations confirm designation of NaVAb/1–226 as an open state that allows permeation of hydrated Na+, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed–open–inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs. PMID:28348242

Ion channel remodeling in vascular smooth muscle during hypertension: Implications for novel therapeutic approaches

PubMed Central

Joseph, Biny K.; Thakali, Keshari M.; Moore, Christopher L.; Rhee, Sung W.

2013-01-01

Ion channels are multimeric, transmembrane proteins that selectively mediate ion flux across the plasma membrane in a variety of cells including vascular smooth muscle cells (VSMCs). The dynamic interplay of Ca2+ and K+ channels on the plasma membrane of VSMCs plays a pivotal role in modulating the vascular tone of small arteries and arterioles. The abnormally-elevated arterial tone observed in hypertension thus points to an aberrant expression and function of Ca2+ and K+ channels in the VSMCs. In this short review, we focus on the three well-studied ion channels in VSMCs, namely the L-type Ca2+ (CaV1.2) channels, the voltage-gated K+ (KV) channels, and the large-conductance Ca2+-activated K+ (BK) channels. First, we provide a brief overview on the physiological role of vascular CaV1.2, KV and BK channels in regulating arterial tone. Second, we discuss the current understanding of the expression changes and regulation of CaV1.2, KV and BK channels in the vasculature during hypertension. Third, based on available proof-of-concept studies, we describe the potential therapeutic approaches targeting these vascular ion channels in order to restore blood pressure to normotensive levels. PMID:23376354

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore*

PubMed Central

El Hiani, Yassine; Linsdell, Paul

2015-01-01

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl− and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl− ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl− ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl− permeation, demonstrating their functional role in maximization of Cl− flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl−. The location of these Cl−-attractive residues suggests that cytoplasmic Cl− ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl− ions from the cytoplasm. PMID:25944907

A Change in the Ion Selectivity of Ligand-Gated Ion Channels Provides a Mechanism to Switch Behavior.

PubMed

Pirri, Jennifer K; Rayes, Diego; Alkema, Mark J

2015-01-01

Behavioral output of neural networks depends on a delicate balance between excitatory and inhibitory synaptic connections. However, it is not known whether network formation and stability is constrained by the sign of synaptic connections between neurons within the network. Here we show that switching the sign of a synapse within a neural circuit can reverse the behavioral output. The inhibitory tyramine-gated chloride channel, LGC-55, induces head relaxation and inhibits forward locomotion during the Caenorhabditis elegans escape response. We switched the ion selectivity of an inhibitory LGC-55 anion channel to an excitatory LGC-55 cation channel. The engineered cation channel is properly trafficked in the native neural circuit and results in behavioral responses that are opposite to those produced by activation of the LGC-55 anion channel. Our findings indicate that switches in ion selectivity of ligand-gated ion channels (LGICs) do not affect network connectivity or stability and may provide an evolutionary and a synthetic mechanism to change behavior.

CFTR: A new horizon in the pathomechanism and treatment of pancreatitis

PubMed Central

Hegyi, Péter; Wilschanski, Michael; Muallem, Shmuel; Lukacs, Gergely; Sahin-Tóth, Miklós; Uc, Aliye; Gray, Michael A.; Rakonczay, Zoltán; Maléth, József

2017-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts chloride and bicarbonate ions across epithelial cell membranes. Mutations in the CFTR gene diminish the ion channel function and lead to impaired epithelial fluid transport in multiple organs such as the lung and the pancreas resulting in cystic fibrosis. Heterozygous carriers of CFTR mutations do not develop cystic fibrosis but exhibit increased risk for pancreatitis and associated pancreatic damage characterized by elevated mucus levels, fibrosis and cyst formation. Importantly, recent studies demonstrated that pancreatitis causing insults, such as alcohol, smoking or bile acids strongly inhibit CFTR function. Furthermore, human studies showed reduced levels of CFTR expression and function in all forms of pancreatitis. These findings indicate that impairment of CFTR is critical in the development of pancreatitis; therefore, correcting CFTR function could be the first specific therapy in pancreatitis. In this review, we summarize recent advances in the field and discuss new possibilities for the treatment of pancreatitis. PMID:26856995

Evans Blue is not a suitable inhibitor of the epithelial sodium channel δ-subunit.

PubMed

Perniss, Alexander; Wolf, Annemarie; Wichmann, Lukas; Schönberger, Matthias; Althaus, Mike

2015-10-23

The Epithelial Sodium Channel (ENaC) is a heterotrimeric ion channel which can be either formed by assembly of its α-, β- and γ-subunits or, alternatively, its δ-, β- and γ-subunits. The physiological function of αβγ-ENaC is well established, but the function of δβγ-ENaC remains elusive. The azo-dye Evans Blue (EvB) has been routinely used to discriminate between the two channel isoforms by decreasing transmembrane currents and amiloride-sensitive current fractions of δβγ-ENaC expressing Xenopus oocytes. Even though these results could be reproduced, it was found by precipitation experiments and spectroscopic methods that the cationic amiloride and the anionic EvB directly interact in solution, forming a strong complex. Thereby a large amount of pharmacologically available amiloride is removed from physiological buffer solutions and the effective amiloride concentration is reduced. This interaction did not occur in the presence of albumin. In microelectrode recordings, EvB was able to abrogate the block of δβγ-ENaC by amiloride or its derivative benzamil. In sum, EvB reduces amiloride-sensitive ion current fractions in electrophysiological experiments. This is not a result of a specific inhibition of δβγ-ENaC but rather represents a pharmacological artefact. EvB should therefore not be used as an inhibitor of δ-ENaC. Copyright © 2015 Elsevier Inc. All rights reserved.

The toxic mode of action of cyclic lipodepsipeptide fusaricidins, produced by Paenibacillus polymyxa, toward mammalian cells.

PubMed

Mikkola, R; Andersson, M A; Grigoriev, P; Heinonen, M; Salkinoja-Salonen, M S

2017-08-01

Toxigenic strains of Paenibacillus polymyxa were isolated from buildings connected with the symptoms of ill health. Our aim was to identify the toxic compounds of Paenibacillus polymyxa and to describe their toxic actions. The toxins of Paenibacillus polymyxa were purified and analysed by HPLC and mass spectrometry. Toxic fusaricidins A and B, and LI-F05a with mass ions at m/z 883·7, 897·6 and 897·6, respectively, were found. The cytotoxicity of purified fusaricidins A and B was measured using boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts. The ion channel forming properties of fusaricidins were studied using the black lipid membrane (BLM) technique. Fusaricidins A and B depolarized the mitochondria of boar sperm, porcine tubular kidney epithelial cells and murine fibroblasts at concentrations of 0·5-1 μg ml -1 and caused nuclear fragmentation and induced apoptosis at concentrations of 2·5-5 μg ml -1 . Furthermore, fusaricidins A and B induced K + permeating single channels. It was concluded that fusaricidins were toxic to mitochondria and induced apoptosis in mammalian cells. It was proposed that the observed toxicity of fusaricidins is due their ion channel forming properties. This paper revealed, for the first time, the mode of action of Paenibacillus polymyxa fusaricidins toxins towards mammalian cells. Fusaricidins, due to their potassium ionophoricity and mitochondria depolarizing impacts, may have contributed to the health damage observed at sites where the producer strains were isolated at high density. © 2017 The Society for Applied Microbiology.

Dendritic ion channelopathy in acquired epilepsy

PubMed Central

Poolos, Nicholas P.; Johnston, Daniel

2012-01-01

Summary Ion channel dysfunction or “channelopathy” is a proven cause of epilepsy in the relatively uncommon genetic epilepsies with Mendelian inheritance. But numerous examples of acquired channelopathy in experimental animal models of epilepsy following brain injury have also been demonstrated. Our understanding of channelopathy has grown due to advances in electrophysiology techniques that have allowed the study of ion channels in the dendrites of pyramidal neurons in cortex and hippocampus. The apical dendrites of pyramidal neurons comprise the vast majority of neuronal surface membrane area, and thus the majority of the neuronal ion channel population. Investigation of dendritic ion channels has demonstrated remarkable plasticity in ion channel localization and biophysical properties in epilepsy, many of which produce hyperexcitability and may contribute to the development and maintenance of the epileptic state. Here we review recent advances in dendritic physiology and cell biology, and their relevance to epilepsy. PMID:23216577

Relevance of quantum mechanics on some aspects of ion channel function

PubMed Central

Roy, Sisir

2010-01-01

Mathematical modeling of ionic diffusion along K ion channels indicates that such diffusion is oscillatory, at the weak non-Markovian limit. This finding leads us to derive a Schrödinger–Langevin equation for this kind of system within the framework of stochastic quantization. The Planck’s constant is shown to be relevant to the Lagrangian action at the level of a single ion channel. This sheds new light on the issue of applicability of quantum formalism to ion channel dynamics and to the physical constraints of the selectivity filter. PMID:19520314

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

PubMed

Farre, Cecilia; Fertig, Niels

2012-06-01

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

Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death.

PubMed

Marks, A R

2001-04-01

Calcium (Ca2+) ions are second messengers in signaling pathways in all types of cells. They regulate muscle contraction, electrical signals which determine the cardiac rhythm and cell growth pathways in the heart. In the past decade cDNA cloning has provided clues as to the molecular structure of the intracellular Ca2+ release channels (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP3R) on the sarcoplasmic and endoplasmic reticulum (SR/ER) and an understanding of how these molecules regulate Ca2+ homeostasis in the heart is beginning to emerge. The intracellular Ca2+ release channels form a distinct class of ion channels distinguished by their structure, size, and function. Both RyRs and IP3Rs have gigantic cytoplasmic domains that serve as scaffolds for modulatory proteins that regulate the channel pore located in the carboxy terminal 10% of the channel sequence. The channels are tetramers comprised of four RyR or IP3R subunits. RyR2 is required for excitation-contraction (EC) coupling in the heart. Using co-sedimentation and co-immunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein mAKAP. We have shown that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (P(o)). In failing human hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

The preference of tryptophan for membrane interfaces: insights from N-methylation of tryptophans in gramicidin channels.

PubMed

Sun, Haiyan; Greathouse, Denise V; Andersen, Olaf S; Koeppe, Roger E

2008-08-08

To better understand the structural and functional roles of tryptophan at the membrane/water interface in membrane proteins, we examined the structural and functional consequences of Trp --> 1-methyl-tryptophan substitutions in membrane-spanning gramicidin A channels. Gramicidin A channels are miniproteins that are anchored to the interface by four Trps near the C terminus of each subunit in a membrane-spanning dimer. We masked the hydrogen bonding ability of individual or multiple Trps by 1-methylation of the indole ring and examined the structural and functional changes using circular dichroism spectroscopy, size exclusion chromatography, solid state (2)H NMR spectroscopy, and single channel analysis. N-Methylation causes distinct changes in the subunit conformational preference, channel-forming propensity, single channel conductance and lifetime, and average indole ring orientations within the membrane-spanning channels. The extent of the local ring dynamic wobble does not increase, and may decrease slightly, when the indole NH is replaced by the non-hydrogen-bonding and more bulky and hydrophobic N-CH(3) group. The changes in conformational preference, which are associated with a shift in the distribution of the aromatic residues across the bilayer, are similar to those observed previously with Trp --> Phe substitutions. We conclude that indole N-H hydrogen bonding is of major importance for the folding of gramicidin channels. The changes in ion permeability, however, are quite different for Trp --> Phe and Trp --> 1-methyl-tryptophan substitutions, indicating that the indole dipole moment and perhaps also ring size and are important for ion permeation through these channels.

Theory and simulation of ion conduction in the pentameric GLIC channel.

PubMed

Zhu, Fangqiang; Hummer, Gerhard

2012-10-09

GLIC is a bacterial member of the large family of pentameric ligand-gated ion channels. To study ion conduction through GLIC and other membrane channels, we combine the one-dimensional potential of mean force for ion passage with a Smoluchowski diffusion model, making it possible to calculate single-channel conductance in the regime of low ion concentrations from all-atom molecular dynamics (MD) simulations. We then perform MD simulations to examine sodium ion conduction through the GLIC transmembrane pore in two systems with different bulk ion concentrations. The ion potentials of mean force, calculated from umbrella sampling simulations with Hamiltonian replica exchange, reveal a major barrier at the hydrophobic constriction of the pore. The relevance of this barrier for ion transport is confirmed by a committor function that rises sharply in the barrier region. From the free evolution of Na(+) ions starting at the barrier top, we estimate the effective diffusion coefficient in the barrier region, and subsequently calculate the conductance of the pore. The resulting diffusivity compares well with the position-dependent ion diffusion coefficient obtained from restrained simulations. The ion conductance obtained from the diffusion model agrees with the value determined via a reactive-flux rate calculation. Our results show that the conformation in the GLIC crystal structure, with an estimated conductance of ~1 picosiemens at 140 mM ion concentration, is consistent with a physiologically open state of the channel.

Ion channels in inflammation.

PubMed

Eisenhut, Michael; Wallace, Helen

2011-04-01

Most physical illness in vertebrates involves inflammation. Inflammation causes disease by fluid shifts across cell membranes and cell layers, changes in muscle function and generation of pain. These disease processes can be explained by changes in numbers or function of ion channels. Changes in ion channels have been detected in diarrhoeal illnesses, pyelonephritis, allergy, acute lung injury and systemic inflammatory response syndromes involving septic shock. The key role played by changes in ion transport is directly evident in inflammation-induced pain. Expression or function of all major categories of ion channels like sodium, chloride, calcium, potassium, transient receptor potential, purinergic receptor and acid-sensing ion channels can be influenced by cyto- and chemokines, prostaglandins, leukotrienes, histamine, ATP, reactive oxygen species and protons released in inflammation. Key pathways in this interaction are cyclic nucleotide, phosphoinositide and mitogen-activated protein kinase-mediated signalling, direct modification by reactive oxygen species like nitric oxide, ATP or protons and disruption of the cytoskeleton. Therapeutic interventions to modulate the adverse and overlapping effects of the numerous different inflammatory mediators on each ion transport system need to target adversely affected ion transport systems directly and locally.

The Voltage-Sensing Domain of K(v)7.2 Channels as a Molecular Target for Epilepsy-Causing Mutations and Anticonvulsants.

PubMed

Miceli, Francesco; Soldovieri, Maria Virginia; Iannotti, Fabio Arturo; Barrese, Vincenzo; Ambrosino, Paolo; Martire, Maria; Cilio, Maria Roberta; Taglialatela, Maurizio

2011-01-01

Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs) has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically determined channelopathies affecting heart rhythm (arrhythmias), neuronal excitability (epilepsy, pain), or skeletal muscle contraction (periodic paralysis). Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function. In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K(+) channels encoded by the K(v)7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by K(v)7.2-K(v)7.5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically determined alterations in K(v)7.2 and K(v)7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of K(v)7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in K(v)7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability.

The Voltage-Sensing Domain of Kv7.2 Channels as a Molecular Target for Epilepsy-Causing Mutations and Anticonvulsants

PubMed Central

Miceli, Francesco; Soldovieri, Maria Virginia; Iannotti, Fabio Arturo; Barrese, Vincenzo; Ambrosino, Paolo; Martire, Maria; Cilio, Maria Roberta; Taglialatela, Maurizio

2010-01-01

Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs) has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically determined channelopathies affecting heart rhythm (arrhythmias), neuronal excitability (epilepsy, pain), or skeletal muscle contraction (periodic paralysis). Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function. In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K+ channels encoded by the Kv7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by Kv7.2–Kv7.5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically determined alterations in Kv7.2 and Kv7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of Kv7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in Kv7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability. PMID:21687499

Unraveling the mechanism of selective ion transport in hydrophobic subnanometer channels

PubMed Central

Li, Hui; Francisco, Joseph S.; Zeng, Xiao Cheng

2015-01-01

Recently reported synthetic organic nanopore (SONP) can mimic a key feature of natural ion channels, i.e., selective ion transport. However, the physical mechanism underlying the K+/Na+ selectivity for the SONPs is dramatically different from that of natural ion channels. To achieve a better understanding of the selective ion transport in hydrophobic subnanometer channels in general and SONPs in particular, we perform a series of ab initio molecular dynamics simulations to investigate the diffusivity of aqua Na+ and K+ ions in two prototype hydrophobic nanochannels: (i) an SONP with radius of 3.2 Å, and (ii) single-walled carbon nanotubes (CNTs) with radii of 3–5 Å (these radii are comparable to those of the biological potassium K+ channels). We find that the hydration shell of aqua Na+ ion is smaller than that of aqua K+ ion but notably more structured and less yielding. The aqua ions do not lower the diffusivity of water molecules in CNTs, but in SONP the diffusivity of aqua ions (Na+ in particular) is strongly suppressed due to the rugged inner surface. Moreover, the aqua Na+ ion requires higher formation energy than aqua K+ ion in the hydrophobic nanochannels. As such, we find that the ion (K+ vs. Na+) selectivity of the (8, 8) CNT is ∼20× higher than that of SONP. Hence, the (8, 8) CNT is likely the most efficient artificial K+ channel due in part to its special interior environment in which Na+ can be fully solvated, whereas K+ cannot. This work provides deeper insights into the physical chemistry behind selective ion transport in nanochannels. PMID:26283377

Gap-junctional channel and hemichannel activity of two recently identified connexin 26 mutants associated with deafness.

PubMed

Dalamon, Viviana; Fiori, Mariana C; Figueroa, Vania A; Oliva, Carolina A; Del Rio, Rodrigo; Gonzalez, Wendy; Canan, Jonathan; Elgoyhen, Ana B; Altenberg, Guillermo A; Retamal, Mauricio A

2016-05-01

Gap-junction channels (GJCs) are formed by head-to-head association of two hemichannels (HCs, connexin hexamers). HCs and GJCs are permeable to ions and hydrophilic molecules of up to Mr ~1 kDa. Hearing impairment of genetic origin is common, and mutations of connexin 26 (Cx26) are its major cause. We recently identified two novel Cx26 mutations in hearing-impaired subjects, L10P and G109V. L10P forms functional GJCs with slightly altered voltage dependence and HCs with decrease ATP/cationic dye selectivity. G109V does not form functional GJCs, but forms functional HCs with enhanced extracellular Ca(2+) sensitivity and subtle alterations in voltage dependence and ATP/cationic dye selectivity. Deafness associated with G109V could result from decreased GJCs activity, whereas deafness associated to L10P may have a more complex mechanism that involves changes in HC permeability.

The N-terminal domain of Slack determines the formation and trafficking of Slick/Slack heteromeric sodium-activated potassium channels.

PubMed

Chen, Haijun; Kronengold, Jack; Yan, Yangyang; Gazula, Valeswara-Rao; Brown, Maile R; Ma, Liqun; Ferreira, Gonzalo; Yang, Youshan; Bhattacharjee, Arin; Sigworth, Fred J; Salkoff, Larry; Kaczmarek, Leonard K

2009-04-29

Potassium channels activated by intracellular Na(+) ions (K(Na)) play several distinct roles in regulating the firing patterns of neurons, and, at the single channel level, their properties are quite diverse. Two known genes, Slick and Slack, encode K(Na) channels. We have now found that Slick and Slack subunits coassemble to form heteromeric channels that differ from the homomers in their unitary conductance, kinetic behavior, subcellular localization, and response to activation of protein kinase C. Heteromer formation requires the N-terminal domain of Slack-B, one of the alternative splice variants of the Slack channel. This cytoplasmic N-terminal domain of Slack-B also facilitates the localization of heteromeric K(Na) channels to the plasma membrane. Immunocytochemical studies indicate that Slick and Slack-B subunits are coexpressed in many central neurons. Our findings provide a molecular explanation for some of the diversity in reported properties of neuronal K(Na) channels.

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

PubMed Central

Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

1996-01-01

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions. Images PMID:8861937

Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

PubMed

Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

1996-08-15

The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions.

Rediscovering sperm ion channels with the patch-clamp technique

PubMed Central

Kirichok, Yuriy; Lishko, Polina V.

2011-01-01

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

Mechanisms Responsible for ω-Pore Currents in Cav Calcium Channel Voltage-Sensing Domains.

PubMed

Monteleone, Stefania; Lieb, Andreas; Pinggera, Alexandra; Negro, Giulia; Fuchs, Julian E; Hofer, Florian; Striessnig, Jörg; Tuluc, Petronel; Liedl, Klaus R

2017-10-03

Mutations of positively charged amino acids in the S4 transmembrane segment of a voltage-gated ion channel form ion-conducting pathways through the voltage-sensing domain, named ω-current. Here, we used structure modeling and MD simulations to predict pathogenic ω-currents in Ca V 1.1 and Ca V 1.3 Ca 2+ channels bearing several S4 charge mutations. Our modeling predicts that mutations of Ca V 1.1-R1 (R528H/G, R897S) or Ca V 1.1-R2 (R900S, R1239H) linked to hypokalemic periodic paralysis type 1 and of Ca V 1.3-R3 (R990H) identified in aldosterone-producing adenomas conducts ω-currents in resting state, but not during voltage-sensing domain activation. The mechanism responsible for the ω-current and its amplitude depend on the number of charges in S4, the position of the mutated S4 charge and countercharges, and the nature of the replacing amino acid. Functional characterization validates the modeling prediction showing that Ca V 1.3-R990H channels conduct ω-currents at hyperpolarizing potentials, but not upon membrane depolarization compared with wild-type channels. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

PubMed Central

Gurnev, Philip A.; Nestorovich, Ekaterina M.

2014-01-01

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications. PMID:25153255

Ion channel stabilization of synthetic alamethicin analogs by rings of inter-helix H-bonds.

PubMed Central

Molle, G; Dugast, J Y; Spach, G; Duclohier, H

1996-01-01

Rings of inter-helix H-bonds due to Gln at position 7, a highly conserved residue in all pore-forming peptaibols, have been suggested to play an important role in the stabilization of alamethicin channels. In an attempt to test this hypothesis, experimental studies have been undertaken on four synthetic alamethicin non-Aib analogs (Alm-dUL) in which the Gln at position 7 (Q7) is substituted by Ala, Asn, or Ser (Q7A, Q7N, or Q7S). Voltage-dependent pore formation by these analogs in planar lipid bilayers is compared at the macroscopic and single-channel conductance levels. As anticipated, the Q7A substitution abolished all channel-forming activity. The voltage dependence of macroscopic current-voltage curves was conserved with the Q7N substitution but reduced in the Q7S analog. Normalized single-channel conductance ratios between substates follow the same pattern, with the Q7S analog yielding the highest unit conductances. Channel lifetimes were the most significantly modulated parameter with markedly faster kinetics when Gln or Asn was replaced by Ser. The effect of the Q7S substitution on channel lifetimes may be explained through a reduced stabilization of bundles by inter-helix H-bonds. PMID:8785325

Multiple modes of a-type potassium current regulation.

PubMed

Cai, Shi-Qing; Li, Wenchao; Sesti, Federico

2007-01-01

Voltage-dependent potassium (K+) channels (Kv) regulate cell excitability by controlling the movement of K+ ions across the membrane in response to changes in the cell voltage. The Kv family, which includes A-type channels, constitute the largest group of K+ channel genes within the superfamily of Na+, Ca2+ and K+ voltage-gated channels. The name "A-type" stems from the typical profile of these currents that results form the opposing effects of fast activation and inactivation. In neuronal cells, A-type currents (I(A)), determine the interval between two consecutive action potentials during repetitive firing. In cardiac muscle, A-type currents (I(to)), control the initial repolarization of the myocardium. Structurally, A-type channels are tetramers of alpha-subunits each containing six putative transmembrane domains including a voltage-sensor. A-type channels can be modulated by means of protein-protein interactions with so-called beta-subunits that control inactivation voltage sensitivity and other properties, and by post-transcriptional modifications such as phosphorylation or oxidation. Recently a new mode of A-type regulation has been discovered in the form of a class of hybrid beta-subunits that posses their own enzymatic activity. Here, we review the biophysical and physiological properties of these multiple modes of A-type channel regulation.

Divalent ions are potential permeating blockers of the non-selective NaK ion channel: combined QM and MD based investigations.

PubMed

Sadhu, Biswajit; Sundararajan, Mahesh; Bandyopadhyay, Tusar

2017-10-18

The bacterial NaK ion channel is distinctly different from other known ion channels due to its inherent non-selective feature. One of the unexplored and rather interesting features is its ability to permeate divalent metal ions (such as Ca 2+ and Ba 2+ ) and not monovalent alkali metal ions. Several intriguing questions about the energetics and structural aspects still remain unanswered. For instance, what causes Ca 2+ to permeate as well as block the selectivity filter (SF) of the NaK ion channel and act as a "permeating blocker"? How and at what energetic cost does another chemical congener, Sr 2+ , as well as Ba 2+ , a potent blocker of the K + ion channel, permeate through the SF of the NaK ion channel? Finally, how do their translocation energetics differ from those of monovalent ions such as K + ? Here, in an attempt to address these outstanding issues, we elucidate the structure, binding and selectivity of divalent ions (Ca 2+ , Sr 2+ and Ba 2+ ) as they permeate through the SF of the NaK ion channel using all-atom molecular dynamics simulations and density functional theory based calculations. We unveil mechanistic insight into this translocation event using well-tempered metadynamics simulations in a polarizable environment using the mean-field model of water and incorporating electronic continuum corrections for ions via charge rescaling. The results show that, akin to K + coordination, Sr 2+ and Ba 2+ bind at the SF in a very similar fashion and remain octa-coordinated at all sites. Interestingly, differing from its local hydration structure, Ca 2+ interacts with eight carbonyls to remain at the middle of the S3 site. Furthermore, the binding of divalent metals at SF binding sites is more favorable than the binding of K + . However, their permeation through the extracellular entrance faces a considerably higher energetic barrier compared to that for K + , which eventually manifests their inherent blocking feature.

Sperm-specific ion channels: targets holding the most potential for male contraceptives in development.

PubMed

Zheng, Li-Ping; Wang, Hua-Feng; Li, Bao-Ming; Zeng, Xu-Hui

2013-10-01

There is a global need for an ideal method of male contraception. However, the development of male contraceptives has not been well successful. Research on sperm-specific ion channels, especially the recent advance obtained from electrophysiological studies, has emphasized the conception that those channels are targets with the most potential to develop non-hormonal male contraceptives. While summarizing the general options for male contraception, this review focuses on the properties and functions of sperm ion channels together with the attempts of utilizing these channels to develop male contraceptives. We believe that a deeper insight into the signaling and molecular mechanisms by which ion channels regulate sperm functions will pave the way for developing novel male-based contraceptives. Copyright © 2013 Elsevier Inc. All rights reserved.

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

PubMed

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

2007-10-01

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

A membrane-embedded pathway delivers general anesthetics to two interacting binding sites in the Gloeobacter violaceus ion channel.

PubMed

Arcario, Mark J; Mayne, Christopher G; Tajkhorshid, Emad

2017-06-09

General anesthetics exert their effects on the central nervous system by acting on ion channels, most notably pentameric ligand-gated ion channels. Although numerous studies have focused on pentameric ligand-gated ion channels, the details of anesthetic binding and channel modulation are still debated. A better understanding of the anesthetic mechanism of action is necessary for the development of safer and more efficacious drugs. Herein, we present a computational study identifying two anesthetic binding sites in the transmembrane domain of the Gloeobacter violaceus ligand-gated ion channel (GLIC) channel, characterize the putative binding pathway, and observe structural changes associated with channel function. Molecular simulations of desflurane reveal a binding pathway to GLIC via a membrane-embedded tunnel using an intrasubunit protein lumen as the conduit, an observation that explains the Meyer-Overton hypothesis, or why the lipophilicity of an anesthetic and its potency are generally proportional. Moreover, employing high concentrations of ligand led to the identification of a second transmembrane site (TM2) that inhibits dissociation of anesthetic from the TM1 site and is consistent with the high concentrations of anesthetics required to achieve clinical effects. Finally, asymmetric binding patterns of anesthetic to the channel were found to promote an iris-like conformational change that constricts and dehydrates the ion pore, creating a 13.5 kcal/mol barrier to ion translocation. Together with previous studies, the simulations presented herein demonstrate a novel anesthetic binding site in GLIC that is accessed through a membrane-embedded tunnel and interacts with a previously known site, resulting in conformational changes that produce a non-conductive state of the channel. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

PubMed

Wright, Joy R; Amisten, Stefan; Goodall, Alison H; Mahaut-Smith, Martyn P

2016-08-01

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288-11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These "channelome" datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

Band gap engineering by swift heavy ions irradiation induced amorphous nano-channels in LiNbO3

DOE PAGES

Sachan, Ritesh; Pakarinen, Olli H.; Liu, Peng; ...

2015-04-01

The irradiation of lithium niobate with swift heavy ions results in the creation of amorphous nano-sized channels along the incident ion path. These nano-channels are on the order of a hundred microns in length and could be useful for photonic applications. However, there are two major challenges in these nano-channels characterization; (i) it is difficult to investigate the structural characteristics of these nano-channels due to their very long length, and (ii) the analytical electron microscopic analysis of individual ion track is complicated due to electron beam sensitive nature of lithium niobate. Here, we report the first high resolution microscopic characterizationmore » of these amorphous nano-channels, widely known as ion-tracks, by direct imaging them at different depths in the material, and subsequently correlating the key characteristics with Se of ions. Energetic Kr ions ( 84Kr 22 with 1.98 GeV energy) are used to irradiate single crystal lithium niobate with a fluence of 2x10 10 ions/cm 2, which results in the formation of individual ion tracks with a penetration depth of ~180 μm. Along the ion path, electron energy loss of the ions, which is responsible for creating the ion tracks, increases with depth under these conditions in LiNbO 3, resulting in increases in track diameter of a factor of ~2 with depth. This diameter increase with electronic stopping power is consistent with predictions of the inelastic thermal spike model. We also show a new method to measure the band gap in individual ion track by using electron energy-loss spectroscopy.« less

Malaria parasite mutants with altered erythrocyte permeability: a new drug resistance mechanism and important molecular tool

PubMed Central

Hill, David A; Desai, Sanjay A

2010-01-01

Erythrocytes infected with plasmodia, including those that cause human malaria, have increased permeability to a diverse collection of organic and inorganic solutes. While these increases have been known for decades, their mechanistic basis was unclear until electrophysiological studies revealed flux through one or more ion channels on the infected erythrocyte membrane. Current debates have centered on the number of distinct ion channels, which channels mediate the transport of each solute and whether the channels represent parasite-encoded proteins or human channels activated after infection. This article reviews the identification of the plasmodial surface anion channel and other proposed channels with an emphasis on two distinct channel mutants generated through in vitro selection. These mutants implicate parasite genetic elements in the parasite-induced permeability, reveal an important new antimalarial drug resistance mechanism and provide tools for molecular studies. We also critically examine the technical issues relevant to the detection of ion channels by electrophysiological methods; these technical considerations have general applicability for interpreting studies of various ion channels proposed for the infected erythrocyte membrane. PMID:20020831

Transient receptor potential canonical 4 and 5 proteins as targets in cancer therapeutics.

PubMed

Gaunt, Hannah J; Vasudev, Naveen S; Beech, David J

2016-10-01

Novel approaches towards cancer therapy are urgently needed. One approach might be to target ion channels mediating Ca 2+ entry because of the critical roles played by Ca 2+ in many cell types, including cancer cells. There are several types of these ion channels, but here we address those formed by assembly of transient receptor potential canonical (TRPC) proteins, particularly those which involve two closely related members of the family: TRPC4 and TRPC5. We focus on these proteins because recent studies point to roles in important aspects of cancer: drug resistance, transmission of drug resistance through extracellular vesicles, tumour vascularisation, and evoked cancer cell death by the TRPC4/5 channel activator (-)-englerin A. We conclude that further research is both justified and necessary before these proteins can be considered as strong targets for anti-cancer cell drug discovery programmes. It is nevertheless already apparent that inhibitors of the channels would be unlikely to cause significant adverse effects, but, rather, have other effects which may be beneficial in the context of cancer and chemotherapy, potentially including suppression of innate fear, visceral pain and pathological cardiac remodelling.

Computational Tools for Interpreting Ion Channel pH-Dependence.

PubMed

Sazanavets, Ivan; Warwicker, Jim

2015-01-01

Activity in many biological systems is mediated by pH, involving proton titratable groups with pKas in the relevant pH range. Experimental analysis of pH-dependence in proteins focusses on particular sidechains, often with mutagenesis of histidine, due to its pKa near to neutral pH. The key question for algorithms that predict pKas is whether they are sufficiently accurate to effectively narrow the search for molecular determinants of pH-dependence. Through analysis of inwardly rectifying potassium (Kir) channels and acid-sensing ion channels (ASICs), mutational effects on pH-dependence are probed, distinguishing between groups described as pH-coupled or pH-sensor. Whereas mutation can lead to a shift in transition pH between open and closed forms for either type of group, only for pH-sensor groups does mutation modulate the amplitude of the transition. It is shown that a hybrid Finite Difference Poisson-Boltzmann (FDPB) - Debye-Hückel continuum electrostatic model can filter mutation candidates, providing enrichment for key pH-coupled and pH-sensor residues in both ASICs and Kir channels, in comparison with application of FDPB alone.

Computational Tools for Interpreting Ion Channel pH-Dependence

PubMed Central

Sazanavets, Ivan; Warwicker, Jim

2015-01-01

Activity in many biological systems is mediated by pH, involving proton titratable groups with pKas in the relevant pH range. Experimental analysis of pH-dependence in proteins focusses on particular sidechains, often with mutagenesis of histidine, due to its pKa near to neutral pH. The key question for algorithms that predict pKas is whether they are sufficiently accurate to effectively narrow the search for molecular determinants of pH-dependence. Through analysis of inwardly rectifying potassium (Kir) channels and acid-sensing ion channels (ASICs), mutational effects on pH-dependence are probed, distinguishing between groups described as pH-coupled or pH-sensor. Whereas mutation can lead to a shift in transition pH between open and closed forms for either type of group, only for pH-sensor groups does mutation modulate the amplitude of the transition. It is shown that a hybrid Finite Difference Poisson-Boltzmann (FDPB) – Debye-Hückel continuum electrostatic model can filter mutation candidates, providing enrichment for key pH-coupled and pH-sensor residues in both ASICs and Kir channels, in comparison with application of FDPB alone. PMID:25915903

Piezo1

PubMed Central

Gottlieb, Philip A.; Sachs, Frederick

2012-01-01

Piezo ion channels have been found to be essential for mechanical responses in cells. These channels were first shown to exist in Neuro2A cells, and the gene was identified by siRNAs that diminished the mechanical response. Piezo channels are approximately 2500 amino acids long, have between 24–32 transmembrane regions, and appear to assemble into tetramers and require no other proteins for activity. They have a reversal potential around 0 mV and show voltage dependent inactivation. The channel is constitutively active in liposomes, indicating that no cytoskeletal elements are required. Heterologous expression of the Piezo protein can create mechanical sensitivity in otherwise insensitive cells. Piezo1 currents in outside-out patches were blocked by the extracellular MSC inhibitor peptide GsMTx4. Both enantiomeric forms of GsMTx4 inhibited channel activity in a manner similar to endogenous mechanical channels. Piezo1 can adopt a tonic (non-inactivating) form with repeated stimulation. The transition to the non-inactivating form generally occurs in large groups of channels, indicating that the channels exist in domains, and once the domain is compromised, the members simultaneously adopt new properties. Piezo proteins are associated with physiological responses in cells, such as the reaction to noxious stimulus of Drosophila larvae. Recent work measuring cell crowding, shows that Piezo1 is essential for the removal of extra cells without apoptosis. Piezo1 mutations have also been linked to the pathological response of red blood cells in a genetic disease called Xerocytosis. These finding suggest that Piezo1 is a key player in cells’ responses to mechanical stimuli. PMID:22790400

Dynamic properties of individual water molecules in a hydrophobic pore lined with acyl chains: a molecular dynamics study.

PubMed

Qi, Z; Sokabe, M

1998-03-30

Recently, a certain class of synthetic molecules has been shown to form ion channels, the pore of which is lined with hydrophobic acyl chains [M. Sokabe, in: F. Oosawa, H. Hayashi, T. Yoshioka (Eds.), Transmembrane Signaling and Sensation, JSSP/VNU Science Press BV, Tokyo, 1984, p. 119; F. Hayashi, M. Sokabe, M. Takagi, K. Hayashi, U. Kishimoto, Biochim. Biophys. Acta, 510 (1978) 305; M.J. Pregel, L. Jullien, J. Canceill, L. Lacombe, J.M. Lehn, J. Chem. Soc. Perkin Trans., 2 (1995) 417; Y. Tanaka, Y. Kobuke, M. Sokabe, Angew. Chem. Int. Ed. Engl., 34 (1995) 693; M. Sokabe, Z. Qi, K. Donowaki, H. Ishida, K. Okubo, Biophys. J., 70 (1996) A201; H. Ishida, K. Donowaki, Y. Inoue, Z. Qi, M. Sokabe, Chem. Lett. (1997) p. 953]. As an initial step towards understanding the physical mechanisms of ion permeation across such a hydrophobic pore, systematic molecular dynamics simulations were performed to investigate dynamic and energetic properties of water molecules inside the pore using a dimer of alanine-N'-acylated cyclic peptide as a channel model. Dynamic energy profiles for water molecules indicated that the energy barrier at the middle region of the pore is approximately 2-3 kcal/mol higher than that in the cap water region which was defined as a vicinity region of the channel entrance. Energetics analyses demonstrated that the mutual interactions among intrapore water molecules are the major factor to give favorable interaction (negative energy contribution) for themselves. The pore, despite being lined with acyl chains, has a favorable van der Waals interaction with intrapore water molecules. These results may help to explain why water-filled channels can be formed by the hydrophobic helices in natural channels.

Structure and assembly mechanism for heteromeric kainate receptors.

PubMed

Kumar, Janesh; Schuck, Peter; Mayer, Mark L

2011-07-28

Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11 nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors. Copyright © 2011 Elsevier Inc. All rights reserved.

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

Laskowski, Lukasz, E-mail: lukasz.laskowski@kik.pcz.pl; Laskowska, Magdalena, E-mail: magdalena.laskowska@onet.pl; Jelonkiewicz, Jerzy, E-mail: jerzy.jelonkiewicz@kik.pcz.pl

The SBA-15 silica thin films containing copper ions anchored inside channels via propyl phosphonate groups are investigated. Such materials were prepared in the form of thin films, with hexagonally arranged pores, laying rectilinear to the substrate surface. However, in the case of our thin films, their free standing form allowed for additional research possibilities, that are not obtainable for typical thin films on a substrate. The structural properties of the samples were investigated by X-ray reflectometry, atomic force microscopy (AFM) and transmission electron microscopy (TEM). The molecular structure was examined by Raman spectroscopy supported by numerical simulations. Magnetic measurements (SQUIDmore » magnetometry and EPR spectroscopy) showed weak antiferromagnetic interactions between active units inside silica channels. Consequently, the pores arrangement was determined and the process of copper ions anchoring by propyl phosphonate groups was verified in unambiguous way. Moreover, the type of interactions between magnetic atoms was determined. - Highlights: • Functionalized free-standing SBA-15 thin films were synthesized for a first time. • Thin films synthesis procedure was described in details. • Structural properties of the films were thoroughly investigated and presented. • Magnetic properties of the novel material was investigated and presented.« less

Moving Fe2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates

PubMed Central

Behera, Rabindra K.; Theil, Elizabeth C.

2014-01-01

Ferritin biominerals are protein-caged metabolic iron concentrates used for iron–protein cofactors and oxidant protection (Fe2+ and O2 sequestration). Fe2+ passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe2+ substrate movement to ferritin enzyme (Fox) sites. Fe2+ and O2 substrates are coupled via a diferric peroxo (DFP) intermediate, λmax 650 nm, which decays to [Fe3+–O–Fe3+] precursors of caged ferritin biominerals. Structural studies show multiple conformations for conserved, carboxylate residues E136 and E57, which are between ferritin ion channel exits and enzymatic sites, suggesting functional connections. Here we show that E136 and E57 are required for ferritin enzyme activity and thus are functional links between ferritin ion channels and enzymatic sites. DFP formation (Kcat and kcat/Km), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe2+ concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe2+ access), Mn2+ << Co2+ < Cu2+ < Zn2+, reflecting metal ion–protein binding stabilities. Fe2+–Cys126 binding in ferritin ion channels, observed as Cu2+–S–Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu2+ inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe2+ movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade that moves external Fe2+ into ferritin enzymatic sites. The results clarify Fe2+ transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations. PMID:24843174

Gating of a pH-sensitive K(2P) potassium channel by an electrostatic effect of basic sensor residues on the selectivity filter.

PubMed

Zúñiga, Leandro; Márquez, Valeria; González-Nilo, Fernando D; Chipot, Christophe; Cid, L Pablo; Sepúlveda, Francisco V; Niemeyer, María Isabel

2011-01-25

K(+) channels share common selectivity characteristics but exhibit a wide diversity in how they are gated open. Leak K(2P) K(+) channels TASK-2, TALK-1 and TALK-2 are gated open by extracellular alkalinization. The mechanism for this alkalinization-dependent gating has been proposed to be the neutralization of the side chain of a single arginine (lysine in TALK-2) residue near the pore of TASK-2, which occurs with the unusual pK(a) of 8.0. We now corroborate this hypothesis by transplanting the TASK-2 extracellular pH (pH(o)) sensor in the background of a pH(o)-insensitive TASK-3 channel, which leads to the restitution of pH(o)-gating. Using a concatenated channel approach, we also demonstrate that for TASK-2 to open, pH(o) sensors must be neutralized in each of the two subunits forming these dimeric channels with no apparent cross-talk between the sensors. These results are consistent with adaptive biasing force analysis of K(+) permeation using a model selectivity filter in wild-type and mutated channels. The underlying free-energy profiles confirm that either a doubly or a singly charged pH(o) sensor is sufficient to abolish ion flow. Atomic detail of the associated mechanism reveals that, rather than a collapse of the pore, as proposed for other K(2P) channels gated at the selectivity filter, an increased height of the energetic barriers for ion translocation accounts for channel blockade at acid pH(o). Our data, therefore, strongly suggest that a cycle of protonation/deprotonation of pH(o)-sensing arginine 224 side chain gates the TASK-2 channel by electrostatically tuning the conformational stability of its selectivity filter.

Gating of a pH-Sensitive K2P Potassium Channel by an Electrostatic Effect of Basic Sensor Residues on the Selectivity Filter

PubMed Central

Zúñiga, Leandro; Márquez, Valeria; González-Nilo, Fernando D.; Chipot, Christophe; Cid, L. Pablo; Sepúlveda, Francisco V.; Niemeyer, María Isabel

2011-01-01

K+ channels share common selectivity characteristics but exhibit a wide diversity in how they are gated open. Leak K2P K+ channels TASK-2, TALK-1 and TALK-2 are gated open by extracellular alkalinization. The mechanism for this alkalinization-dependent gating has been proposed to be the neutralization of the side chain of a single arginine (lysine in TALK-2) residue near the pore of TASK-2, which occurs with the unusual pKa of 8.0. We now corroborate this hypothesis by transplanting the TASK-2 extracellular pH (pHo) sensor in the background of a pHo-insensitive TASK-3 channel, which leads to the restitution of pHo-gating. Using a concatenated channel approach, we also demonstrate that for TASK-2 to open, pHo sensors must be neutralized in each of the two subunits forming these dimeric channels with no apparent cross-talk between the sensors. These results are consistent with adaptive biasing force analysis of K+ permeation using a model selectivity filter in wild-type and mutated channels. The underlying free-energy profiles confirm that either a doubly or a singly charged pHo sensor is sufficient to abolish ion flow. Atomic detail of the associated mechanism reveals that, rather than a collapse of the pore, as proposed for other K2P channels gated at the selectivity filter, an increased height of the energetic barriers for ion translocation accounts for channel blockade at acid pHo. Our data, therefore, strongly suggest that a cycle of protonation/deprotonation of pHo-sensing arginine 224 side chain gates the TASK-2 channel by electrostatically tuning the conformational stability of its selectivity filter. PMID:21283586

Different structural requirements for functional ion pore transplantation suggest different gating mechanisms of NMDA and kainate receptors.

PubMed

Villmann, Carmen; Hoffmann, Jutta; Werner, Markus; Kott, Sabine; Strutz-Seebohm, Nathalie; Nilsson, Tanja; Hollmann, Michael

2008-10-01

Although considerable progress has been made in characterizing the physiological function of the high-affinity kainate (KA) receptor subunits KA1 and KA2, no homomeric ion channel function has been shown. An ion channel transplantation approach was employed in this study to directly test if homomerically expressed KA1 and KA2 pore domains are capable of conducting currents. Transplantation of the ion pore of KA1 or KA2 into GluR6 generated perfectly functional ion channels that allowed characterization of those electrophysiological and pharmacological properties that are determined exclusively by the ion pore of KA1 or KA2. This demonstrates for the first time that KA1 and KA2 ion pore domains are intrinsically capable of conducting ions even in homomeric pore assemblies. NMDA receptors, similar to KA1- or KA2-containing receptors, function only as heteromeric complexes. They are composed of NR1 and NR2 subunits, which both are non-functional when expressed homomerically. In contrast to NR1, the homomeric NR2B ion pore failed to translate ligand binding into pore opening when transplanted into GluR6. Similarly, heteromeric coexpression of the ion channel domains of both NR1 and NR2 inserted into GluR6 failed to produce functional channels. Therefore, we conclude that the mechanism underlying the ion channel opening in the obligatorily heterotetrameric NMDA receptors differs significantly from that in the facultatively heterotetrameric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and KA receptors.

Bio-inspired voltage-dependent calcium channel blockers.

PubMed

Yang, Tingting; He, Lin-Ling; Chen, Ming; Fang, Kun; Colecraft, Henry M

2013-01-01

Ca(2+) influx via voltage-dependent CaV1/CaV2 channels couples electrical signals to biological responses in excitable cells. CaV1/CaV2 channel blockers have broad biotechnological and therapeutic applications. Here we report a general method for developing novel genetically encoded calcium channel blockers inspired by Rem, a small G-protein that constitutively inhibits CaV1/CaV2 channels. We show that diverse cytosolic proteins (CaVβ, 14-3-3, calmodulin and CaMKII) that bind pore-forming α1-subunits can be converted into calcium channel blockers with tunable selectivity, kinetics and potency, simply by anchoring them to the plasma membrane. We term this method 'channel inactivation induced by membrane-tethering of an associated protein' (ChIMP). ChIMP is potentially extendable to small-molecule drug discovery, as engineering FK506-binding protein into intracellular sites within CaV1.2-α1C permits heterodimerization-initiated channel inhibition with rapamycin. The results reveal a universal method for developing novel calcium channel blockers that may be extended to develop probes for a broad cohort of unrelated ion channels.

Role of potassium ion channels in detrusor smooth muscle function and dysfunction

PubMed Central

Petkov, Georgi V.

2013-01-01

Contraction and relaxation of the detrusor smooth muscle (DSM), which makes up the wall of the urinary bladder, facilitates the storage and voiding of urine. Several families of K+ channels, including voltage-gated K+ (KV) channels, Ca2+-activated K+ (KCa) channels, inward-rectifying ATP-sensitive K+ (Kir, KATP) channels, and two-pore-domain K+ (K2P) channels, are expressed and functional in DSM. They control DSM excitability and contractility by maintaining the resting membrane potential and shaping the action potentials that determine the phasic nature of contractility in this tissue. Defects in DSM K+ channel proteins or in the molecules involved in their regulatory pathways may underlie certain forms of bladder dysfunction, such as overactive bladder. K+ channels represent an opportunity for novel pharmacological manipulation and therapeutic intervention in human DSM. Modulation of DSM K+ channels directly or indirectly by targeting their regulatory mechanisms has the potential to control urinary bladder function. This Review summarizes our current state of knowledge of the functional role of K+ channels in DSM in health and disease, with special emphasis on current advancements in the field. PMID:22158596

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

PubMed

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

2011-06-01

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

Activation of the mechanosensitive ion channel MscL by mechanical stimulation of supported Droplet-Hydrogel bilayers

PubMed Central

Rosholm, Kadla R.; Baker, Matthew A. B.; Ridone, Pietro; Nakayama, Yoshitaka; Rohde, Paul R.; Cuello, Luis G.; Lee, Lawrence K.; Martinac, Boris

2017-01-01

The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform. PMID:28345591

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

Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme

PubMed Central

Zhou, Jing-Min; Zhou, De-Min; Takagi, Yasuomi; Kasai, Yasuhiro; Inoue, Atsushi; Baba, Tadashi; Taira, Kazunari

2002-01-01

The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn2+ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na+ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses. PMID:12034824

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions.

PubMed

Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A; Bukauskas, Feliksas F

2018-01-01

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pH i ) and cytosolic magnesium ion concentration ([Mg 2+ ] i ), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pH i and [Mg 2+ ] i affect junctional conductance (g j ) in an interdependent manner; in other words, intracellular acidification cause increase or decay in g j depending on whether [Mg 2+ ] i is high or low, respectively, and intracellular alkalization cause reduction in g j independently of [Mg 2+ ] i . Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pH i and [Mg 2+ ] i . Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pH i and [Mg 2+ ] i . Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg 2+ ] i , while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36 * E8Q lost the initial increase of g j at low [Mg 2+ ] i and double mutation lost the sensitivity to high [Mg 2+ ] i . These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg 2+ and H + ions.

Using iPSC Models to Probe Regulation of Cardiac Ion Channel Function.

PubMed

Bruyneel, Arne A N; McKeithan, Wesley L; Feyen, Dries A M; Mercola, Mark

2018-05-25

Cardiovascular disease is the leading contributor to mortality and morbidity. Many deaths of heart failure patients can be attributed to sudden cardiac death due primarily to ventricular arrhythmia. Currently, most anti-arrhythmics modulate ion channel conductivity or β-adrenergic signaling, but these drugs have limited efficacy for some indications, and can potentially be proarrhythmic. Recent studies have shown that mutations in proteins other than cardiac ion channels may confer susceptibility to congenital as well as acquired arrhythmias. Additionally, ion channels themselves are subject to regulation at the levels of channel expression, trafficking and post-translational modification; thus, research into the regulation of ion channels may elucidate disease mechanisms and potential therapeutic targets for future drug development. This review summarizes the current knowledge of the molecular mechanisms of arrhythmia susceptibility and discusses technological advances such as induced pluripotent stem cell-derived cardiomyocytes, gene editing, functional genomics, and physiological screening platforms that provide a new paradigm for discovery of new therapeutic targets to treat congenital and acquired diseases of the heart rhythm.

A Secondary Structural Transition in the C-helix Promotes Gating of Cyclic Nucleotide-regulated Ion Channels*

PubMed Central

Puljung, Michael C.; Zagotta, William N.

2013-01-01

Cyclic nucleotide-regulated ion channels bind second messengers like cAMP to a C-terminal domain, consisting of a β-roll, followed by two α-helices (B- and C-helices). We monitored the cAMP-dependent changes in the structure of the C-helix of a C-terminal fragment of HCN2 channels using transition metal ion FRET between fluorophores on the C-helix and metal ions bound between histidine pairs on the same helix. cAMP induced a change in the dimensions of the C-helix and an increase in the metal binding affinity of the histidine pair. cAMP also caused an increase in the distance between a fluorophore on the C-helix and metal ions bound to the B-helix. Stabilizing the C-helix of intact CNGA1 channels by metal binding to a pair of histidines promoted channel opening. These data suggest that ordering of the C-helix is part of the gating conformational change in cyclic nucleotide-regulated channels. PMID:23525108

Molecular pathophysiology and pharmacology of the voltage-sensing module of neuronal ion channels

PubMed Central

Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; De Maria, Michela; Manocchio, Laura; Medoro, Alessandro; Taglialatela, Maurizio

2015-01-01

Voltage-gated ion channels (VGICs) are membrane proteins that switch from a closed to open state in response to changes in membrane potential, thus enabling ion fluxes across the cell membranes. The mechanism that regulate the structural rearrangements occurring in VGICs in response to changes in membrane potential still remains one of the most challenging topic of modern biophysics. Na+, Ca2+ and K+ voltage-gated channels are structurally formed by the assembly of four similar domains, each comprising six transmembrane segments. Each domain can be divided into two main regions: the Pore Module (PM) and the Voltage-Sensing Module (VSM). The PM (helices S5 and S6 and intervening linker) is responsible for gate opening and ion selectivity; by contrast, the VSM, comprising the first four transmembrane helices (S1–S4), undergoes the first conformational changes in response to membrane voltage variations. In particular, the S4 segment of each domain, which contains several positively charged residues interspersed with hydrophobic amino acids, is located within the membrane electric field and plays an essential role in voltage sensing. In neurons, specific gating properties of each channel subtype underlie a variety of biological events, ranging from the generation and propagation of electrical impulses, to the secretion of neurotransmitters and to the regulation of gene expression. Given the important functional role played by the VSM in neuronal VGICs, it is not surprising that various VSM mutations affecting the gating process of these channels are responsible for human diseases, and that compounds acting on the VSM have emerged as important investigational tools with great therapeutic potential. In the present review we will briefly describe the most recent discoveries concerning how the VSM exerts its function, how genetically inherited diseases caused by mutations occurring in the VSM affects gating in VGICs, and how several classes of drugs and toxins selectively target the VSM. PMID:26236192

Molecular pathophysiology and pharmacology of the voltage-sensing module of neuronal ion channels.

PubMed

Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; De Maria, Michela; Manocchio, Laura; Medoro, Alessandro; Taglialatela, Maurizio

2015-01-01

Voltage-gated ion channels (VGICs) are membrane proteins that switch from a closed to open state in response to changes in membrane potential, thus enabling ion fluxes across the cell membranes. The mechanism that regulate the structural rearrangements occurring in VGICs in response to changes in membrane potential still remains one of the most challenging topic of modern biophysics. Na(+), Ca(2+) and K(+) voltage-gated channels are structurally formed by the assembly of four similar domains, each comprising six transmembrane segments. Each domain can be divided into two main regions: the Pore Module (PM) and the Voltage-Sensing Module (VSM). The PM (helices S5 and S6 and intervening linker) is responsible for gate opening and ion selectivity; by contrast, the VSM, comprising the first four transmembrane helices (S1-S4), undergoes the first conformational changes in response to membrane voltage variations. In particular, the S4 segment of each domain, which contains several positively charged residues interspersed with hydrophobic amino acids, is located within the membrane electric field and plays an essential role in voltage sensing. In neurons, specific gating properties of each channel subtype underlie a variety of biological events, ranging from the generation and propagation of electrical impulses, to the secretion of neurotransmitters and to the regulation of gene expression. Given the important functional role played by the VSM in neuronal VGICs, it is not surprising that various VSM mutations affecting the gating process of these channels are responsible for human diseases, and that compounds acting on the VSM have emerged as important investigational tools with great therapeutic potential. In the present review we will briefly describe the most recent discoveries concerning how the VSM exerts its function, how genetically inherited diseases caused by mutations occurring in the VSM affects gating in VGICs, and how several classes of drugs and toxins selectively target the VSM.

Analysis of the Electrohydrodynamic Flow in a Symmetric System of Electrodes by the Method of Dynamic Current-Voltage Characteristics

NASA Astrophysics Data System (ADS)

Stishkov, Yu. K.; Zakir'yanova, R. E.

2018-04-01

We have solved the problem of injection-type through electrohydrodynamic (EHD) flow in a closed channel. We have considered a model of a liquid with four types of ions. It is shown that a through EHD flow without internal vortices in the electrode gap is formed for the ratio 2 : 1 of the initial injection current from the electrodes in the channel. The structure of the flow in different parts of the channel and the integral characteristics of the flow have been analyzed. It is shown that for a quadratic function of injection at the electrodes, the current-voltage characteristic of the flow is also quadratic.

Experiments with crystal deflectors for high energy ion beams: Electromagnetic dissociation probability for well channeled ions

NASA Astrophysics Data System (ADS)

Scandale, W.; Taratin, A. M.; Kovalenko, A. D.

2013-01-01

The paper presents the current status with the use of the crystal defectors for high energy ion beams. The channeling properties of multicharged ions are discussed. The results of the experiments on the deflection and extraction (collimation) of high energy ion beams with bent crystals performed in the accelerator centers are shortly considered. The analysis of the recent collimation experiment with a Pb nuclei of 270GeV/c per charge at the CERN Super Proton Synchrotron showed that the channeling efficiency was as large as about 90%. For Pb ions of the LHC energies a new mechanism, which can reduce the channeling efficiency, appears. The electromagnetic dissociation (ED) becomes possible for well channeled particles. However, the estimations performed in the paper show that the ED probability is small and should not visibly reduce the collimation efficiency. On the other hand, the aligned crystal gives the possibility to study the ED processes of heavy nuclei in the conditions when nuclear interactions are fully suppressed.

Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration.

PubMed

Franklin, Brandon M; Voss, S Randal; Osborn, Jeffrey L

2017-08-01

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K V 2.1, K V 2.2, L-type Ca V channels and H/K ATPases) or completely (GlyR, GABA A R, K V 1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K + channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H + pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair. Copyright © 2017 Elsevier B.V. All rights reserved.

CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane

PubMed Central

Gupta, Ankit; Balabaskaran-Nina, Praveen; Nguitragool, Wang; Saggu, Gagandeep S.; Schureck, Marc A.

2018-01-01

ABSTRACT Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. These proteins lack the multiple transmembrane domains typically present in channel-forming proteins, raising doubts about their precise roles. Using the virulent human Plasmodium falciparum parasite, we report that CLAG3 undergoes self-association and that this protein’s expression determines channel phenotype quantitatively. We overcame epigenetic silencing of clag3 paralogs and engineered parasites that express two CLAG3 isoforms simultaneously. Stoichiometric expression of these isoforms yielded intermediate channel phenotypes, in agreement with observed trafficking of both proteins to the host membrane. Coimmunoprecipitation and surface labeling revealed formation of CLAG3 oligomers. In vitro selections applied to these transfectant lines yielded distinct mutants with correlated changes in channel activity. These findings support involvement of the identified oligomers in PSAC formation and parasite nutrient acquisition. PMID:29739907

Atomic determinants of BK channel activation by polyunsaturated fatty acids

PubMed Central

Tian, Yutao; Aursnes, Marius; Hansen, Trond Vidar; Tungen, Jørn Eivind; Galpin, Jason D.; Leisle, Lilia; Ahern, Christopher A.; Xu, Rong; Heinemann, Stefan H.; Hoshi, Toshinori

2016-01-01

Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA–channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion–dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery. PMID:27849612

Metal-Assisted Channel Stabilization: Disposition of a Single Histidine on the N-terminus of Alamethicin Yields Channels with Extraordinarily Long Lifetimes

PubMed Central

Noshiro, Daisuke; Asami, Koji; Futaki, Shiroh

2010-01-01

Abstract Alamethicin, a member of the peptaibol family of antibiotics, is a typical channel-forming peptide with a helical structure. The self-assembly of the peptide in the membranes yields voltage-dependent channels. In this study, three alamethicin analogs possessing a charged residue (His, Lys, or Glu) on their N-termini were designed with the expectation of stabilizing the transmembrane structure. A slight elongation of channel lifetime was observed for the Lys and Glu analogs. On the other hand, extensive stabilization of certain channel open states was observed for the His analog. This stabilization was predominantly observed in the presence of metal ions such as Zn2+, suggesting that metal coordination with His facilitates the formation of a supramolecular assembly in the membranes. Channel stability was greatly diminished by acetylation of the N-terminal amino group, indicating that the N-terminal amino group also plays an important role in metal coordination. PMID:20441743

Electrotonic potentials in Aloe vera L.: Effects of intercellular and external electrodes arrangement.

PubMed

Volkov, Alexander G; Nyasani, Eunice K; Tuckett, Clayton; Scott, Jessenia M; Jackson, Mariah M Z; Greeman, Esther A; Greenidge, Ariane S; Cohen, Devin O; Volkova, Maia I; Shtessel, Yuri B

2017-02-01

Electrostimulation of plants can induce plant movements, activation of ion channels, ion transport, gene expression, enzymatic systems activation, electrical signaling, plant-cell damage, enhanced wound healing, and influence plant growth. Here we found that electrical networks in plant tissues have electrical differentiators. The amplitude of electrical responses decreases along a leaf and increases by decreasing the distance between polarizing Pt-electrodes. Intercellular Ag/AgCl electrodes inserted in a leaf and extracellular Ag/AgCl electrodes attached to the leaf surface were used to detect the electrotonic potential propagation along a leaf of Aloe vera. There is a difference in duration and amplitude of electrical potentials measured by electrodes inserted in a leaf and those attached to a leaf's surface. If the external reference electrode is located in the soil near the root, it changes the amplitude and duration of electrotonic potentials due to existence of additional resistance, capacitance, ion channels and ion pumps in the root. The information gained from this study can be used to elucidate extracellular and intercellular communication in the form of electrical signals within plants. Copyright © 2016 Elsevier B.V. All rights reserved.

Metastability of isoformyl ions in collisions with helium and hydrogen. [in interstellar molecular clouds

NASA Technical Reports Server (NTRS)

Green, S.

1984-01-01

The stability of HOC(+) ions under conditions in interstellar molecular clouds is considered. In particular, the possibility that collisions with helium or hydrogen will induce isomerization to the stable HCO(+) form is examined theoretically. Portions of the electronic potential energy surfaces for interaction with He and H atoms are obtained from standard quantum mechanical calculations. Collisions with He atoms are found to be totally ineffective for inducing isomerization. Collisions with H atoms are found to be ineffective at low interstellar temperatures owing to a small (about 500 K) barrier in the entrance channel; at higher temperatures where this barrier can be overcome, however, collisions with hydrogen atoms do result in conversion to the stable HCO(+) form. Although detailed calculations are not presented, it is argued that low-energy collisions with H2 molecules are also ineffective in destroying the metastable ion.

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque.

PubMed

Terauchi, Takashi; Terashima, Hiroyuki; Kojima, Seiji; Homma, Michio

2011-08-01

Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H(+) or Na(+)) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na(+)-driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H(+)-type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na(+) to exhibit swimming, but this effect was suppressed by additional mutations, PomA-N194Y or PomB-S27T. Moreover, the motility of the PomB-F22Y mutant was resistant to phenamil, a specific inhibitor for the Na(+) channel. When PomB-F22 was changed to other amino acids and the effects on swimming ability were investigated, replacement with a hydrophilic residue decreased the maximum swimming speed and conferred strong resistance to phenamil. From these results, we speculate that the Na(+) flux is reduced by the PomB-F22Y mutation, and that PomB-F22 is important for the effective release of Na(+) from PomB-D24.

Trails of Kilovolt Ions Created by Subsurface Channeling

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

Redinger, Alex; Standop, Sebastian; Michely, Thomas

2010-02-19

Using scanning tunneling microscopy, we observe the damage trails produced by keV noble-gas ions incident at glancing angles onto Pt(111). Surface vacancies and adatoms aligned along the ion trajectory constitute the ion trails. Atomistic simulations reveal that these straight trails are produced by nuclear (elastic) collisions with surface layer atoms during subsurface channeling of the projectiles. In a small energy window around 5 keV, Xe{sup +} ions create vacancy grooves that mark the ion trajectory with atomic precision. The asymmetry of the adatom production on the two sides of the projectile path is traced back to the asymmetry of themore » ion's subsurface channel.« less

Insights into channel dysfunction from modelling and molecular dynamics simulations.

PubMed

Musgaard, Maria; Paramo, Teresa; Domicevica, Laura; Andersen, Ole Juul; Biggin, Philip C

2018-04-01

Developments in structural biology mean that the number of different ion channel structures has increased significantly in recent years. Structures of ion channels enable us to rationalize how mutations may lead to channelopathies. However, determining the structures of ion channels is still not trivial, especially as they necessarily exist in many distinct functional states. Therefore, the use of computational modelling can provide complementary information that can refine working hypotheses of both wild type and mutant ion channels. The simplest but still powerful tool is homology modelling. Many structures are available now that can provide suitable templates for many different types of ion channels, allowing a full three-dimensional interpretation of mutational effects. These structural models, and indeed the structures themselves obtained by X-ray crystallography, and more recently cryo-electron microscopy, can be subjected to molecular dynamics simulations, either as a tool to help explore the conformational dynamics in detail or simply as a means to refine the models further. Here we review how these approaches have been used to improve our understanding of how diseases might be linked to specific mutations in ion channel proteins. This article is part of the Special Issue entitled 'Channelopathies.' Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Multiple-channel detection of cellular activities by ion-sensitive transistors

NASA Astrophysics Data System (ADS)

Machida, Satoru; Shimada, Hideto; Motoyama, Yumi

2018-04-01

An ion-sensitive field-effect transistor to record cellular activities was demonstrated. This field-effect transistor (bio transistor) includes cultured cells on the gate insulator instead of gate electrode. The bio transistor converts a change in potential underneath the cells into variation of the drain current when ion channels open. The bio transistor has high detection sensitivity to even minute variations in potential utilizing a subthreshold swing region. To open ion channels, a reagent solution (acetylcholine) was added to a human-originating cell cultured on the bio transistor. The drain current was successfully decreased with the addition of acetylcholine. Moreover, we attempted to detect the opening of ion channels using a multiple-channel measurement circuit containing several bio transistors. As a consequence, the drain current distinctly decreased only after the addition of acetylcholine. We confirmed that this measurement system including bio transistors enables to observation of cellular activities sensitively and simultaneously.

Fundamental properties of local anesthetics: half-maximal blocking concentrations for tonic block of Na+ and K+ channels in peripheral nerve.

PubMed

Bräu, M E; Vogel, W; Hempelmann, G

1998-10-01

Local anesthetics suppress excitability by interfering with ion channel function. Ensheathment of peripheral nerve fibers, however, impedes diffusion of drugs to the ion channels and may influence the evaluation of local anesthetic potencies. Investigating ion channels in excised membrane patches avoids these diffusion barriers. We investigated the effect of local anesthetics with voltage-dependent Na+ and K+ channels in enzymatically dissociated sciatic nerve fibers of Xenopus laevis using the patch clamp method. The outside-out configuration was chosen to apply drugs to the external face of the membrane. Local anesthetics reversibly blocked the transient Na+ inward current, as well as the steady-state K+ outward current. Half-maximal tonic inhibiting concentrations (IC50), as obtained from concentration-effect curves for Na+ current block were: tetracaine 0.7 microM, etidocaine 18 microM, bupivacaine 27 microM, procaine 60 microM, mepivacaine 149 microM, and lidocaine 204 microM. The values for voltage-dependent K+ current block were: bupivacaine 92 microM, etidocaine 176 microM, tetracaine 946 microM, lidocaine 1118 microM, mepivacaine 2305 microM, and procaine 6302 microM. Correlation of potencies with octanol:buffer partition coefficients (logP0) revealed that ester-bound local anesthetics were more potent in blocking Na+ channels than amide drugs. Within these groups, lipophilicity governed local anesthetic potency. We conclude that local anesthetic action on peripheral nerve ion channels is mediated via lipophilic drug-channel interactions. Half-maximal blocking concentrations of commonly used local anesthetics for Na+ and K+ channel block were determined on small membrane patches of peripheral nerve fibers. Because drugs can directly diffuse to the ion channel in this model, these data result from direct interactions of the drugs with ion channels.

Sub-cellular distribution and translocation of TRP channels.

PubMed

Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

2011-01-01

Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

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

PubMed

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

2015-09-01

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

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel.

PubMed

Vaisey, George; Miller, Alexandria N; Long, Stephen B

2016-11-22

Cytoplasmic calcium (Ca 2+ ) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca 2+ -independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca 2+ activation and ion selectivity. A "Ca 2+ clasp" within the channel's intracellular region acts as a sensor of cytoplasmic Ca 2+ . Alanine substitutions within a hydrophobic "neck" of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca 2+ . We conclude that the primary function of the neck is as a "gate" that controls chloride permeation in a Ca 2+ -dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel's ion selectivity. We find that mutation of a cytosolic "aperture" of the pore does not perturb the Ca 2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single "selectivity filter," in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

PubMed

El Hiani, Yassine; Linsdell, Paul

2015-06-19

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Smooth Muscle Ion Channels and Regulation of Vascular Tone in Resistance Arteries and Arterioles

PubMed Central

Tykocki, Nathan R.; Boerman, Erika M.; Jackson, William F.

2017-01-01

Vascular tone of resistance arteries and arterioles determines peripheral vascular resistance, contributing to the regulation of blood pressure and blood flow to, and within the body’s tissues and organs. Ion channels in the plasma membrane and endoplasmic reticulum of vascular smooth muscle cells (SMCs) in these blood vessels importantly contribute to the regulation of intracellular Ca2+ concentration, the primary determinant of SMC contractile activity and vascular tone. Ion channels provide the main source of activator Ca2+ that determines vascular tone, and strongly contribute to setting and regulating membrane potential, which, in turn, regulates the open-state-probability of voltage gated Ca2+ channels (VGCCs), the primary source of Ca2+ in resistance artery and arteriolar SMCs. Ion channel function is also modulated by vasoconstrictors and vasodilators, contributing to all aspects of the regulation of vascular tone. This review will focus on the physiology of VGCCs, voltage-gated K+ (KV) channels, large-conductance Ca2+-activated K+ (BKCa) channels, strong-inward-rectifier K+ (KIR) channels, ATP-sensitive K+ (KATP) channels, ryanodine receptors (RyRs), inositol 1,4,5-trisphosphate receptors (IP3Rs), and a variety of transient receptor potential (TRP) channels that contribute to pressure-induced myogenic tone in resistance arteries and arterioles, the modulation of the function of these ion channels by vasoconstrictors and vasodilators, their role in the functional regulation of tissue blood flow and their dysfunction in diseases such as hypertension, obesity, and diabetes. PMID:28333380

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

Ngo, Van; Wang, Yibo; Haas, Stephan

Crystal structures of several bacterial Na v channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Na v channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial Na vAb channel. This approach provided new insight into the mechanism of selective ion permeation inmore » bacterial Nav channels. The non-equilibrium simulations indicate that two or three extracellular K + ions can block the entrance to the selectivity filter of Na vAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K+ ions that can be ‘locked’ in place bymodest applied forces. In contrast to K +, three Na + ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na + ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K + block is equivalent to large applied potentials experimentally measured for two bacterial Na v channels to induce inward currents of K + ions. Here, these results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.« less

Influence of the narrow {111} planes on axial and planar ion channeling.

PubMed

Motapothula, M; Dang, Z Y; Venkatesan, T; Breese, M B H; Rana, M A; Osman, A

2012-05-11

We report channeling patterns where clearly resolved effects of the narrow {111} planes are observed in axial and planar alignments for 2 MeV protons passing through a 55 nm [001] silicon membrane. At certain axes, such as <213> and <314>, the offset in atomic rows forming the narrow {111} planes results in shielding from the large potential at the wide {111} planes, producing a region of shallow, asymmetric potential from which axial channeling patterns have no plane of symmetry. At small tilts from such axes, different behavior is observed from the wide and narrow {111} planes. At planar alignment, distinctive channeling effects due to the narrow planes are observed. As a consequence of the shallow potential well at the narrow planes, incident protons suffer dechanneled trajectories which are excluded from channeling within the wide planes, resulting in an anomalously large scattered beam at {111} alignment.

Combining molecular dynamics and an electrodiffusion model to calculate ion channel conductance

NASA Astrophysics Data System (ADS)

Wilson, Michael A.; Nguyen, Thuy Hien; Pohorille, Andrew

2014-12-01

Establishing the relation between the structures and functions of protein ion channels, which are protein assemblies that facilitate transmembrane ion transport through water-filled pores, is at the forefront of biological and medical sciences. A reliable way to determine whether our understanding of this relation is satisfactory is to reproduce the measured ionic conductance over a broad range of applied voltages. This can be done in molecular dynamics simulations by way of applying an external electric field to the system and counting the number of ions that traverse the channel per unit time. Since this approach is computationally very expensive we develop a markedly more efficient alternative in which molecular dynamics is combined with an electrodiffusion equation. This alternative approach applies if steady-state ion transport through channels can be described with sufficient accuracy by the one-dimensional diffusion equation in the potential given by the free energy profile and applied voltage. The theory refers only to line densities of ions in the channel and, therefore, avoids ambiguities related to determining the surface area of the channel near its endpoints or other procedures connecting the line and bulk ion densities. We apply the theory to a simple, model system based on the trichotoxin channel. We test the assumptions of the electrodiffusion equation, and determine the precision and consistency of the calculated conductance. We demonstrate that it is possible to calculate current/voltage dependence and accurately reconstruct the underlying (equilibrium) free energy profile, all from molecular dynamics simulations at a single voltage. The approach developed here applies to other channels that satisfy the conditions of the electrodiffusion equation.

Inversion of membrane surface charge by trivalent cations probed with a cation-selective channel

PubMed Central

Gurnev, Philip A.; Bezrukov, Sergey M.

2014-01-01

We demonstrate that the cation-selective channel formed by gramicidin A can be used as a reliable sensor for studying the multivalent ion accumulation at the surfaces of charged lipid membranes and the “charge inversion” phenomenon. In asymmetrically charged membranes with the individual leaflets formed from pure negative and positive lipids bathed by 0.1 M CsCl solutions the channel exhibits current rectification which is comparable to that of a typical n/p semiconductor diode. We show that even at these highly asymmetrical conditions the channel conductance can be satisfactorily described by the electrodiffusion equation in the constant field approximation but, due to predictable limitations, only when the applied voltages do not exceed 50 mV. Analysis of the changes in the voltage-dependent channel conductance upon addition of trivalent cations allows us to gauge their interactions with the membrane surface. The inversion of the sign of the effective surface charge takes place at the concentrations which correlate with the cation size. Specifically, these concentrations are close to 0.05 mM for lanthanum, 0.25 mM for hexaamminecobalt, and 4 mM for spermidine. PMID:23088396

Inversion of membrane surface charge by trivalent cations probed with a cation-selective channel.

PubMed

Gurnev, Philip A; Bezrukov, Sergey M

2012-11-13

We demonstrate that the cation-selective channel formed by gramicidin A can be used as a reliable sensor for studying the multivalent ion accumulation at the surfaces of charged lipid membranes and the "charge inversion" phenomenon. In asymmetrically charged membranes with the individual leaflets formed from pure negative and positive lipids bathed by 0.1 M CsCl solutions the channel exhibits current rectification, which is comparable to that of a typical n/p semiconductor diode. We show that even at these highly asymmetrical conditions the channel conductance can be satisfactorily described by the electrodiffusion equation in the constant field approximation but, due to predictable limitations, only when the applied voltages do not exceed 50 mV. Analysis of the changes in the voltage-dependent channel conductance upon addition of trivalent cations allows us to gauge their interactions with the membrane surface. The inversion of the sign of the effective surface charge takes place at the concentrations, which correlate with the cation size. Specifically, these concentrations are close to 0.05 mM for lanthanum, 0.25 mM for hexaamminecobalt, and 4 mM for spermidine.

Modulation of A-type potassium channels by a family of calcium sensors.

PubMed

An, W F; Bowlby, M R; Betty, M; Cao, J; Ling, H P; Mendoza, G; Hinson, J W; Mattsson, K I; Strassle, B W; Trimmer, J S; Rhodes, K J

2000-02-03

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

Human Digital Meissner Corpuscles Display Immunoreactivity for the Multifunctional Ion Channels Trpc6 and Trpv4.

PubMed

Alonso-González, Paula; Cabo, Roberto; San José, Isabel; Gago, Angel; Suazo, Iván C; García-Suárez, Olivia; Cobo, Juan; Vega, José A

2017-06-01

Ion channels are at the basis of the sensory processes including mechanosensing. Some members of the transient receptor potential (TRP) ion channel superfamily have been proposed as mechanosensors, but their putative role in mechanotransduction is controversial. Among them there are TRP canonical 6 (TRPC6) and TRP vanilloid 4 (TRPV4) ion channels, which are known to cooperate in mechanical hyperalgesia. Here, we investigated the occurrence, distribution, and possible colocalization of TRPC6 and TRPV4 in human digital Meissner sensory corpuscles using immunohistochemistry and double immunofluorescence (associate with markers for specific corpuscular constituents). TRPC6 immunoreactivity was restricted to the axon of Meissner corpuscles, whereas TRPV4 was detected in the axon but also in the lamellar cells. Moreover, axonal colocalization of TRPV4 and TRPC6 was found in the digital Meissner corpuscles. Present results demonstrate for the first time the occurrence and colocalization of two ion channels candidates to mechanosensors in human cutaneous mechanoreceptors. The functional significance of these ion channels in that place remains to be clarified, but should be related to different properties of mechanosensitivity. Anat Rec, 300:1022-1031, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Stochastic modelling of a single ion channel: an alternating renewal approach with application to limited time resolution.

PubMed

Milne, R K; Yeo, G F; Edeson, R O; Madsen, B W

1988-04-22

Stochastic models of ion channels have been based largely on Markov theory where individual states and transition rates must be specified, and sojourn-time densities for each state are constrained to be exponential. This study presents an approach based on random-sum methods and alternating-renewal theory, allowing individual states to be grouped into classes provided the successive sojourn times in a given class are independent and identically distributed. Under these conditions Markov models form a special case. The utility of the approach is illustrated by considering the effects of limited time resolution (modelled by using a discrete detection limit, xi) on the properties of observable events, with emphasis on the observed open-time (xi-open-time). The cumulants and Laplace transform for a xi-open-time are derived for a range of Markov and non-Markov models; several useful approximations to the xi-open-time density function are presented. Numerical studies show that the effects of limited time resolution can be extreme, and also highlight the relative importance of the various model parameters. The theory could form a basis for future inferential studies in which parameter estimation takes account of limited time resolution in single channel records. Appendixes include relevant results concerning random sums and a discussion of the role of exponential distributions in Markov models.

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

PubMed

Kajma, Anna; Szewczyk, Adam

2012-10-01

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

In silico assessment of the conduction mechanism of the Ryanodine Receptor 1 reveals previously unknown exit pathways.

PubMed

Heinz, Leonard P; Kopec, Wojciech; de Groot, Bert L; Fink, Rainer H A

2018-05-02

The ryanodine receptor 1 is a large calcium ion channel found in mammalian skeletal muscle. The ion channel gained a lot of attention recently, after multiple independent authors published near-atomic cryo electron microscopy data. Taking advantage of the unprecedented quality of structural data, we performed molecular dynamics simulations on the entire ion channel as well as on a reduced model. We calculated potentials of mean force for Ba 2+ , Ca 2+ , Mg 2+ , K + , Na + and Cl - ions using umbrella sampling to identify the key residues involved in ion permeation. We found two main binding sites for the cations, whereas the channel is strongly repulsive for chloride ions. Furthermore, the data is consistent with the model that the receptor achieves its ion selectivity by over-affinity for divalent cations in a calcium-block-like fashion. We reproduced the experimental conductance for potassium ions in permeation simulations with applied voltage. The analysis of the permeation paths shows that ions exit the pore via multiple pathways, which we suggest to be related to the experimental observation of different subconducting states.

Movement of Potassium Ions inside KcsA in the High Concentration Regime using a Molecular Dynamics Simulation

NASA Astrophysics Data System (ADS)

Kim, Myojeong; Jo, Byeong Chul; Yoon, Hyun Jung; Wu, Sangwook; Thangappan, Jayaraman; Eun, Changsun

2018-05-01

The selectivity and conduction specificity of the KcsA channel toward potassium ions is crucial to the activity of this protein and this channel is intricately associated with several osmotic regulation and neuronal signaling processes. Despite multi-ion characteristics, the selective conduction behavior of KcsA is controlled by the size and distance specific electrostatic interaction between the selected residues and the potassium ions. The mechanism describing the movement of potassium ions in the channel and the conformational changes to KcsA that facilitate ion movement were investigated by a molecular dynamics (MD) simulation. In this study, we analyze the movement of potassium ions and water molecules at various time intervals during a 90 ns molecular dynamics simulation in the high potassium ion concentration regime and in the absence of the voltage. Examination of specific (3.6, 17.3, 43.38 and 43.44 ns) simulation periods revealed that key residues in the selectivity filter of KcsA influence the movement of potassium ions in the channel.

α1- and α2-Adrenergic responsiveness in human skeletal muscle feed arteries: the role of TRPV ion channels in heat-induced sympatholysis

PubMed Central

Gifford, Jayson R.; Ives, Stephen J.; Park, Song-Young; Andtbacka, Robert H. I.; Hyngstrom, John R.; Mueller, Michelle T.; Treiman, Gerald S.; Ward, Christopher; Trinity, Joel D.

2014-01-01

The purpose of this study was to determine if heat inhibits α2-adrenergic vasocontraction, similarly to α1-adrenergic contraction, in isolated human skeletal muscle feed arteries (SMFA) and elucidate the role of the temperature-sensitive vanilloid-type transient receptor potential (TRPV) ion channels in this response. Isolated SMFA from 37 subjects were studied using wire myography. α1 [Phenylephrine (PE)]- and α2 [dexmedetomidine (DEX)]-contractions were induced at 37 and 39°C with and without TRPV family and TRPV4-specific inhibition [ruthenium red (RR) and RN-1734, respectively]. Endothelial function [acetylcholine (ACh)] and smooth muscle function [sodium nitroprusside (SNP) and potassium chloride (KCl)] were also assessed under these conditions. Heat and TRPV inhibition was further examined in endothelium-denuded arteries. Contraction data are reported as a percentage of maximal contraction elicited by 100 mM KCl (LTmax). DEX elicited a small and variable contractile response, one-fifth the magnitude of PE, which was not as clearly attenuated when heated from 37 to 39°C (12 ± 4 to 6 ± 2% LTmax; P = 0.18) as were PE-induced contractions (59 ± 5 to 24 ± 4% LTmax; P < 0.05). Both forms of TRPV inhibition restored PE-induced contraction at 39°C (P < 0.05) implicating these channels, particularly the TRPV4 channels, in the heat-induced attenuation of α1-adrenergic vasocontraction. TRPV inhibition significantly blunted ACh relaxation while denudation prevented heat-induced sympatholysis without having an additive effect when combined with TRPV inhibition. In conclusion, physiological increases in temperature elicit a sympatholysis-like inhibition of α1-adrenergic vasocontraction in human SMFA that appears to be mediated by endothelial TRPV4 ion channels. PMID:25172894

The Effect of Silver Chloride Formation on the Kinetics of Silver Dissolution in Chloride Solution

PubMed Central

Ha, Hung; Payer, Joe

2011-01-01

The precipitation and growth of AgCl on silver in physiological NaCl solution were investigated. AgCl was found to form at bottom of scratches on the surface which may be the less effective sites for diffusion or the favorable sites for heterogeneous nucleation. Patches of silver chloride expanded laterally on the substrate until a continuous film formed. The ionic transport path through this newly formed continuous film was via spaces between AgCl patches. As the film grew, the spaces between AgCl patches closed and ion transport was primarily via micro-channels running through AgCl patches. The decrease of AgCl layer conductivity during film growth were attributed to the clogging of micro-channels or decrease in charge carrier concentration inside the micro-channels. Under thin AgCl layer, i.e. on the order of a micrometer, the dissolution of silver substrate was under mixed activation-Ohmic control. Under thick AgCl layer, i.e. on the order of tens of micrometers, the dissolution of silver substrate was mediated by the Ohmic resistance of AgCl layer. PMID:21516171

Evaluation of microtransplantation of rat brain neurolemma into Xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage-sensitive ion channels.

PubMed

Murenzi, Edwin; Toltin, Abigail C; Symington, Steven B; Morgan, Molly M; Clark, John M

2017-05-01

Microtransplantation of mammalian brain neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Na v 1.1-Na v 1.9) was determined in neurolemma using automated western blotting, with the predominant isoforms expressed being Na v 1.2 and Na v 1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE was without effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs in Xenopus oocytes and with many other electrophysiological approaches, validating the use of the microtransplantation procedure as a toxicologically-relevant ex vivo assay. Once fully characterized, it is likely that this approach could be expanded to study the role of environmental toxicants and contaminants on various target tissues (e.g. neural, reproductive, developmental) from many species. Copyright © 2016 Elsevier B.V. All rights reserved.

Thin membrane sensor with biochemical switch

NASA Technical Reports Server (NTRS)

Worley, III, Jennings F. (Inventor); Case, George D. (Inventor)

1994-01-01

A modular biosensor system for chemical or biological agent detection utilizes electrochemical measurement of an ion current across a gate membrane triggered by the reaction of the target agent with a recognition protein conjugated to a channel blocker. The sensor system includes a bioresponse simulator or biochemical switch module which contains the recognition protein-channel blocker conjugate, and in which the detection reactions occur, and a transducer module which contains a gate membrane and a measuring electrode, and in which the presence of agent is sensed electrically. In the poised state, ion channels in the gate membrane are blocked by the recognition protein-channel blocker conjugate. Detection reactions remove the recognition protein-channel blocker conjugate from the ion channels, thus eliciting an ion current surge in the gate membrane which subsequently triggers an output alarm. Sufficiently large currents are generated that simple direct current electronics are adequate for the measurements. The biosensor has applications for environmental, medical, and industrial use.

K + block is the mechanism of functional asymmetry in bacterial Na v channels

DOE PAGES

Ngo, Van; Wang, Yibo; Haas, Stephan; ...

2016-01-04

Crystal structures of several bacterial Na v channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Na v channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial Na vAb channel. This approach provided new insight into the mechanism of selective ion permeation inmore » bacterial Nav channels. The non-equilibrium simulations indicate that two or three extracellular K + ions can block the entrance to the selectivity filter of Na vAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K+ ions that can be ‘locked’ in place bymodest applied forces. In contrast to K +, three Na + ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na + ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K + block is equivalent to large applied potentials experimentally measured for two bacterial Na v channels to induce inward currents of K + ions. Here, these results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.« less

K+ Block Is the Mechanism of Functional Asymmetry in Bacterial Nav Channels

PubMed Central

Ngo, Van; Wang, Yibo; Haas, Stephan; Noskov, Sergei Y.; Farley, Robert A.

2016-01-01

Crystal structures of several bacterial Nav channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Nav channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial NavAb channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Nav channels. The non-equilibrium simulations indicate that two or three extracellular K+ ions can block the entrance to the selectivity filter of NavAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K+ ions that can be ‘locked’ in place by modest applied forces. In contrast to K+, three Na+ ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na+ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K+ block is equivalent to large applied potentials experimentally measured for two bacterial Nav channels to induce inward currents of K+ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes. PMID:26727271

Method for manufacturing compound semiconductor field-effect transistors with improved DC and high frequency performance

DOEpatents

Zolper, John C.; Sherwin, Marc E.; Baca, Albert G.

2000-01-01

A method for making compound semiconductor devices including the use of a p-type dopant is disclosed wherein the dopant is co-implanted with an n-type donor species at the time the n-channel is formed and a single anneal at moderate temperature is then performed. Also disclosed are devices manufactured using the method. In the preferred embodiment n-MESFETs and other similar field effect transistor devices are manufactured using C ions co-implanted with Si atoms in GaAs to form an n-channel. C exhibits a unique characteristic in the context of the invention in that it exhibits a low activation efficiency (typically, 50% or less) as a p-type dopant, and consequently, it acts to sharpen the Si n-channel by compensating Si donors in the region of the Si-channel tail, but does not contribute substantially to the acceptor concentration in the buried p region. As a result, the invention provides for improved field effect semiconductor and related devices with enhancement of both DC and high-frequency performance.

Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel

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

Li, Minghui; Zhang, Wei K.; Benvin, Nicole M.

The activities of organellar ion channels are often regulated by Ca2+ and H+, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca2+/pH regulation of TRPML1, a Ca2+-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore loop. Cysteine cross-linking and cryo-EMmore » analyses confirmed that this architecture occurs in the full-length channel. Structure–function studies demonstrated that Ca2+ and H+ interact with the luminal pore and exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal-domain structure and cause TRPML1 mislocalization. Our study reveals the structural underpinnings of TRPML1's regulation, assembly and pathogenesis.« less

Molecular dynamics study of ion transport through an open model of voltage-gated sodium channel.

PubMed

Li, Yang; Sun, Ruining; Liu, Huihui; Gong, Haipeng

2017-05-01

Voltage-gated sodium (Na V ) channels are critical in the signal transduction of excitable cells. In this work, we modeled the open conformation for the pore domain of a prokaryotic Na V channel (Na V Rh), and used molecular dynamics simulations to track the translocation of dozens of Na + ions through the channel in the presence of a physiological transmembrane ion concentration gradient and a transmembrane electrical field that was closer to the physiological one than previous studies. Channel conductance was then estimated from simulations on the wide-type and DEKA mutant of Na V Rh. Interestingly, the conductivity predicted from the DEKA mutant agrees well with experimental measurement on eukaryotic Na V 1.4 channel. Moreover, the wide-type and DEKA mutant of Na V Rh exhibited markedly distinct ion permeation patterns, which thus implies the mechanistic difference between prokaryotic and eukaryotic Na V channels. Copyright © 2017 Elsevier B.V. All rights reserved.

Modulation of Brain Hemichannels and Gap Junction Channels by Pro-Inflammatory Agents and Their Possible Role in Neurodegeneration

PubMed Central

Sáez, Pablo J.; Shoji, Kenji F.; Schalper, Kurt A.; Palacios–Prado, Nicolás; Velarde, Victoria; Giaume, Christian; Bennett, Michael V.L.; Sáez, Juan C.

2009-01-01

Abstract In normal brain, neurons, astrocytes, and oligodendrocytes, the most abundant and active cells express pannexins and connexins, protein subunits of two families forming membrane channels. Most available evidence indicates that in mammals endogenously expressed pannexins form only hemichannels and connexins form both gap junction channels and hemichannels. Whereas gap junction channels connect the cytoplasm of contacting cells and coordinate electric and metabolic activity, hemichannels communicate the intra- and extracellular compartments and serve as a diffusional pathway for ions and small molecules. A subthreshold stimulation by acute pathological threatening conditions (e.g., global ischemia subthreshold for cell death) enhances neuronal Cx36 and glial Cx43 hemichannel activity, favoring ATP release and generation of preconditioning. If the stimulus is sufficiently deleterious, microglia become overactivated and release bioactive molecules that increase the activity of hemichannels and reduce gap junctional communication in astroglial networks, depriving neurons of astrocytic protective functions, and further reducing neuronal viability. Continuous glial activation triggered by low levels of anomalous proteins expressed in several neurodegenerative diseases induce glial hemichannel and gap junction channel disorders similar to those of acute inflammatory responses triggered by ischemia or infectious diseases. These changes are likely to occur in diverse cell types of the CNS and contribute to neurodegeneration during inflammatory process. Antiox. Redox Signal. 11, 369–399. PMID:18816186

Computer simulations of transport through membranes: passive diffusion, pores, channels and transporters.

PubMed

Tieleman, D Peter

2006-10-01

A key function of biological membranes is to provide mechanisms for the controlled transport of ions, nutrients, metabolites, peptides and proteins between a cell and its environment. We are using computer simulations to study several processes involved in transport. In model membranes, the distribution of small molecules can be accurately calculated; we are making progress towards understanding the factors that determine the partitioning behaviour in the inhomogeneous lipid environment, with implications for drug distribution, membrane protein folding and the energetics of voltage gating. Lipid bilayers can be simulated at a scale that is sufficiently large to study significant defects, such as those caused by electroporation. Computer simulations of complex membrane proteins, such as potassium channels and ATP-binding cassette (ABC) transporters, can give detailed information about the atomistic dynamics that form the basis of ion transport, selectivity, conformational change and the molecular mechanism of ATP-driven transport. This is illustrated in the present review with recent simulation studies of the voltage-gated potassium channel KvAP and the ABC transporter BtuCD.

Channel-resolved photo- and Auger-electron spectroscopy of halogenated hydrocarbons

NASA Astrophysics Data System (ADS)

Ablikim, Utuq; Kaderiya, B.; Kumarapan, V.; Kushawaha, R.; Rudenko, A.; Rolles, D.; Xiong, H.; Berrah, N.; Bomme, C.; Savelyev, E.; Kilcoyne, D.

2016-05-01

Inner-shell photoelectron and Auger electron spectra of polyatomic molecules such as halogenated hydrocarbons are typically hard to interpret and assign due to many overlapping states that form broad bands even in high-resolution measurements. With the help of electron-ion-ion coincidence measurements performed using the velocity map imaging technique, we are able to detect high-energy (<= 150 eV) photo- and Auger electrons in coincidence with two- or many-body ionic fragmentation channels. Such channel-resolved measurements allow disentangling the overlapping electronic structures and help assigning individual components of the electron spectra to specific potential surfaces and final states. In this work, we present measurements on CH3 I, CH2 IBr, and CH2 ICl molecules in the gas-phase using soft x-ray light provided by the Advanced Light Source at LBNL. This project is supported by the DOE, Office of Science, BES, Division of Chemical, Geological and Biological Sciences under Award Number DE-FG02-86ER13491 (U.A., B.K., V.K., A.R., D.R.) and Award Number DE-SC0012376 (H.X., N.B.).

When a transmembrane channel isn't, or how biophysics and biochemistry (mis)communicate.

PubMed

Reviakine, Ilya

2018-02-12

Annexins are a family of soluble proteins that bind to acidic phospholipids such as phosphatidylserine in a calcium-dependent manner. The archetypical member of the annexin family is annexin A5. For many years, its function remained unknown despite the availability of a high-resolution structure. This, combined with the observations of specific ion conductance in annexin-bound membranes, fueled speculations about the possible membrane-spanning forms of annexins that functioned as ion channels. The channel hypothesis remained controversial and did not gather sufficient evidence to become accepted. Yet, it continues to draw attention as a framework for interpreting indirect (e.g., biochemical) data. The goal of the mini-review is to examine the data on annexin-lipid interactions from the last ~30 years from the point of view of the controversy between the two lines of inquiry: the well-characterized peripheral assembly of the annexins at membranes vs. their putative transmembrane insertion. In particular, the potential role of lipid rearrangements induced by annexin binding is highlighted. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation of Connexin-Based Channels by Fatty Acids

PubMed Central

Puebla, Carlos; Retamal, Mauricio A.; Acuña, Rodrigo; Sáez, Juan C.

2017-01-01

In this mini-review, we briefly summarize the current knowledge about the effects of fatty acids (FAs) on connexin-based channels, as well as discuss the limited information about the impact FAs may have on pannexins (Panxs). FAs regulate diverse cellular functions, some of which are explained by changes in the activity of channels constituted by connexins (Cxs) or Panxs, which are known to play critical roles in maintaining the functional integrity of diverse organs and tissues. Cxs are transmembrane proteins that oligomerize into hexamers to form hemichannels (HCs), which in turn can assemble into dodecamers to form gap junction channels (GJCs). While GJCs communicate the cytoplasm of contacting cells, HCs serve as pathways for the exchange of ions and small molecules between the intra and extracellular milieu. Panxs, as well as Cx HCs, form channels at the plasma membrane that enable the interchange of molecules between the intra and extracellular spaces. Both Cx- and Panx-based channels are controlled by several post-translational modifications. However, the mechanism of action of FAs on these channels has not been described in detail. It has been shown however that FAs frequently decrease GJC-mediated cell-cell communication. The opposite effect also has been described for HC or Panx-dependent intercellular communication, where, the acute FA effect can be reversed upon washout. Additionally, changes in GJCs mediated by FAs have been associated with post-translational modifications (e.g., phosphorylation), and seem to be directly related to chemical properties of FAs (e.g., length of carbon chain and/or degree of saturation), but this possible link remains poorly understood. PMID:28174541

Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels

PubMed Central

2017-01-01

TMEM16A and TMEM16B encode for Ca2+-activated Cl− channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B. PMID:28046119

Robustness, Death of Spiral Wave in the Network of Neurons under Partial Ion Channel Block

NASA Astrophysics Data System (ADS)

Ma, Jun; Huang, Long; Wang, Chun-Ni; Pu, Zhong-Sheng

2013-02-01

The development of spiral wave in a two-dimensional square array due to partial ion channel block (Potassium, Sodium) is investigated, the dynamics of the node is described by Hodgkin—Huxley neuron and these neurons are coupled with nearest neighbor connection. The parameter ratio xNa (and xK), which defines the ratio of working ion channel number of sodium (potassium) to the total ion channel number of sodium (and potassium), is used to measure the shift conductance induced by channel block. The distribution of statistical variable R in the two-parameter phase space (parameter ratio vs. poisoning area) is extensively calculated to mark the parameter region for transition of spiral wave induced by partial ion channel block, the area with smaller factors of synchronization R is associated the parameter region that spiral wave keeps alive and robust to the channel poisoning. Spiral wave keeps alive when the poisoned area (potassium or sodium) and degree of intoxication are small, distinct transition (death, several spiral waves coexist or multi-arm spiral wave emergence) occurs under moderate ratio xNa (and xK) when the size of blocked area exceeds certain thresholds. Breakup of spiral wave occurs and multi-arm of spiral waves are observed when the channel noise is considered.

On the estimation of cooperativity in ion channel kinetics: activation free energy and kinetic mechanism of Shaker K+ channel.

PubMed

Banerjee, Kinshuk; Das, Biswajit; Gangopadhyay, Gautam

2013-04-28

In this paper, we have explored generic criteria of cooperative behavior in ion channel kinetics treating it on the same footing with multistate receptor-ligand binding in a compact theoretical framework. We have shown that the characterization of cooperativity of ion channels in terms of the Hill coefficient violates the standard Hill criteria defined for allosteric cooperativity of ligand binding. To resolve the issue, an alternative measure of cooperativity is proposed here in terms of the cooperativity index that sets a unified criteria for both the systems. More importantly, for ion channel this index can be very useful to describe the cooperative kinetics as it can be readily determined from the experimentally measured ionic current combined with theoretical modelling. We have analyzed the correlation between the voltage value and slope of the voltage-activation curve at the half-activation point and consequently determined the standard free energy of activation of the ion channel using two well-established mechanisms of cooperativity, namely, Koshland-Nemethy-Filmer (KNF) and Monod-Wyman-Changeux (MWC) models. Comparison of the theoretical results for both the models with appropriate experimental data of mutational perturbation of Shaker K(+) channel supports the experimental fact that the KNF model is more suitable to describe the cooperative behavior of this class of ion channels, whereas the performance of the MWC model is unsatisfactory. We have also estimated the mechanistic performance through standard free energy of channel activation for both the models and proposed a possible functional disadvantage in the MWC scheme.

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.

Ion Transport in Confined Geometries below the Nanoscale: Access Resistance Dominates Protein Channel Conductance in Diluted Solutions.

PubMed

Alcaraz, Antonio; López, M Lidón; Queralt-Martín, María; Aguilella, Vicente M

2017-10-24

Synthetic nanopores and mesoscopic protein channels have common traits like the importance of electrostatic interactions between the permeating ions and the nanochannel. Ion transport at the nanoscale occurs under confinement conditions so that the usual assumptions made in microfluidics are challenged, among others, by interfacial effects such as access resistance (AR). Here, we show that a sound interpretation of electrophysiological measurements in terms of channel ion selective properties requires the consideration of interfacial effects, up to the point that they dominate protein channel conductance in diluted solutions. We measure AR in a large ion channel, the bacterial porin OmpF, by means of single-channel conductance measurements in electrolyte solutions containing varying concentrations of high molecular weight PEG, sterically excluded from the pore. Comparison of experiments performed in charged and neutral planar membranes shows that lipid surface charges modify the ion distribution and determine the value of AR, indicating that lipid molecules are more than passive scaffolds even in the case of large transmembrane proteins. We also found that AR may reach up to 80% of the total channel conductance in diluted solutions, where electrophysiological recordings register essentially the AR of the system and depend marginally on the pore characteristics. These findings may have implications for several low aspect ratio biological channels that perform their physiological function in a low ionic strength and macromolecule crowded environment, just the two conditions enhancing the AR contribution.

Mechanosensitive Piezo Channels in the Gastrointestinal Tract

PubMed Central

Alcaino, C.; Farrugia, G.; Beyder, A.

2017-01-01

Sensation of mechanical forces is critical for normal function of the gastrointestinal (GI) tract and abnormalities in mechanosensation are linked to GI pathologies. In the GI tract there are several mechanosensitive cell types—epithelial enterochromaffin cells, intrinsic and extrinsic enteric neurons, smooth muscle cells and interstitial cells of Cajal. These cells use mechanosensitive ion channels that respond to mechanical forces by altering transmembrane ionic currents in a process called mechanoelectrical coupling. Several mechanosensitive ionic conductances have been identified in the mechano-sensory GI cells, ranging from mechanosensitive voltage-gated sodium and calcium channels to the mechanogated ion channels, such as the two-pore domain potassium channels K2P (TREK-1) and nonselective cation channels from the transient receptor potential family. The recently discovered Piezo channels are increasingly recognized as significant contributors to cellular mechanosensitivity. Piezo1 and Piezo2 are nonselective cationic ion channels that are directly activated by mechanical forces and have well-defined biophysical and pharmacologic properties. The role of Piezo channels in the GI epithelium is currently under investigation and their role in the smooth muscle syncytium and enteric neurons is still not known. In this review, we outline the current state of knowledge on mechanosensitive ion channels in the GI tract, with a focus on the known and potential functions of the Piezo channels. PMID:28728818

Ion channels in artificial bolaamphiphile membranes deposited on sensor chips: optical detection in an ion-channel-based biosensor

NASA Astrophysics Data System (ADS)

Schalkhammer, Thomas G. M.; Weiss-Wichert, Christof; Smetazko, Michaela M.; Valina-Saba, Miriam

1997-06-01

Signal amplification using labels should be replaced by a technique monitoring the biochemical binding event directly. The use of a ligand coupled to an artificial gated membrane ion channel is a new promising strategy. Binding of protein- or DNA/RNA-analytes at ligand modified peptide channels results in an on/off-response of the channel current due to channel closure or distortion. The sensor consists of stable transmembrane channels with a ligand bound covalently at the peptide channel entrance, a sensor chip with a photostructurized hydrophobic polymer frame, a hydrophilic ion conducting membrane support, a lipid membrane incorporating the engineered ion channels, and a current amplifier or a sensitive fluorescence monitor. Detection of channel opening or closure can ether be obtained by directly monitoring membrane conductivity or a transient change of pH or ion concentration within the membrane compartment. This change can be induced by electrochemical or optical means and its decay is directly correlated to the permeability of the membrane. The ion concentration in the sub membrane compartment was monitored by incorporation of fluorescent indicator dyes. To obtain the stable sensor membrane the lipid layer had to be attached on a support and the floating of the second lipid membrane on top of the first one had to be prevented. Both problems do not occur using our new circular C44-C76 bolaamphiphilic lipids consisting of a long hydrophobic core region and two hydrophilic heads. Use of maleic ester-head groups enabled us to easily modify the lipids with amines, thioles, alcohols, phosphates, boronic acid as well as fluorescent dyes. The properties of these membranes were studied using LB and fluorescence techniques. Based on this detection principle miniaturized sensor chips with significantly enhanced sensitivity and large multi analyte arrays are under construction.

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

Koenig, Xaver; Kovar, Michael; Rubi, Lena

The plant alkaloid ibogaine has promising anti-addictive properties. Albeit not licenced as a therapeutic drug, and despite hints that ibogaine may perturb the heart rhythm, this alkaloid is used to treat drug addicts. We have recently reported that ibogaine inhibits human ERG (hERG) potassium channels at concentrations similar to the drugs affinity for several of its known brain targets. Thereby the drug may disturb the heart's electrophysiology. Here, to assess the drug's cardiac ion channel profile in more detail, we studied the effects of ibogaine and its congener 18-Methoxycoronaridine (18-MC) on various cardiac voltage-gated ion channels. We confirmed that heterologouslymore » expressed hERG currents are reduced by ibogaine in low micromolar concentrations. Moreover, at higher concentrations, the drug also reduced human Na{sub v}1.5 sodium and Ca{sub v}1.2 calcium currents. Ion currents were as well reduced by 18-MC, yet with diminished potency. Unexpectedly, although blocking hERG channels, ibogaine did not prolong the action potential (AP) in guinea pig cardiomyocytes at low micromolar concentrations. Higher concentrations (≥ 10 μM) even shortened the AP. These findings can be explained by the drug's calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaine's inhibitory effects on human ion channels in a computer model of a ventricular cardiomyocyte, on the other hand, suggested that ibogaine does prolong the AP in the human heart. We conclude that therapeutic concentrations of ibogaine have the propensity to prolong the QT interval of the electrocardiogram in humans. In some cases this may lead to cardiac arrhythmias. - Highlights: • We study effects of anti-addiction drug ibogaine on ionic currents in cardiomyocytes. • We assess the cardiac ion channel profile of ibogaine. • Ibogaine inhibits hERG potassium, sodium and calcium channels. • Ibogaine’s effects on ion channels are a potential source of cardiac arrhythmias. • 18-Methoxycoronaridine has a lower affinity for cardiac ion channels than ibogaine.« less

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

Lolicato, Marco; Arrigoni, Cristina; Mori, Takahiro

Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK1 subfamily generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses1, 2, 3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit4, 5, 6. In contrast to other potassium channels, K2P channels use a selectivity filter ‘C-type’ gate7, 8, 9, 10 as the principal gating site. Despite recent advances3, 11, 12, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a classmore » of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators—an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402—define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation–π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.« less

Molecular mechanism of acetylcholine receptor-controlled ion translocation across cell membranes

PubMed Central

Cash, Derek J.; Hess, George P.

1980-01-01

Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of 86Rb+ and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of Electrophorus electricus in eel Ringer's solution at pH 7.0 and 1°C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec-1. Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec-1 and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine investigated. The values of the constants in the rate equation form the basis for predicting receptor-controlled changes in the transmembrane potential of cells and the conditions leading to transmission of signals between cells. PMID:6928684

X-ray structures define human P2X3 receptor gating cycle and antagonist action

PubMed Central

Mansoor, Steven E.; Lü, Wei; Oosterheert, Wout; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

2016-01-01

Summary P2X receptors are trimeric, non-selective cation channels activated by ATP that play important roles in cardiovascular, neuronal and immune systems. Despite their central function in human physiology and as potential targets of therapeutic agents, there are no structures of human P2X receptors. Mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structure of the pore-forming transmembrane domains remain unclear. We report x-ray crystal structures of human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/desensitized and antagonist-bound closed states. The open state structure harbors an intracellular motif we term the “cytoplasmic cap”, that stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. Competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements underpinning P2X receptor gating and provide a foundation for development of new pharmacologic agents. PMID:27626375

Ion Selectivity Mechanism in a Bacterial Pentameric Ligand-Gated Ion Channel

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

Fritsch, Sebastian M; Ivanov, Ivaylo N; Wang, Hailong

2011-01-01

The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor (nAChR) that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential of mean force (PMF) profiles for sodium and chloride ions inside the transmembrane region. Our calculationsmore » reveal that the GLIC channel is open for a sodium ion to transport, but presents a ~10 kcal/mol free energy barrier for a chloride ion, which arises primarily from the unfavorable interactions with a ring of negatively charged glutamate residues (E-2 ) at the intracellular end and a ring of hydrophobic residues (I9 ) in the middle of the transmembrane domain. Our collective findings further suggest that the charge selection mechanism can, to a large extent, be attributed to the narrow intracellular end and a ring of glutamate residues in this position their strong negative electrostatics and ability to bind cations. By contrast, E19 at the extracellular entrance only plays a minor role in ion selectivity of GLIC. In addition to electrostatics, both ion hydration and protein dynamics are found to be crucial for ion conduction as well, which explains why a chloride ion experiences a much greater barrier than a sodium ion in the hydrophobic region of the pore.« less

BRAIN NETWORKS. Correlated gene expression supports synchronous activity in brain networks.

PubMed

Richiardi, Jonas; Altmann, Andre; Milazzo, Anna-Clare; Chang, Catie; Chakravarty, M Mallar; Banaschewski, Tobias; Barker, Gareth J; Bokde, Arun L W; Bromberg, Uli; Büchel, Christian; Conrod, Patricia; Fauth-Bühler, Mira; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny; Heinz, Andreas; Lemaître, Hervé; Mann, Karl F; Martinot, Jean-Luc; Nees, Frauke; Paus, Tomáš; Pausova, Zdenka; Rietschel, Marcella; Robbins, Trevor W; Smolka, Michael N; Spanagel, Rainer; Ströhle, Andreas; Schumann, Gunter; Hawrylycz, Mike; Poline, Jean-Baptiste; Greicius, Michael D

2015-06-12

During rest, brain activity is synchronized between different regions widely distributed throughout the brain, forming functional networks. However, the molecular mechanisms supporting functional connectivity remain undefined. We show that functional brain networks defined with resting-state functional magnetic resonance imaging can be recapitulated by using measures of correlated gene expression in a post mortem brain tissue data set. The set of 136 genes we identify is significantly enriched for ion channels. Polymorphisms in this set of genes significantly affect resting-state functional connectivity in a large sample of healthy adolescents. Expression levels of these genes are also significantly associated with axonal connectivity in the mouse. The results provide convergent, multimodal evidence that resting-state functional networks correlate with the orchestrated activity of dozens of genes linked to ion channel activity and synaptic function. Copyright © 2015, American Association for the Advancement of Science.

A new dual-channel optical signal probe for Cu2+ detection based on morin and boric acid.

PubMed

Wang, Peng; Yuan, Bin Fang; Li, Nian Bing; Luo, Hong Qun

2014-01-01

In this work we utilized the common analytical reagent morin to develop a new a dual-channel, cost-effective, and sensitive method for determination of Cu(2+). It is found that morin is only weakly fluorescent by itself, but forms highly fluorescent complexes with boric acid. Moreover, the fluorescence of complexes of morin with boric acid is quenched linearly by Cu(2+) in a certain concentration range. Under optimum conditions, the fluorescence quenching efficiency was linearly proportional to the concentration of cupric ions in the range of 0.5-25 μM with high sensitivity, and the detection limit for Cu(2+) was 0.38 μM. The linear range was 1-25 μM determined by spectrophotometry, and the detection limit for cupric ions was 0.8 μM. Furthermore, the mechanism of sensitive fluorescence quenching response of morin to Cu(2+) is discussed.

A study of quantum mechanical probabilities in the classical Hodgkin-Huxley model.

PubMed

Moradi, N; Scholkmann, F; Salari, V

2015-03-01

The Hodgkin-Huxley (HH) model is a powerful model to explain different aspects of spike generation in excitable cells. However, the HH model was proposed in 1952 when the real structure of the ion channel was unknown. It is now common knowledge that in many ion-channel proteins the flow of ions through the pore is governed by a gate, comprising a so-called "selectivity filter" inside the ion channel, which can be controlled by electrical interactions. The selectivity filter (SF) is believed to be responsible for the selection and fast conduction of particular ions across the membrane of an excitable cell. Other (generally larger) parts of the molecule such as the pore-domain gate control the access of ions to the channel protein. In fact, two types of gates are considered here for ion channels: the "external gate", which is the voltage sensitive gate, and the "internal gate" which is the selectivity filter gate (SFG). Some quantum effects are expected in the SFG due to its small dimensions, which may play an important role in the operation of an ion channel. Here, we examine parameters in a generalized model of HH to see whether any parameter affects the spike generation. Our results indicate that the previously suggested semi-quantum-classical equation proposed by Bernroider and Summhammer (BS) agrees strongly with the HH equation under different conditions and may even provide a better explanation in some cases. We conclude that the BS model can refine the classical HH model substantially.

SIMULATION OF ION CONDUCTION IN α-HEMOLYSIN NANOPORES WITH COVALENTLY ATTACHED β-CYCLODEXTRIN BASED ON BOLTZMANN TRANSPORT MONTE CARLO MODEL

PubMed Central

Toghraee, Reza; Lee, Kyu-Il; Papke, David; Chiu, See-Wing; Jakobsson, Eric; Ravaioli, Umberto

2009-01-01

Ion channels, as natures’ solution to regulating biological environments, are particularly interesting to device engineers seeking to understand how natural molecular systems realize device-like functions, such as stochastic sensing of organic analytes. What’s more, attaching molecular adaptors in desired orientations inside genetically engineered ion channels, enhances the system functionality as a biosensor. In general, a hierarchy of simulation methodologies is needed to study different aspects of a biological system like ion channels. Biology Monte Carlo (BioMOCA), a three-dimensional coarse-grained particle ion channel simulator, offers a powerful and general approach to study ion channel permeation. BioMOCA is based on the Boltzmann Transport Monte Carlo (BTMC) and Particle-Particle-Particle-Mesh (P3M) methodologies developed at the University of Illinois at Urbana-Champaign. In this paper, we have employed BioMOCA to study two engineered mutations of α-HL, namely (M113F)6(M113C-D8RL2)1-β-CD and (M113N)6(T117C-D8RL3)1-β-CD. The channel conductance calculated by BioMOCA is slightly higher than experimental values. Permanent charge distributions and the geometrical shape of the channels gives rise to selectivity towards anions and also an asymmetry in I-V curves, promoting a rectification largely for cations. PMID:20938493

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

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

Disease-associated changes in the expression of ion channels, ion receptors, ion exchangers and Ca{sup 2+}-handling proteins in heart hypertrophy

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

Zwadlo, Carolin; Borlak, Juergen

2005-09-15

The molecular pathology of cardiac hypertrophy is multifactorial with transcript regulation of ion channels, ion exchangers and Ca{sup 2+}-handling proteins being speculative. We therefore investigated disease-associated changes in gene expression of various ion channels and their receptors as well as ion exchangers, cytoskeletal proteins and Ca{sup 2+}-handling proteins in normotensive and spontaneously hypertensive (SHR) rats. We also compared experimental findings with results from hypertrophic human hearts, previously published (Borlak, J., and Thum, T., 2003. Hallmarks of ion channel gene expression in end-stage heart failure. FASEB J. 17, 1592-1608). We observed significant (P < 0.05) induction in transcript level of ATP-drivenmore » ion exchangers (Atp1A1, NCX-1, SERCA2a), ion channels (L-type Ca{sup 2+}-channel, K{sub ir}3.4, Na{sub v}1.5) and RyR-2 in hypertrophic hearts, while gene expression was repressed in diseased human hearts. Further, the genes coding for calreticulin and calmodulin, PMCA 1 and 4 as well as {alpha}-skeletal actin were significantly (P < 0.05) changed in hypertrophic human heart, but were unchanged in hypertrophic left ventricles of the rat heart. Notably, transcript level of {alpha}- and {beta}-MHC, calsequestrin, K{sub ir}6.1 (in the right ventricle only), phospholamban as well as troponin T were repressed in both diseased human and rat hearts. Our study enabled an identification of disease-associated candidate genes. Their regulation is likely to be the result of an imbalance between pressure load/stretch force and vascular tonus and the observed changes may provide a rational for the rhythm disturbances observed in patients with cardiac hypertrophy.« less

IA channels: diverse regulatory mechanisms.

PubMed

Carrasquillo, Yarimar; Nerbonne, Jeanne M

2014-04-01

In many peripheral and central neurons, A-type K(+) currents, IA, have been identified and shown to be key determinants in shaping action potential waveforms and repetitive firing properties, as well as in the regulation of synaptic transmission and synaptic plasticity. The functional properties and physiological roles of native neuronal IA, however, have been shown to be quite diverse in different types of neurons. Accumulating evidence suggests that this functional diversity is generated by multiple mechanisms, including the expression and subcellular distributions of IA channels encoded by different voltage-gated K(+) (Kv) channel pore-forming (α) subunits, interactions of Kv α subunits with cytosolic and/or transmembrane accessory subunits and regulatory proteins and post-translational modifications of channel subunits. Several recent reports further suggest that local protein translation in the dendrites of neurons and interactions between IA channels with other types of voltage-gated ion channels further expands the functional diversity of native neuronal IA channels. Here, we review the diverse molecular mechanisms that have been shown or proposed to underlie the functional diversity of native neuronal IA channels.

AtALMT9 is a malate-activated vacuolar chloride channel required for stomatal opening in Arabidopsis

PubMed Central

De Angeli, Alexis; Zhang, Jingbo; Meyer, Stefan; Martinoia, Enrico

2013-01-01

Water deficit strongly affects crop productivity. Plants control water loss and CO2 uptake by regulating the aperture of the stomatal pores within the leaf epidermis. Stomata aperture is regulated by the two guard cells forming the pore and changing their size in response to ion uptake and release. While our knowledge about potassium and chloride fluxes across the plasma membrane of guard cells is advanced, little is known about fluxes across the vacuolar membrane. Here we present the molecular identification of the long-sought-after vacuolar chloride channel. AtALMT9 is a chloride channel activated by physiological concentrations of cytosolic malate. Single-channel measurements demonstrate that this activation is due to a malate-dependent increase in the channel open probability. Arabidopsis thaliana atalmt9 knockout mutants exhibited impaired stomatal opening and wilt more slowly than the wild type. Our findings show that AtALMT9 is a vacuolar chloride channel having a major role in controlling stomata aperture. PMID:23653216

Gene replacement therapy for retinal CNG channelopathies.

PubMed

Schön, Christian; Biel, Martin; Michalakis, Stylianos

2013-10-01

Visual phototransduction relies on the function of cyclic nucleotide-gated channels in the rod and cone photoreceptor outer segment plasma membranes. The role of these ion channels is to translate light-triggered changes in the second messenger cyclic guanosine 3'-5'-monophosphate levels into an electrical signal that is further processed within the retinal network and then sent to higher visual centers. Rod and cone photoreceptors express distinct CNG channels. The rod photoreceptor CNG channel is composed of one CNGB1 and three CNGA1 subunits, whereas the cone channel is formed by one CNGB3 and three CNGA3 subunits. Mutations in any of these channel subunits result in severe and currently untreatable retinal degenerative diseases like retinitis pigmentosa or achromatopsia. In this review, we provide an overview of the human diseases and relevant animal models of CNG channelopathies. Furthermore, we summarize recent results from preclinical gene therapy studies using adeno-associated viral vectors and discuss the efficacy and translational potential of these gene therapeutic approaches.

The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells.

PubMed

Gao, Yong-Qiang; Wu, Wei-Hua; Wang, Yi

2017-11-01

Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K + channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K + channels in maize guard cells is limited. In the present study, we identified two KAT1-like Shaker K + channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K + (K in ) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K + currents. However, KZM2 can interact with KZM3 forming heteromeric K in channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2-KZM3 heteromeric channel became slower than the KZM3 channel. Patch-clamping results showed that the inward K + currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the K in channels in maize guard cells. KZM2 and KZM3 may form heteromeric K in channel and control stomatal opening in maize. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

[Architecture of receptor-operated ionic channels of biological membranes].

PubMed

Bregestovski, P D

2011-01-01

Ion channels of biological membranes are the key proteins, which provide bioelectric functioning of living systems. These proteins are homo- or heterooligomers assembled from several identical or different subunits. Understanding the architectural organization and functioning of ion channels has been significantly extended due to resolving the crystal structure of several types of voltage-gated and receptor-operated channels. This review summarizes the information obtained from crystal structures of potassium, nicotinic acetylcholine receptor, P2X, and other ligand-gated ion channels. Despite the differences in the function, topology, ionic selectivity, and the subunit stoichiometry, a high similarity in the principles of organization of these macromolecular complexes has been revealed.

Long-pore Electrostatics in Inward-rectifier Potassium Channels

PubMed Central

Robertson, Janice L.; Palmer, Lawrence G.; Roux, Benoît

2008-01-01

Inward-rectifier potassium (Kir) channels differ from the canonical K+ channel structure in that they possess a long extended pore (∼85 Å) for ion conduction that reaches deeply into the cytoplasm. This unique structural feature is presumably involved in regulating functional properties specific to Kir channels, such as conductance, rectification block, and ligand-dependent gating. To elucidate the underpinnings of these functional roles, we examine the electrostatics of an ion along this extended pore. Homology models are constructed based on the open-state model of KirBac1.1 for four mammalian Kir channels: Kir1.1/ROMK, Kir2.1/IRK, Kir3.1/GIRK, and Kir6.2/KATP. By solving the Poisson-Boltzmann equation, the electrostatic free energy of a K+ ion is determined along each pore, revealing that mammalian Kir channels provide a favorable environment for cations and suggesting the existence of high-density regions in the cytoplasmic domain and cavity. The contribution from the reaction field (the self-energy arising from the dielectric polarization induced by the ion's charge in the complex geometry of the pore) is unfavorable inside the long pore. However, this is well compensated by the electrostatic interaction with the static field arising from the protein charges and shielded by the dielectric surrounding. Decomposition of the static field provides a list of residues that display remarkable correspondence with existing mutagenesis data identifying amino acids that affect conduction and rectification. Many of these residues demonstrate interactions with the ion over long distances, up to 40 Å, suggesting that mutations potentially affect ion or blocker energetics over the entire pore. These results provide a foundation for understanding ion interactions in Kir channels and extend to the study of ion permeation, block, and gating in long, cation-specific pores. PMID:19001143

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

Emelianov, A. V., E-mail: emmsowton@gmail.com; Romashkin, A. V.; Tsarik, K. A.

This study is devoted to the fabrication of molecular semiconductor channels based on polymer molecules with nanoscale electrodes made of single-walled carbon nanotubes. A reproducible technology for forming nanoscale gaps in carbon nanotubes using a focused Ga{sup +} ion beam is proposed. Polyaniline molecules are deposited into nanogaps up to 30 nm wide between nanotubes by electrophoresis from N-methyl-2-pyrrolidone solution. As a result, molecular organic transistors are fabricated, in which the field effect is studied and the molecular-channel mobility is determined as 0.1 cm{sup 2}/(V s) at an on/off current ratio of 5 × 10{sup 2}.

Profiling neuronal ion channelopathies with non-invasive brain imaging and dynamic causal models: Case studies of single gene mutations

PubMed Central

Gilbert, Jessica R.; Symmonds, Mkael; Hanna, Michael G.; Dolan, Raymond J.; Friston, Karl J.; Moran, Rosalyn J.

2016-01-01

Clinical assessments of brain function rely upon visual inspection of electroencephalographic waveform abnormalities in tandem with functional magnetic resonance imaging. However, no current technology proffers in vivo assessments of activity at synapses, receptors and ion-channels, the basis of neuronal communication. Using dynamic causal modeling we compared electrophysiological responses from two patients with distinct monogenic ion channelopathies and a large cohort of healthy controls to demonstrate the feasibility of assaying synaptic-level channel communication non-invasively. Synaptic channel abnormality was identified in both patients (100% sensitivity) with assay specificity above 89%, furnishing estimates of neurotransmitter and voltage-gated ion throughput of sodium, calcium, chloride and potassium. This performance indicates a potential novel application as an adjunct for clinical assessments in neurological and psychiatric settings. More broadly, these findings indicate that biophysical models of synaptic channels can be estimated non-invasively, having important implications for advancing human neuroimaging to the level of non-invasive ion channel assays. PMID:26342528

Ion transport through lipid bilayers by synthetic ionophores: modulation of activity and selectivity.

PubMed

De Riccardis, Francesco; Izzo, Irene; Montesarchio, Daniela; Tecilla, Paolo

2013-12-17

The ion-coupled processes that occur in the plasma membrane regulate the cell machineries in all the living organisms. The details of the chemical events that allow ion transport in biological systems remain elusive. However, investigations of the structure and function of natural and artificial transporters has led to increasing insights about the conductance mechanisms. Since the publication of the first successful artificial system by Tabushi and co-workers in 1982, synthetic chemists have designed and constructed a variety of chemically diverse and effective low molecular weight ionophores. Despite their relative structural simplicity, ionophores must satisfy several requirements. They must partition in the membrane, interact specifically with ions, shield them from the hydrocarbon core of the phospholipid bilayer, and transport ions from one side of the membrane to the other. All these attributes require amphipathic molecules in which the polar donor set used for ion recognition (usually oxygens for cations and hydrogen bond donors for anions) is arranged on a lipophilic organic scaffold. Playing with these two structural motifs, donor atoms and scaffolds, researchers have constructed a variety of different ionophores, and we describe a subset of interesting examples in this Account. Despite the ample structural diversity, structure/activity relationships studies reveal common features. Even when they include different hydrophilic moieties (oxyethylene chains, free hydroxyl, etc.) and scaffolds (steroid derivatives, neutral or polar macrocycles, etc.), amphipathic molecules, that cannot span the entire phospholipid bilayer, generate defects in the contact zone between the ionophore and the lipids and increase the permeability in the bulk membrane. Therefore, topologically complex structures that span the entire membrane are needed to elicit channel-like and ion selective behaviors. In particular the alternate-calix[4]arene macrocycle proved to be a versatile platform to obtain 3D-structures that can form unimolecular channels in membranes. In these systems, the selection of proper donor groups allows us to control the ion selectivity of the process. We can switch from cation to anion transport by substituting protonated amines for the oxygen donors. Large and stable tubular structures with nanometric sized transmembrane nanopores that provide ample internal space represent a different approach for the preparation of synthetic ion channels. We used the metal-mediated self-assembly of porphyrin ligands with Re(I) corners as a new method for producing to robust channel-like structures. Such structures can survive in the complex membrane environment and show interesting ionophoric behavior. In addition to the development of new design principles, the selective modification of the biological membrane permeability could lead to important developments in medicine and technology.

Superstatistics analysis of the ion current distribution function: Met3PbCl influence study.

PubMed

Miśkiewicz, Janusz; Trela, Zenon; Przestalski, Stanisław; Karcz, Waldemar

2010-09-01

A novel analysis of ion current time series is proposed. It is shown that higher (second, third and fourth) statistical moments of the ion current probability distribution function (PDF) can yield new information about ion channel properties. The method is illustrated on a two-state model where the PDF of the compound states are given by normal distributions. The proposed method was applied to the analysis of the SV cation channels of vacuolar membrane of Beta vulgaris and the influence of trimethyllead chloride (Met(3)PbCl) on the ion current probability distribution. Ion currents were measured by patch-clamp technique. It was shown that Met(3)PbCl influences the variance of the open-state ion current but does not alter the PDF of the closed-state ion current. Incorporation of higher statistical moments into the standard investigation of ion channel properties is proposed.

Fragile X mental retardation protein controls ion channel expression and activity.

PubMed

Ferron, Laurent

2016-10-15

Fragile X-associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA-binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage-gated potassium channels (K v 3.1 and K v 4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium-activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single-channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary β4 subunit of the calcium-activated potassium (BK) channel; this interaction increases calcium-dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage-gated calcium channel, Ca v 2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X-associated disorders. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Atomic force microscopy study on crystal growth of Cu 2+-doped L-arginine phosphate monohydrate crystals

NASA Astrophysics Data System (ADS)

Geng, Y. L.; Xu, D.; Wang, Y. L.; Du, W.; Liu, H. Y.; Zhang, G. H.; Wang, X. Q.; Sun, D. L.

2005-01-01

Sub-steps and defects of the {1 0 0} planes of Cu 2+-doped L-arginine phosphate monohydrate (LAP) crystals are observed by atomic force microscopy. Formation of sub-steps is not due to the stacking faults but a result of single LAP: Cu 2+ molecule acting as growth unit. Two-dimensional (2D) nuclei with the same height as sub-steps occur on the step-edges. Impurities of Cu 2+ ions cause steps bunch and macrosteps formation. Liquid inclusions in the form of long channels form when the macrosteps lose their stability. Numerous small 3D growth hillocks are found in the channels. The extra stress induced by the 3D islands can result in dislocations and steps mismatches.

Crystal structure and association behaviour of the GluR2 amino-terminal domain

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

Jin, Rongsheng; Singh, Satinder K.; Gu, Shenyan

2009-09-02

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanismsmore » by which the ATD guides subfamily-specific receptor assembly.« less

Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits.

PubMed

Molinarolo, Steven; Granata, Daniele; Carnevale, Vincenzo; Ahern, Christopher A

2018-02-21

Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

PubMed Central

Li, Boxing; Jie, Wei; Huang, Lianyan; Wei, Peng; Li, Shuji; Luo, Zhengyi; Friedman, Allyson K.; Meredith, Andrea L.; Han, Ming-Hu; Zhu, Xin-Hong; Gao, Tian-Ming

2014-01-01

Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal ion channels in the regulation of Ca2+ is well established. In contrast, surprisingly less is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally blockade of nuclear BK channels (nBK channels) induces NE-derived Ca2+ release, nucleoplasmic Ca2+ elevation, and cAMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+-sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+-dependent manner. These results suggest that nBK channel functions as a molecular linker between neuronal activity and nuclear Ca2+ to convey the signals from synapse to nucleus and is a new modulator for synaptic activity-dependent neuronal functions at the NE level. PMID:24952642

Hepatitis C virus p7 mediates membrane-to-membrane adhesion.

PubMed

Lee, Gi Young; Lee, Sora; Lee, Hye-Ra; Yoo, Young Do

2016-09-01

Viroporin p7 of the hepatitis C virus (HCV) acts as an ion channel for pH equilibration to stabilize HCV particles; most studies of p7 have focused on this role. However, pH equilibration by p7 via its ion channel activity does not fully explain the importance of p7 in HCV particle production. Indeed, several researchers have suggested p7 to have an unidentified ion channel-independent function. Here, we show that p7 has a novel role as a lipid raft adhesion factor, which is independent of its ion channel activity. We found that p7 targets not only the liquid-disordered (Ld) phase, but also the negatively-charged liquid-ordered (Lo) phase that can be represented as a lipid raft. p7 clusters at the phase boundary of the neutral Ld phase and the negatively-charged Lo phase. Interestingly, p7 targeting the Lo phase facilitates membrane-to-membrane adhesion, and this activity is not inhibited by p7 ion channel inhibitors. Our results demonstrated that HCV p7 has dual roles as a viroporin and as a lipid raft adhesion factor. This ion channel-independent function of p7 might be an attractive target for development of anti-HCV compounds. Copyright © 2016 Elsevier B.V. All rights reserved.