Ionic Channels In Excitable Membranes Research Articles

A new standard: A review of Handbook of Ion Channels.

Handbook of Ion Channels (2015) Jie Zheng and Matthew Trudeau, editors CRC Press, 691 pp. The publication in 1984 of Bertil Hille’s Ionic Channels in Excitable Membranes ([Hille, 1984][1]) marked a milestone and turning point in the field of ion channel biophysics and physiology. Not only did

Exponential integrators for a Markov chain model of the fast sodium channel of cardiomyocytes.

The modern Markov chain models of ionic channels in excitable membranes are numerically stiff. The popular numerical methods for these models require very small time steps to ensure stability. Our objective is to formulate and test two methods addressing this issue, so that the timestep can be chosen based on accuracy rather than stability. Both proposed methods extend Rush-Larsen technique, which was originally developed to Hogdkin-Huxley type gate models. One method, "matrix Rush-Larsen" (MRL) uses a matrix reformulation of the Rush-Larsen scheme, where the matrix exponentials are calculated using precomputed tables of eigenvalues and eigenvectors. The other, "hybrid operator splitting" (HOS) method exploits asymptotic properties of a particular Markov chain model, allowing explicit analytical expressions for the substeps. We test both methods on the Clancy and Rudy (2002) I(Na)Markov chain model. With precomputed tables for functions of the transmembrane voltage, both methods are comparable to the forward Euler method in accuracy and computational cost, but allow longer time steps without numerical instability. We conclude that both methods are of practical interest. MRL requires more computations than HOS, but is formulated in general terms which can be readily extended to other Markov chain channel models, whereas the utility of HOS depends on the asymptotic properties of a particular model. The significance of the methods is that they allow a considerable speed-up of large-scale computations of cardiac excitation models by increasing the time step, while maintaining acceptable accuracy and preserving numerical stability.

TRPM Channels Modulate Epileptic-like Convulsions via Systemic Ion Homeostasis

Neuronal networks operate over a wide range of activity levels, with both neuronal and nonneuronal cells contributing to the balance of excitation and inhibition. Activity imbalance within neuronal networks underlies many neurological diseases, such as epilepsy. The Caenorhabditis elegans locomotor circuit operates via coordinated activity of cholinergic excitatory and GABAergic inhibitory transmission. We have previously shown that a gain-of-function mutation in a neuronal acetylcholine receptor, acr-2(gf), causes an epileptic-like convulsion behavior. Here we report that the behavioral and physiological effects of acr-2(gf) require the activity of the TRPM channel GTL-2 in nonneuronal tissues. Loss of gtl-2 function does not affect baseline synaptic transmission but can compensate for the excitation-inhibition imbalance caused by acr-2(gf). The compensatory effects of removing gtl-2 are counterbalanced by another TRPM channel, GTL-1, and can be recapitulated by acute treatment with divalent cation chelators, including those specific for Zn(2+). Together, these data reveal an important role for ion homeostasis in the balance of neuronal network activity and a novel function of nonneuronal TRPM channels in the fine-tuning of this network activity.

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.

Tunable Calcium Current through TRPV1 Receptor Channels

TRPV1 receptors are polymodal cation channels that open in response to diverse stimuli including noxious heat, capsaicin, and protons. Because Ca2+ is vital for TRPV1 signaling, we sought to precisely measure its contribution to TRPV1 responses and discovered that the Ca2+ current was tuned by the mode of activation. Using patch clamp photometry, we found that the fraction of the total current carried by Ca2+ (called the Pf%) was significantly smaller for TRPV1 currents evoked by protons than for those evoked by capsaicin. Using site-directed mutagenesis, we discovered that the smaller Pf% was due to protonation of three acidic amino acids (Asp646, Glu648, and Glu651) that are located in the mouth of the pore. Thus, in keeping with recent reports of time-dependent changes in the ionic permeability of some ligand-gated ion channels, we now show for the first time that the physiologically important Ca2+ current of the TRPV1 receptor is also dynamic and depends on the mode of activation. This current is significantly smaller when the receptor is activated by a change in pH, owing to atomic scale interactions of H+ and Ca2+ with the fixed negative charge of side chains in the pore.

Identification of Pore Residues Engaged in Determining Divalent Cationic Permeation in Transient Receptor Potential Melastatin Subtype Channel 2

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca2+ and Mg2+ permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg2+ permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca2+ permeability along with concentration-dependent inhibition of Na+-carrying currents by Ca2+. Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca2+-permeable TRPM channels, substantially increased Ca2+ permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca2+ permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

On the Role of the First Transmembrane Domain in Cation Permeability and Flux of the ATP-gated P2X2 Receptor

P2X receptors are a family of seven ligand-gated ion channels (P2X1-P2X7) that open in the presence of ATP. We used alanine-scanning mutagenesis and patch clamp photometry to study the role of the first transmembrane domain of the rat P2X2 receptor in cation permeability and flux. Three alanine-substituted mutants did not respond to ATP, and 19 of the 22 functional receptors resembled the wild-type receptor with regard to the fraction of the total ATP-gated current carried by calcium or the permeability of calcium relative to cesium. The remaining three mutants showed modest changes in calcium dynamics. Two of these occurred at sites (Gly30 and Phe44) that are unlikely to interact with permeating cations in a meaningful way. The third was a conserved tyrosine (Tyr43) that may form an inter-pore binding site for calcium. The data suggest that, with the possible exception of Tyr43, the first transmembrane domain contributes little to the permeation properties of the P2X2 receptor.

HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.

A Family of Acetylcholine-gated Chloride Channel Subunits in Caenorhabditis elegans

The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors.

Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking.

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively. Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1-S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.

Single-walled Carbon Nanotubes Are a New Class of Ion Channel Blockers

Here we identify a novel class of biological membrane ion channel blockers called single-walled carbon nanotubes (SWNTs). SWNTs with diameter distributions peaked at approximately 0.9 and 1.3 nm, C60 fullerenes, multi wall nanotubes (MWNTs), and hyperfullerenes (nano-"onions") were synthesized by several techniques and applied to diverse channel types heterologously expressed in mammalian cells. External as-fabricated and purified SWNTs blocked K+ channel subunits in a dose-dependent manner. Blockage was dependent on the shape and dimensions of the nanoparticles used and did not require any electrochemical interaction. SWNTs were more effective than the spherical fullerenes and, for both, diameter was the determining factor. These findings postulate new uses for SWNTs in biological applications and provide unexpected insights into the current view of mechanisms governing the interaction of ion channels with blocking molecules.

Characterization of a novel radiolabeled peptide selective for a subpopulation of voltage-gated potassium channels in mammalian brain.

BgK, a 37-amino acid voltage-gated potassium (Kv) 1 channel blocker isolated from the sea anemone Bunodosoma granulifera, can be modified at certain positions to alter its pharmacological profile (Alessandri-Haber, N., Lecoq, A., Gasparini, S., Grangier-Macmath, G., Jacquet, G., Harvey, A. L., de Medeiros, C., Rowan, E. G., Gola, M., Ménez, A., and Crest, M. (1999) J. Biol. Chem. 274, 35653-35661). In the present study, we report the design of two BgK analogs that have been radiolabeled with (125)INa. Whereas BgK(W5Y/Y26F) and its radiolabeled derivative, (125)I-BgK(W5Y/Y26F), bind to Kv1.1, Kv1.2, and Kv1.6 channels with potencies similar to those for the parent peptide, BgK, BgK(W5Y/F6A/Y26F) and its monoiodo-tyrosine derivative, (125)I-BgK(W5Y/F6A/Y26F), display a distinctive and unique pharmacological profile; they bind with high affinity to homomultimeric Kv1.1 and Kv1.6 channels, but not to Kv1.2 channels. Interaction of BgK(W5Y/F6A/Y26F) with potassium channels depends on the nature of a residue in the mouth of the channel, at a position that determines channel sensitivity to external tetraethylammonium. In native brain tissue, (125)I-BgK(W5Y/F6A/Y26F) binds to a population of Kv1 channels that appear to consist of at least two sensitive (Kv1.1 and/or Kv1.6) subunits, in adjacent position. Given its unique pharmacological properties, (125)I-BgK(W5Y/F6A/Y26F) represents a new tool for studying subpopulations of Kv1 channels in native tissues.

Spatial Control of Ca2+ Signaling by Nicotinic Acid Adenine Dinucleotide Phosphate Diffusion and Gradients

Intracellular Ca(2+) is able to control numerous cellular responses through complex spatiotemporal organization. Ca(2+) waves mediated by inositol trisphosphate or ryanodine receptors propagate by Ca(2+)-induced Ca(2+) release and therefore do not have an absolute requirement for a gradient in either inositol trisphosphate or cyclic ADP-ribose, respectively. In contrast, we report that although Ca(2+) increases induced by nicotinic acid adenine dinucleotide phosphate (NAADP) are amplified by Ca(2+)-induced Ca(2+) release locally, Ca(2+) waves mediated by NAADP have an absolute requirement for an NAADP gradient. If NAADP is increased such that its concentration is spatially uniform in one region of an egg, the Ca(2+) increase occurs simultaneously throughout this area, and only where there is diffusion out of this area to establish an NAADP gradient is there a Ca(2+) wave. A local increase in NAADP results in a Ca(2+) increase that spreads by NAADP diffusion. NAADP diffusion is restricted at low but not high concentrations of NAADP, indicating that NAADP diffusion is strongly influenced by binding to immobile and saturable sites, probably the NAADP receptor itself. Thus, the range of action of NAADP can be tuned by its concentration from that of a local messenger, like Ca(2+), to that of a global messenger, like IP(3) or cyclic ADP-ribose.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

Computing with Lipids, Proteins, and Ions

Computing with Lipids, Proteins, and Ions

Molecular Mechanism of Diltiazem Interaction with L-type Ca2+ Channels

Benzothiazepine Ca2+ antagonists (such as (+)-cis-diltiazem) interact with transmembrane segments IIIS6 and IVS6 in the alpha1 subunit of L-type Ca2+ channels. We investigated the contribution of individual IIIS6 amino acid residues for diltiazem sensitivity by employing alanine scanning mutagenesis in a benzothiazepine-sensitive alpha1 subunit chimera (ALDIL) expressed in Xenopus laevis oocytes. The most dramatic decrease of block by 100 microM diltiazem (ALDIL 45 +/- 4.8% inhibition) during trains of 100-ms pulses (0.1 Hz, -80 mV holding potential) was found after mutation of adjacent IIIS6 residues Phe1164(21 +/- 3%) and Val1165 (8.5 +/- 1.4%). Diltiazem delayed current recovery by promoting a slowly recovering current component. This effect was similar in ALDIL and F1164A but largely prevented in V1165A. Both mutations slowed inactivation kinetics during a pulse. The reduced diltiazem block can therefore be explained by slowing of inactivation kinetics (F1164A and V1165A) and accelerated recovery from drug block (V1165A). The bulkier diltiazem derivative benziazem still efficiently blocked V1165A. From these functional and from additional radioligand binding studies with the dihydropyridine (+)-[3H]isradipine we propose a model in which Val1165 controls dissociation of the bound diltiazem molecule, and where bulky substituents on the basic nitrogen of diltiazem protrude toward the adjacent dihydropyridine binding domain.

Human Saphenous Vein Endothelial Cells Express a Tetrodotoxin-resistant, Voltage-gated Sodium Current

Whole-cell patch-clamp electrophysiological investigation of endothelial cells cultured from human saphenous vein (HSVECs) has identified a voltage-gated Na+ current with a mean peak magnitude of -595 +/- 49 pA (n = 75). This current was inhibited by tetrodotoxin (TTX) in a concentration-dependent manner, with an IC50 value of 4.7 microM, suggesting that it was of the TTX-resistant subtype. An antibody directed against the highly conserved intracellular linker region between domains III and IV of known Na+ channel alpha-subunits was able to retard current inactivation when applied intracellularly. This antibody identified a 245-kDa protein from membrane lysates on Western blotting and positively immunolabeled both cultured HSVECs and intact venous endothelium. HSVECs were also shown by reverse transcription-polymerase chain reaction to contain transcripts of the hH1 sodium channel gene. The expression of Na+ channels by HSVECs was shown using electrophysiology and cell-based enzyme-linked immunosorbent assay to be dependent on the concentration and source of human serum. Together, these results suggest that TTX-resistant Na+ channels of the hH1 isoform are expressed in human saphenous vein endothelium and that the presence of these channels is controlled by a serum factor.

Investigative Urology: An Analysis of the Maxi-K sup + (k sub Ca) Channel in Cultured Human Corporal Smooth Muscle Cells

Previous studies have demonstrated that cultured corporal smooth muscle cells have prominent outward K currents composed of several different K channel subtypes. The goals of the present investigation were (1) to assert the nature of these channels and to evaluate the characteristics of the most predominant of these channel subtypes, the Maxi-K+ (KCa) channel, and (2) to compare KCa channel behavior in cultured corporal smooth muscle cells derived from the human corpus cavernosum of two distinct patient populations. The patient population was subdivided into two broad diagnostic categories: Group 1: 4 patients without evidence of organic disease of the corpus cavernosum, 3 of whom had documented erections; and Group 2: 4 patients with organic erectile dysfunction. Consistent with previous observations, 3 different K channel subtypes were detected in both patient populations, with corresponding conductances of 180, 100 and 40 pS, respectively. The approximately 183 pS channel was identified as the KCa channel based on its selective permeability to K+ and the fact that its open probability was modulated by both membrane potential and intracellular calcium levels. Specifically, the relative permeability of the 183 pS KCa channel to K+, Rb+, and NH4+ was 1.00:0.64:0.46. The channel was virtually impermeable to Na+ and Li+ (relative permeability < 0.02). In addition, the KCa channel was responsible for more than 90% of the outward K+ current passed through the cell membrane when depolarized. Furthermore, pharmacological studies using the K channel blocker tetraethylammonium ion (TEA) revealed that the sensitivity of KCa channels to TEA inhibition (as judged by the [TEA] required to block one-half of the outward whole cell current induced by a 90 mV depolarizing pulse) in cells from Group 1 patients was 1.05 +/- 0.22 mM. (n = 10 cells), while in sharp contrast the observed value for cells from Group 2 patients was 12.7 +/- 3.8 (n = 9 cells). The difference between the two groups was highly significant. These observations confirm and extend our previous studies to suggest that the KCa channel plays an important role in corporal smooth muscle physiology and, moreover, that alterations in the function/regulation of KCa channels may be an important feature of organic erectile dysfunction. As such, altered KCa channel behavior may contribute to an impaired hyperpolarizing ability of corporal smooth muscle, possibly altering intracellular calcium homeostasis and, perhaps, corporal smooth muscle reactivity and tone.

Ionic Channels of Excitable Membranes, 2nd Ed.

Ionic Channels of Excitable Membranes, 2nd Ed.

BOOK REVIEWS

Journal Article BOOK REVIEWS Get access Ionic Channels of Excitable Membranes. By Bertil Hille 1984. Pp. 426. Sunderland, MA. Sinauer Distributed in UK by Blackwell. Price £29.50. Voltage and Patch Clamping with Microelectrodes. Edited by Thomas G. Smith, Harold Lecar, Steven J Radman and Peter W Gage. 1985 Pp. 260Bethesda: American Physiological Society. Price $39.50. T. A. SEARS T. A. SEARS Search for other works by this author on: Oxford Academic PubMed Google Scholar Brain, Volume 109, Issue 5, October 1986, Pages 1067–1068, https://doi.org/10.1093/brain/109.5.1067 Published: 01 October 1986