Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K+ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage sensitive channels. Single ATP-sensitive K⁺ (K-ATP) channel currents were recorded in acutely dissociated rat neocortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intracellular ATP, but also by membrane potential (V_m), and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and diversity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activation-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary conductance of about 80 pS in detail (n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane potentials. This property of the K-ATP channels may be related to their pathophysiological function. Project supported by the National Natural Science Foundation of China and Natural Science Foundation of Guangdong Province     

Double-gating mechanism and diversity of an adenosine triphosphate (ATP)-sensitive K~ channel in neurons acutely dissociated from rat neocortex

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage-sensitive channels. Single ATP-sensitive K (K-ATP) channel currents were recorded in acutely dissociated rat neo-cortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intra-cellular ATP, but also by membrane potential ( Vm) , and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and di-versity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activa-tion-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary con-ductance of about 80 pS in detail ( n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane poten     

Ion channel inhibitors block caspase activation by mechanisms other than restoring intracellular potassium concentration

Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na⁺ and loss of K⁺ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA⁺) and 4-aminopyridine-sensitive K⁺ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K⁺] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K⁺]_i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K⁺ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K⁺]_i associated with apoptosis.     

An overview of the potassium channel family

Potassium channels, tetrameric integral membrane proteins that form aqueous pores through which K⁺ can flow, are found in virtually all organisms; the genomes of humans, Drosophila, and Caenorhabditis elegans contain 30-100 K⁺ channel genes each. The structure of a bacterial K⁺ channel, sequence comparisons with other channels and electrophysiological measurements have enabled conclusions about the mechanism of gating and ion flow to be drawn for many other channels.     

Electrophysiological Properties of Ion Channels in Ascaris suum Tissue Incorporated into Planar Lipid Bilayers

Ion channels are important targets of anthelmintic agents. In this study, we identified 3 types of ion channels in Ascaris suum tissue incorporated into planar lipid bilayers using an electrophysiological technique. The most frequent channel was a large-conductance cation channel (209 pS), which accounted for 64.5% of channels incorporated (n=60). Its open-state probability (P_o) was ~0.3 in the voltage range of −60~+60 mV. A substate was observed at 55% of the main-state. The permeability ratio of Cl⁻ to K⁺ (P_Cl/P_K) was ~0.5 and P_Na/P_K was 0.81 in both states. Another type of cation channel was recorded in 7.5% of channels incorporated (n=7) and discriminated from the large-conductance cation channel by its smaller conductance (55.3 pS). Its Po was low at all voltages tested (~0.1). The third type was an anion channel recorded in 27.9% of channels incorporated (n=26). Its conductance was 39.0 pS and P_Cl/P_K was 8.6±0.8. P_o was ~1.0 at all tested potentials. In summary, we identified 2 types of cation and 1 type of anion channels in Ascaris suum. Gating of these channels did not much vary with voltage and their ionic selectivity is rather low. Their molecular nature, functions, and potentials as anthelmintic drug targets remain to be studied further.     

Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit

Ethanol often causes critical health problems by altering the neuro-nal activities of the central and peripheral nerve systems. One of the cellular targets of ethanol is the plasma membrane proteins including ion channels and receptors. Recently, we reported that ethanol elevates membrane excitability in sympathetic neurons by inhibiting Kv7.2/7.3 channels in a cell type-specific manner. Even though our studies revealed that the inhibitory effects of ethanol on the Kv7.2/7.3 channel was diminished by the increase of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI (4,5)P₂), the molecular mechanism of ethanol on Kv7.2/7.3 channel inhibition remains unclear. By investigating the kinetics of Kv7.2/7.3 current in high K⁺ solution, we found that ethanol inhibited Kv7.2/7.3 channels through a mechanism distinct from that of tetraethylammonium (TEA) which enters into the pore and blocks the gate of the channels. Using a non-stationary noise analysis (NSNA), we demonstrated that the inhibitory effect of ethanol is the result of reduction of open probability (P_O) of the Kv7.2/7.3 channel, but not of a single channel current (i) or channel number (N). Finally, ethanol selectively facilitated the kinetics of Kv7.2 current suppression by voltage-sensing phosphatase (VSP)-induced PI(4,5)P₂ depletion, while it slowed down Kv7.2 current recovery from the VSP-induced inhibition. Together our results suggest that ethanol regulates neuronal activity through the reduction of open probability and PI(4,5)P₂ sensitivity of Kv7.2/7.3 channels.     

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na⁺ channels facilitate the transport of Na⁺ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na⁺ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.Many ion channels are silent at rest and are activated in response to a variety of factors, including membrane potential, external ligands, and intracellular signaling processes. The ENaC² has evolved as a channel that is thought to reside primarily in an active state, facilitating the bulk movement of Na⁺ out of renal tubular or airway lumens. The regulated insertion and retrieval of channels at the plasma membrane have important roles in modulating ENaC-dependent Na⁺ transport (1). A number of factors also have a role in regulating ENaC activity via changes in channel P_o or gating. In this regard, it has become increasingly apparent that proteolysis of ENaC subunits has a key role in this process (2). This minireview addresses several questions regarding the role of ENaC subunit proteolysis in regulating channel gating. (i) Where are ENaC subunits cleaved? (ii) Which proteases mediate ENaC cleavage? (iii) Why are channels activated by proteolysis? (iv) Is proteolysis responsible, in part, for the highly variable channel P_o that has been noted for ENaC? (v) Why have ENaCs evolved as channels that require proteolysis for activation?     

Muscarinic Cholinergic Excitation of Smooth Muscle: Signal Transduction and Single Cationic Channel Properties

Acetylcholine, the principal neurotransmitter of the parasympathetic nervous system, evokes smooth muscle excitation and contraction by acting at the muscarinic receptors which, in many tissues, including the gastrointestinal tract, are comprised of the M₂ and M₃ subtypes. The opening of ion channels selective for monovalent cations (e.g., Na⁺ and K⁺) is the major mechanism of cholinergic excitation. We have studied signal transduction pathways and single cationic channel properties using patch-clamp recording and Ca²⁺ imaging techniques in guinea-pig single ileal myocytes. Cationic channels were found to couple to both M₂ and M₃ receptors via the GTP-bound G_oα and phospholipase C activation, respectively. When these primarily signaling links are established, cationic channel opening can be further potentiated by membrane depolarization and an increase in the intracellular Ca²⁺ concentration. A strong synergism exists between the receptor occupancy by the agonist and intrinsic voltage dependence of the current as the former can effectively modulate the voltage range of cationic channel activation, while membrane depolarization produces a strong sensitizing effect. However, at potentials close to 0 mV ion flux is terminated by channel flickery block, while further depolarization induces long-lasting channel inactivation. Channel flicker is not caused by intracellular Mg²⁺, polyamines, or any other freely diffusible molecule and is confined to potentials around 0 mV irrespective of the driving force. Thus, it appears to be an intrinsic channel property of physiological importance as it improves conditions for the action potential discharge and propagation. Similarly, intracellular Ca²⁺-dependent facilitation of channel opening is counteracted by a slower desensitization. Further, the most intriguing negative control was discovered in the experiments whereby all cellular G proteins were non-selectively and persistently activated by GTPγS infusion, in which case, over time, carbachol instead of activation caused strong and almost irreversible inhibition of the cationic current. In cell-attached and outside-out membrane patches exposed to 50 μM carbachol or 200 μM internal GTPγS, the activity of three types of cationic channels was observed. They had dissimilar conductances (10, 50, and 130 pS), voltage dependence, and kinetics. The properties of the 50 pS channel are consistent with the whole-cell current behavior, at least when [Ca²⁺] _i is “clamped” at 100 nM. The voltage-independent component of the cationic conductance, which appears at higher levels of [Ca²⁺] _i , is likely mediated by the 130 pS channel, while the role of the 10 pS channel at present is unclear. Thus, smooth muscle cationic channels can uniquely detect and integrate many of the most important physiological signals such as the active conformation of two different muscarinic receptors, their associated G proteins and enzymes, as well as membrane potential and [Ca²⁺] _i levels. Moreover, some signals act in synergy, while most of them, depending on the intensity, can be either stimulatory or inhibitory.     

An analysis of the change in K-permeability on depolarization in terms of affinity and numbers of total channels.

A theoretical relation between permeability and ionic concentrations in a bathing solution has been derived by assuming that only channels unoccupied by a competing non-permeable ion can transport ions specific for that channel. The affinities of the channel to the ion and the competitor are expressed by dissociation constants of the ion-site and competitor-site complexes in the channel.Analyses of the relation of K permeability to [K]_o obtained from myelinated nerve fibres and Nitella cells revealed that the affinity of sites in K channels was independent of membrane potential, whereas K conductivity increased with depolarization. The value of the dissociation constant of the K⁺-site complex, K₁, was estimated as 1244 mm for myelinated nerve, and $\text{K}{}_1\text{exp(ψ}{}_0\text{F}\text{RT}$) for Nitella was 17.5 mm (ψ₀ is the surface potential at the outer surface of membrane). The dependence on voltage of the total number of K channels was estimated from the dependence of K conductance on membrane potential at [K]_o = [K]₁ (obtained from the theoretical magnitude of K current computed by using the dissociation constants described above). It should be noted that when the channels are partially saturated with K⁺, neither the chord conductance nor the “permeability coefficient”, as defined in the Goldman and Hodgkin-Katz formulation, correctly represents the dependence on membrane potential of the total number of channels.     

Action potential duration and activation of ATP-sensitive potassium current in isolated guinea-pig ventricular myocytes

It is difficult to associate the ATP-sensitive potassium (K-ATP) channel of cardiac muscle with hypoxia/ischemia induced action potential shortening because this occurs before intracellular ATP falls to levels associated in vitro with channel opening. This leaves the cardiac K-ATP channel without any obvious physiological function. We have quantitatively examined the relationship between action potential duration and K-ATP channel activity in enzymatically isolated ventricular myocytes of the guinea-pig. In whole-cell voltage-clamp recording experiments when the K-ATP channel opener SR 44866 (2-10 microM) stimulated an outward membrane current greater than 50 pA at 0 mV membrane potential (the equivalent of 30 open K-ATP channels or 1% of the cell K-ATP channel population) action potential duration was reduced by more than 50%. In the majority of cell-attached membrane patch recordings metabolic inhibition stimulated K-ATP channel open probability of 1-2% which continued for long periods (7-25 min) before cell contracture and coincident major K-ATP channel activation (open probability 65%). Our quantitative analysis thus shows that physiologically relevant activity of K-ATP channels in cardiac muscle is confined to a very small percentage of the possible cell K-ATP current and thus intracellular ATP would not have to fall very far before the opening of K-ATP channels would influence cardiac excitability.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca²⁺ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca²⁺]_cyt is increased by Ca²⁺ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca²⁺-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca²⁺-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca²⁺-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca²⁺ channels, the genetic counterparts of specific Ca²⁺-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca²⁺ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca²⁺ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La³⁺ and nifedipine, and their increased activity as [Ca²⁺]_cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca²⁺-permeable) K⁺ channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

Ion conductance and ion selectivity of potassium channels in snail neurones

Summary Delayed potassium channels were studied in internally perfused neurone somata from land snails. Relaxation and fluctuation analysis of this class of ion channels revealed Hodgkin-Huxley type K channels with an average single channel conductance ( _K) of 2.40±0.15 pS. The conductance of open channels is independent of voltage and virtually all K channels seem to be open at maximum K conductance (g _K) of the membrane. Voltage dependent time constants of activation ofg _K, calculated from K current relaxation and from cut-off frequencies of power spectra, are very similar indicating dominant first-order kinetics. Ion selectivity of K channels was studied by ion substitution in the external medium and exhibited the following sequence: T1⁺>K⁺>Rb⁺>Cs⁺>NH ₄ ⁺ >Li⁺>Na⁺. The sequence of the alkali cations does not conform to any of the sequences predicted by Eisenman's theory. However, the data are well accommodated by a new theory assuming a single rate-limiting barrier that governs ion movement through the channel.This paper is dedicated to the memory of Walther Wilbrandt.     

The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel

Voltage-gated potassium (K_V) channels are membrane proteins that respond to changes in membrane potential by enabling K⁺ ion flux across the membrane. Polyunsaturated fatty acids (PUFAs) induce channel opening by modulating the voltage-sensitivity, which can provide effective treatment against refractory epilepsy by means of a ketogenic diet. While PUFAs have been reported to influence the gating mechanism by electrostatic interactions to the voltage-sensor domain (VSD), the exact PUFA-protein interactions are still elusive. In this study, we report on the interactions between the Shaker K_V channel in open and closed states and a PUFA-enriched lipid bilayer using microsecond molecular dynamics simulations. We determined a putative PUFA binding site in the open state of the channel located at the protein-lipid interface in the vicinity of the extracellular halves of the S3 and S4 helices of the VSD. In particular, the lipophilic PUFA tail covered a wide range of non-specific hydrophobic interactions in the hydrophobic central core of the protein-lipid interface, while the carboxylic head group displayed more specific interactions to polar/charged residues at the extracellular regions of the S3 and S4 helices, encompassing the S3-S4 linker. Moreover, by studying the interactions between saturated fatty acids (SFA) and the Shaker K_V channel, our study confirmed an increased conformational flexibility in the polyunsaturated carbon tails compared to saturated carbon chains, which may explain the specificity of PUFA action on channel proteins.     

Ionic Blockage of Sodium Channels in Nerve

Increasing the hydrogen ion concentration of the bathing medium reversibly depresses the sodium permeability of voltage-clamped frog nerves. The depression depends on membrane voltage: changing from pH 7 to pH 5 causes a 60% reduction in sodium permeability at +20 mV, but only a 20% reduction at +180 mV. This voltage-dependent block of sodium channels by hydrogen ions is explained by assuming that hydrogen ions enter the open sodium channel and bind there, preventing sodium ion passage. The voltage dependence arises because the binding site is assumed to lie far enough across the membrane for bound ions to be affected by part of the potential difference across the membrane. Equations are derived for the general case where the blocking ion enters the channel from either side of the membrane. For H⁺ ion blockage, a simpler model, in which H⁺ enters the channel only from the bathing medium, is found to be sufficient. The dissociation constant of H⁺ ions from the channel site, 3.9 x 10^-6 M (pK_a 5.4), is like that of a carboxylic acid. From the voltage dependence of the block, this acid site is about one-quarter of the way across the membrane potential from the outside. In addition to blocking as described by the model, hydrogen ions also shift the responses of sodium channel "gates" to voltage, probably by altering the surface potential of the nerve. Evidence for voltage-dependent blockage by calcium ions is also presented.     

Sodium Conductance Activation without Inactivation in Pronase-perfused Axons

A controversy of long standing in membrane electrophysio-logy is whether the sodium ion current (I_Na) and potassium ion current (I_K) pass through the membrane in separate channels, or through a single set of channels which conduct first I_Na and then I_K. In support of the latter hypothesis it has been noted that the sodium conductance (g_Na) decline, called inactivation, proceeds with about the same time course as the potassium conductance (g_K) increase. This could mean that Na⁺ selective channels are being converted into K⁺ selective channels. The hypothesis is especially interesting because of the possibility that the carrier postulated in active transport is convertible from Na⁺ to K⁺ selectivity¹. An explicit statement of the single channel hypothesis and the means for disproving it were given by Mullins². Because a single channel could not simultaneously conduct I_Na and I_K, disproof requires that membrane conductance (g_m) be made somehow to exceed the maximum value of g_Na or g_K. We report here that inactivation of g_Na can be destroyed fairly selectively by the action from inside the axon of the unspecific proteolytic enzymes of pronase. In many cases g_m after pronase treatment is greater than maximum g_K before treatment, making untenable the single channel hypothesis.     

Transmembrane Topologies of Ca2+-permeable Mechanosensitive Channels MCA1 and MCA2 in Arabidopsis thaliana

Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca²⁺-permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca²⁺ in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood. Arabidopsis MCA1 and MCA2 form a homotetramer and exhibit Ca²⁺-permeable MS channel activity; however, their structures have only been partially elucidated. The transmembrane topologies of these ion channels need to be determined in more detail to elucidate the underlying regulatory mechanisms. We herein determined the topologies of MCA1 and MCA2 using two independent methods, the Suc2C reporter and split-ubiquitin yeast two-hybrid methods, and found that both proteins are single-pass type I integral membrane proteins with extracellular N termini and intracellular C termini. These results imply that an EF hand-like motif, coiled-coil motif, and plac8 motif are all present in the cytoplasm. Thus, the activities of both channels can be regulated by intracellular Ca²⁺ and protein interactions.     

The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl^?) channels. The EAATs couple the transport of glutamate to the co-transport of three Na⁺ ions and one H⁺ ion into the cell, and the counter-transport of one K⁺ ion out of the cell. The EAAT Cl^? channel is activated by the binding of glutamate and Na⁺, but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, Glt_Ph, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl^? permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl^? permeation and the mechanisms that underlie their split personality.