Function Of Voltage-gated Ion Channels Research Articles

Voltage gated ion channel function: gating, conduction, and the role of water and protons.

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.

Contributions of Conserved Negative Surface Potentials in S2 and S3 to Voltage-Gating of Sodium Channels

The question of how S4 charges are stabilized in the hydrophobic membrane environment is of critical importance to understand the function of voltage-gated ion channels (VGICs). Besides lipids, proteinaceous interactions are thought to play an important role and, indeed, the electronegative surface potentials of aromatic side chains and the net negative charge of acidic side chains are well suited to fulfill this role. Interestingly, a single aromatic side chain in S2 and a number of acidic side chains in S2 and S3 are highly conserved among VGICs. These residues have been extensively studied in potassium channels but little is known about their role in sodium channels. Here we incorporate unnatural amino acids to probe their contributions to channel gating in the skeletal sodium channel isoform (Nav1.4). Replacing the S2 aromatic in domains I-IV with fluorinated Phe derivatives does not affect channel gating, ruling out a cation-pi interaction with S4 charges. Trp substitutions in this position lead to a significant right-shift in the GV in domain I only, highlighting not only the functional differences between Nav domains, but also sodium and potassium channels, as the latter show a dramatic left-shift in the GV when Trp is introduced at the equivalent position in S2. Furthermore, we replaced Glu side chains with the neutral isosteric Glu analog nitrohomolalanine (Nha). Our data show that neutralizing a negative charge in S2 near the extracellular end of S4 leads to a significant right-shift in the GV in domain I, while neutralizing negative charges in S2 and S3 near the intracellular end of S4 result in small or no shifts in the GV. Together, these results shed new light on our understanding of voltage-gating in sodium channels.

Hydrophobic anions potently and uncompetitively antagonize GABAA receptor function in the absence of a conventional binding site

A 'lock-and-key' binding site typically accounts for the effect of receptor antagonists. However, sulphated neurosteroids are potent non-competitive antagonists of GABA(A) receptors without a clear structure-activity relationship. To gain new insights, we tested two structurally unrelated hydrophobic anions with superficially similar properties to sulphated neurosteroids. We used voltage-clamp techniques in Xenopus oocytes and hippocampal neurons to characterize dipicrylamine (DPA) and tetraphenylborate (TPB), compounds previously used to probe membrane structure and voltage-gated ion channel function. Both DPA and TPB potently antagonized GABA(A) receptors. DPA exhibited an IC₅₀ near 60 nM at half-maximal GABA concentration and antagonism with features indistinguishable from pregnenolone sulphate antagonism, including sensitivity to a point mutation in transmembrane domain 2 of the α1 subunit. Bovine serum albumin, which scavenges free membrane-associated DPA, accelerated both capacitance offset and antagonism washout. Membrane interactions and antagonism were explored using the voltage-dependent movement of DPA between membrane leaflets. Washout of DPA antagonism was strongly voltage-dependent, paralleling DPA membrane loss, although steady-state antagonism lacked voltage dependence. At antagonist concentrations, DPA failed to affect inhibitory post-synaptic current (IPSC) amplitude or decay, but DPA accelerated pharmacologically prolonged IPSCs. Neurosteroid-like GABA(A) receptor antagonism appears to lacks a conventional binding site. These features highlight key roles of membrane interactions in antagonism. Because its membrane mobility can be controlled, DPA may be a useful probe of GABA(A) receptors, but its effects on excitability via GABA(A) receptors raise caveats for its use in monitoring neuronal activity.

The Mechanisms of Decreasing Voltage-Gated Sodium Current by Nanosecond Electric Pulses

The application of high-voltage nanosecond electric pulses (nsEP) causes the formation of nanopores in plasma membrane of mammalian cells, modifies function of voltage-gated ion channels. However, it is not known if nsEPs affect ion channels directly, or these effects are mediated in alternate method. We used the whole-cell patch-clamp technique to explore the effect of 300-nsEP on voltage-gated sodium current (INa) in NG108 neuroblastoma cells. Our data have shown that a single nsEP decreased the INa in dose-dependent manners; in parallel nsEP exposures induced a non-inactivating, voltage-sensitive inward current due to nanopore formation. At the same time, the recovery of INa after nsEP exposure took significantly longer than nanopore resealing. To check if the inflow of Na+ through nanopores was efficient enough to overcome the buffering capacity of the pipette, we measured changes of Na+ concentration in “patched” cells using the Na+-sensitive fluorescent dye (Sodium Green). These experiments showed that opening of nanopores increases the Na+ concentration in patched cells; however, the maximum increase the Na+ content, even with the most intense exposure (5.3 kV/cm), was only 2.7mM, which could unlikely cause INa inhibition. The measurement of submembrane fluorescence intensity of Sodium Green by nsEP didn’t show significant increase of submembrane Na+ concentration too. The another potential pathway of reducing the INa is increasing the intracellular Ca2+ concentration after nsEP and Ca2+ mediated inhibition of INa. Our data showed that nsEP exposure in presence of high concentration Ca2+ buffer - BAPTA (20mM) in the intracellular solution caused reduce INa in NG108 cells. Our finding suggest that decreasing of INa by nsEP was not resulted of inward Na+ leakage through nanopores, as well as Ca2+ release from intracellular stores after nsEP exposure.

Prolog to "Growing Cells Atop Microelectronic Chips: Interfacing Electrogenic Cells In Vitro With CMOS-Based Microelectrode Arrays"

To grasp the challenges and potentials faced, an overview of the fundamentals of recording electrical cell activity is in order. Many types of cells can undergo a transient electrical depolarization and repolarization, triggered by external mechanisms or by intracellular, spontaneous mechanisms. Cells that can generate electrical signals are called electrogenic cells. The most common types of electrogenic cells are brain cells (neurons) and heart cells (cardiomyocytes). The biology of electrogenic cells is complex, a symphonic interdependence of electrical, electrophysiological and biochemical processes. This requires an understanding of mechanisms underlying the functioning of voltage-gated ion channels, the generation of action potentials, and the conduction of action potentials through a cellular network.

Sialic Acids Attached to O-Glycans Modulate Voltage-gated Potassium Channel Gating

Neuronal, cardiac, and skeletal muscle action potentials are produced and conducted through the highly regulated activity of several types of voltage-gated ion channels. Voltage-gated potassium (K(v)) channels are responsible for action potential repolarization. Glycans can be attached to glycoproteins through N- and O-linkages. Previous reports described the impact of N-glycans on voltage-gated ion channel function. Here, we show that sialic acids attached through O-linkages modulate gating of K(v)2.1, K(v)4.2, and K(v)4.3. The conductance-voltage (G-V) relationships for each isoform were shifted uniquely by a depolarizing 8-16 mV under conditions of reduced sialylation. The data indicate that sialic acids modulate K(v) channel activation through apparent electrostatic mechanisms that promote channel activity. Voltage-dependent steady-state inactivation was unaffected by changes in sialylation. N-Linked sialic acids cannot be responsible for the G-V shifts because K(v)4.2 and K(v)4.3 cannot be N-glycosylated, and immunoblot analysis confirmed K(v)2.1 is not N-glycosylated. Glycosidase gel shift analysis suggested that K(v)2.1, K(v)4.2, and K(v)4.3 were O-glycosylated and sialylated. To confirm this, azide-modified sugar residues involved specifically in O-glycan and sialic acid biosynthesis were shown to incorporate into all three K(v) channel isoforms using Cu(I)-catalyzed cycloaddition chemistry. Together, the data indicate that sialic acids attached to O-glycans uniquely modulate gating of three K(v) channel isoforms that are not N-glycosylated. These data provide the first evidence that external O-glycans, with core structures distinct from N-glycans in type and number of sugar residues, can modulate K(v) channel function and thereby contribute to changes in electrical signaling that result from regulated ion channel expression and/or O-glycosylation.

Learning to Encode Timing: Mechanisms of Plasticity in the Auditory Brainstem

Mechanisms of plasticity have traditionally been ascribed to higher-order sensory processing areas such as the cortex, whereas early sensory processing centers have been considered largely hard-wired. In agreement with this view, the auditory brainstem has been viewed as a nonplastic site, important for preserving temporal information and minimizing transmission delays. However, recent groundbreaking results from animal models and human studies have revealed remarkable evidence for cellular and behavioral mechanisms for learning and memory in the auditory brainstem.

Polyunsaturated Fatty Acid Modulation of Voltage-Gated Ion Channels

Arachidonic acid (AA) was found to inhibit the function of whole-cell voltage-gated (VG) calcium currents nearly 16 years ago. There are now numerous examples demonstrating that AA and other polyunsaturated fatty acids (PUFAs) modulate the function of VG ion channels, primarily in neurons and muscle cells. We will review and extract some common features about the modulation by PUFAs of VG calcium, sodium, and potassium channels and discuss the impact of this modulation on the excitability of neurons and cardiac myocytes. We will describe the fatty acid nature of the membrane, how fatty acids become available to function as modulators of VG channels, and the physiologic importance of this type of modulation. We will review the evidence for molecular mechanisms and assess our current understanding of the structural basis for modulation. With guidance from research on the structure of fatty acid binding proteins, the role of lipids in gating mechanosensitive (MS) channels, and the impact of membrane lipid composition on membrane-embedded proteins, we will highlight some avenues for future investigations.

Four and a half LIM protein 1: a partner for KCNA5 in human atrium

Protein-protein interactions are critical for the normal membrane trafficking, localization, and function of voltage-gated ion channels. In human heart, the Shaker-related voltage-gated K(+) channel KCNA5 alpha-subunit forms the major basis of an atrial-specific, ultra-rapid delayed rectifier K(+) current, I(Kur). We sought to identify proteins that interact with KCNA5 in human atrium and investigate their role in the I(Kur) complex. Using a glutathione-S-transferase (GST)-KCNA5 C-terminal fusion protein and mass spectrometry-based methods, the scaffolding protein four and a half LIM (for Lin-11, Isl-1, and Mec3) protein 1 (FHL1) was identified as a potential protein partner for KCNA5. Immunoprecipitation experiments confirmed a physical interaction of FHL1 with the K(+) channel complex in human atrium, as well as in Chinese hamster ovary (CHO) cells transfected with both KCNA5 and FHL1. In cotransfected cells, confocal microscopy demonstrated areas of colocalization after immunolabelling both proteins. To investigate the functional effects of this interaction, K(+) currents were recorded in CHO cells transfected with KCNA5 in the absence and presence of FHL1 coexpression. With coexpression of FHL1, K(+) current density was markedly increased, compared with cells expressing KCNA5 alone. This effect was associated with a shift in the voltage dependence of K(+) channel activation to more positive potentials, consistent with findings of I(Kur) in atrial myocytes. FHL1 also increased the extent and speed of K(+) current slow inactivation, with additional effects on the voltage dependence and recovery of this process. These results support a role of FHL1 as a key molecular component in the I(Kur) complex in human atrium, where it likely regulates functional expression of KCNA5.

Effect of Local Anesthetic Lidocaine on Electrostatic Properties of a Lipid Bilayer

The influence of the local anesthetic lidocaine on electrostatic properties of a lipid membrane bilayer was studied by molecular dynamics simulations. The electrostatic dipole potential, charge densities, and orientations of the headgroup angle have been examined in the presence of different amounts of charged or uncharged forms of lidocaine. Important changes in the membrane properties caused by the presence of both forms of lidocaine are presented and discussed. Our simulations have shown that both charged and uncharged lidocaine cause almost the same increase in the electrostatic potential in the middle of the membrane, although for different reasons. The increase, ∼90 mV for 9 mol % of lidocaine and 220 mV for 28 mol % of lidocaine, is of a size that may affect the functioning of voltage-gated ion channels.

Heterogeneous Expression of NO-Activated Soluble Guanylyl Cyclase in Mammalian Heart: Implications for NO- and Redox-Mediated Indirect Versus Direct Regulation of Cardiac Ion Channel Function

Nitrogen oxides exert significant but diverse regulatory effects on cardiac myocytes. Many of these effects are due to modulation of voltage-sensitive ion channel function. The redox-status of NO-related compounds is a critical factor in determining whether indirect (cGMP-dependent) versus direct (cGMP-independent) effects are dominant. However, molecular mechanisms by which different cardiac myocyte types, and associated different ion channel types expressed within them, could achieve selectivity between NO-related indirect versus direct effects are unclear We have previously demonstrated heterogeneous expression gradients of Type III NO synthase (eNOS) and sarcolemmal superoxide dismutase (ECSOD) in ferret and human ventricle, with both enzymes being highly expressed in right ventricle and left ventricular subepicardium but markedly reduced in left ventricular subendocardium. In this study we extend this previous analysis by analyzing NO-activated soluble guanylyl cyclase (sGC) expression in the heart (ferret and human). We demonstrate that, at both tissue and single myocyte levels, sGC protein expression is heterogeneous, being high in sinoatrial node, right atrium, right ventricle and left ventricular subepicardium, but markedly reduced to absent in left atrium and left ventricular subendocardium. Thus, there is a significant overlap in expression gradients of sGC, eNOS, and ECSOD among distinct cardiac tissue and myocyte types. These gradients positively correlate with both: i) experimentally measured basal NO production levels; and ii) expression gradients of specific voltage-gated ion channels (particularly Kv1 and Kv4 channels). Our results provide the first demonstration in the heart of an expressed coupled multienzymatic system for selective regulation of indirect (sGC-dependent) versus direct (sGC-independent) NO- and redox-related modulation of voltage-gated ion channel function in different myocyte types. Our results also have functional implications for NO. / redox - related modulation of ion channels expressed in other cell types, including neurons, skeletal muscle and smooth muscle.

The S4-S5 Linker Directly Couples Voltage Sensor Movement to the Activation Gate in the Human Ether-á-go-go-related Gene (hERG) K+ Channel

A key unresolved question regarding the basic function of voltage-gated ion channels is how movement of the voltage sensor is coupled to channel opening. We previously proposed that the S4-S5 linker couples voltage sensor movement to the S6 domain in the human ether-a'-go-go-related gene (hERG) K+ channel. The recently solved crystal structure of the voltage-gated Kv1.2 channel reveals that the S4-S5 linker is the structural link between the voltage sensing and pore domains. In this study, we used chimeras constructed from hERG and ether-a'-go-go (EAG) channels to identify interactions between residues in the S4-S5 linker and S6 domain that were critical for stabilizing the channel in a closed state. To verify the spatial proximity of these regions, we introduced cysteines in the S4-S5 linker and at the C-terminal end of the S6 domain and then probed for the effect of oxidation. The D540C-L666C channel current decreased in an oxidizing environment in a state-dependent manner consistent with formation of a disulfide bond that locked the channel in a closed state. Disulfide bond formation also restricted movement of the voltage sensor, as measured by gating currents. Taken together, these data confirm that the S4-S5 linker directly couples voltage sensor movement to the activation gate. Moreover, rather than functioning simply as a mechanical lever, these findings imply that specific interactions between the S4-S5 linker and the activation gate stabilize the closed channel conformation.

The other side of the engram: experience-driven changes in neuronal intrinsic excitability.

Modern theories of memory storage have largely focused on persistent, experience-dependent changes in synaptic function such as long-term potentiation and depression. But in addition to these synaptic changes, certain learning tasks produce enduring changes in the intrinsic excitability of neurons by changing the function of voltage-gated ion channels, a change that can produce broader, even neuron-wide changes in synaptic throughput. We will consider the evidence for persistent changes in intrinsic neuronal excitability — what we will call intrinsic plasticity — that is produced by training in behaving animals and by artificial patterns of activation in brain slices and neuronal cultures. These intrinsic changes might function as part of the engram itself, or as a related phenomenon such as a trigger for the consolidation or adaptive generalization of memories.

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

Alterations in Outward K+ Currents on Removal of External Ca2+ in Human Atrial Myocytes

External divalent cations are known to play an important role in the function of voltage-gated ion channels. The purpose of this study was to examine the sensitivity of the voltage-gated K+ currents of human atrial myocytes to external Ca2+ ions. Myocytes were isolated by collagenase digestion of atrial appendages taken from patients undergoing coronary artery-bypass surgery. Currents were recorded from single isolated myocytes at 37°C using the whole-cell patch-clamp technique. With 0.5 mM external Ca2+, voltage pulses positive to −20 mV (holding potential = −60 mV) activated outward currents which very rapidly reached a peak (Ipeak) and subsequently inactivated (τ = 7.5 ± 0.7 msec at +60 mV) to a sustained level, demonstrating the contribution of both rapidly inactivating transient (Ito1) and non-inactivating sustained (Iso) outward currents. The Ito1 component of Ipeak, but not Iso, showed voltage-dependent inactivation using 100 msec prepulses (V1/2 = −35.2 ± 0.5 mV). The K+ channel blocker, 4-aminopyridine (4-AP, 2 mM), inhibited Ito1 by ∼76% and reduced Iso by ∼33%. Removal of external Ca2+ had several effects: (i) Ipeak was reduced in a manner consistent with an ∼13 mV shift to negative voltages in the voltage-dependent inactivation of Ito1. (ii) Iso was increased over the entire voltage range and this was associated with an increase in a non-inactivating 4-AP-sensitive current. (iii) In 79% cells (11/14), a slowly inactivating component was revealed such that the time-dependent inactivation was described by a double exponential time course (τ1 = 7.0 ± 0.7, τ2 = 90 ± 21 msec at +60 mV) with no effect on the fast time constant. Removal of external Ca2+ was associated with an additional component to the voltage-dependent inactivation of Ipeak and Iso (V1/2 = −20.5 ± 1.5 mV). The slowly inactivating component was seen only in the absence of external Ca2+ ions and was insensitive to 4-AP (2 mM). Experiments with Cs+-rich pipette solutions suggested that the Ca2+-sensitive currents were carried predominantly by K+ ions. External Ca2+ ions are important to voltage-gated K+ channel function in human atrial myocytes and removal of external Ca2+ ions affects Ito1 and 4-AP-sensitive Iso in distinct ways.

Structure and function of voltage-gated ion channels.

Voltage-gated ion channels are key molecules for the generation of electrical signals in cells. They are integral membrane proteins which are activated by a depolarized membrane potential resulting in a conformational change, allowing ions to permeate. Voltage-gated ion channels can either be inactivated from this open state by an additional conformational change which leads to a nonconducting state of the channel, or they may be deactivated by a repolarized membrane potential. Following the first successful cloning of voltage-gated ion channels in 1984 the combination of molecular biological and electrophysiological techniques has been very fruitful in the investigation of the structure and function of these membrane proteins. From these studies a molecular picture of the structural elements important for the activity of voltage-gated ion channels has been established. This has assisted in clarifying the molecular basis of the electrical excitability of cells.

Channel-specific effects of n-alkyl sulphate anions on three shaker-related potassium channels expressed in Xenopus oocytes.

n-Alkyl sulphate anions have been shown to reversibly affect the functioning of voltage-gated ion channels in a variety of preparations. They are suggested to exert their effects by increasing the magnitude of the negative surface potential at the external face of the membrane. Here we report the effects of n-octyl sulphate (OS-) and n-dodecyl sulphate (DDS-) on RCK1 (Kv1.1), RCK4 (Kv1.4) and Shaker B potassium channels exogenously expressed in Xenopus oocytes. Both OS- and DDS- produced a hyperpolarising shift in the activation voltage dependence of all three channels, consistent with an increased negative external surface potential. Similar kinetic changes were also observed, the kinetics of both activation and inactivation being accelerated in the presence of OS- and DDS-. However, we also found that while 10 mM OS- and 50 microM DDS- significantly increased the maximum conductance of RCK1 and Shaker B channels, 5 mM OS- and 15 microM DDS- produced a large decrease in RCK4 conductance; the possible involvement of RCK4 residue K533 in this effect is discussed. Our data indicate that n-alkyl sulphate anions can perturb ion channel function in a variety of ways and that their effects are complex and channel specific.

Structure and Function of Voltage-Gated Ion Channels

Structure and Function of Voltage-Gated Ion Channels

Structure and function of voltage-gated ion channels.

The principal subunits of the voltage-gated Na+, Ca2+ and K+ channels are members of a related gene family and are functionally autonomous in voltage-dependent activation, ion conductance and inactivation. In this article, recent work locating the structural elements that are responsible for these three basic functions of the voltage-gated ion channels is reviewed. These studies reveal strong functional analogies among the different ion channels and suggest that the striking differences in their properties arise as variations on a common structural and functional theme.