Modification of hERG1 channel gating by Cd2+

Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd²⁺ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd²⁺ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd²⁺ shifted the half-point (V_1/2) for the voltage dependence of the OFF gating charge–voltage (Q_OFF-V) relationship with an EC₅₀ of 171 µM; at 0.3 mM, V_1/2 was shifted by +50 mV. Cd²⁺ also induced an as of yet unrecognized small outward current (I_Cd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd²⁺ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd²⁺ in an outward direction to produce I_Cd-out. Co²⁺, Zn²⁺, and La³⁺ at concentrations that caused ∼+35-mV shifts in the Q_OFF-V relationship did not induce a current similar to I_Cd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd²⁺. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd²⁺.     

Effects of intracellular magnesium on Kv1.5 and Kv2.1 potassium channels

We characterized the effects of intracellular Mg²⁺ (Mg²⁺_i) on potassium currents mediated by the Kv1.5 and Kv2.1 channels expressed in Xenopus oocytes. Increase in Mg²⁺_i caused a voltage-dependent block of the current amplitude, apparent acceleration of the current kinetics (explained by a corresponding shift in the steady-state activation) and leftward shifts in activation and inactivation dependencies for both channels. The voltage-dependent block was more potent for Kv2.1 [dissociation constant at 0 mV, K_d(0), was ~70 mM and the electric distance of the Mg²⁺ binding site, , was 0.2] than for the Kv1.5 channel [K_d(0)~40 mM and =0.1]. Similar shifts in the voltage-dependent parameters for both channels were described by the Gouy-Chapman formalism with the negative charge density of 1 e^–/100 Ų. Additionally, Mg²⁺_i selectively reduced a non-inactivating current and increased the accumulation of inactivation of the Kv1.5, but not the Kv2.1 channel. A potential functional role of the differential effects of Mg²⁺_i on the Kv channels is discussed.     

Divalent cations as probes for structure-function relationships of cloned voltage-dependent sodium channels

1. Several cloned sodium channels were expressed in oocytes and compared with respect to their sensitivity to internal Mg²⁺ concerning the open-channel block and to external Ca²⁺ concerning open-channel block and shifts in steady-state activation. 2. A quantitative comparison between wild-type II channels and a mutant with a positive charge in the S4 segment of repeat I neutralized (K226Q) revealed no significant differences in the Mg²⁺ block. 3. The blocking effect of extracellular Ca²⁺ ions on single-channel inward currents was studied for type II, mutant K226Q and type III. A quantitative comparison showed that all three channel types differ significantly in their Ca⁺ sensitivity. 4. The influence of extracellular Ca²⁺ on the voltage dependence of steady-state activation of macroscopic currents was compared for type II and K226Q channels. Extracellular Ca⁺ increases the voltage of half-maximal activation, V_1/2, more for K226Q than for wild-type II channels; a plot of V _1/2 against [Ca] _o , is twice as steep for the mutant K226Q as for the wild-type on a logarithmic concentration scale. 5. The differential effects of extracellular Ca⁺ and intracellular Mg²⁺ on wild-type II and K226Q channels are discussed in terms of structural models of the Na⁺ channel protein.Abbreviations [Na] _i intracellular Na⁺ concentration - [Mg] intracellular Mg²⁺ concentration - [Ca] _o extracellular Ca²⁺ concentration     

Fine-tuning of Voltage Sensitivity of the Kv1.2 Potassium Channel by Interhelix Loop Dynamics

Many proteins function by changing conformation in response to ligand binding or changes in other factors in their environment. Any change in the sequence of a protein, for example during evolution, which alters the relative free energies of the different functional conformations changes the conditions under which the protein will function. Voltage-gated ion channels are membrane proteins that open and close an ion-selective pore in response to changes in transmembrane voltage. The charged S4 transmembrane helix transduces changes in transmembrane voltage into a change in protein internal energy by interacting with the rest of the channel protein through a combination of non-covalent interactions between adjacent helices and covalent interactions along the peptide backbone. However, the structural basis for the wide variation in the V₅₀ value between different voltage-gated potassium channels is not well defined. To test the role of the loop linking the S3 helix and the S4 helix in voltage sensitivity, we have constructed a set of mutants of the rat K_v1.2 channel that vary solely in the length and composition of the extracellular loop that connects S4 to S3. We evaluated the effect of these different loop substitutions on the voltage sensitivity of the channel and compared these experimental results with molecular dynamics simulations of the loop structures. Here, we show that this loop has a significant role in setting the precise V₅₀ of activation in K_v1 family channels.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K⁺ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K⁺ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd²⁺ and protons on Arabidopsis thaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd²⁺ at 300 µM depolarizes the V₅₀ of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V₅₀ by 49 mV. Regulation of KAT1 by Cd²⁺ is state dependent and consistent with Cd²⁺ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd²⁺ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd²⁺ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K⁺ channels.     

Computing transient gating charge movement of voltage-dependent ion channels

The opening of voltage-gated sodium, potassium, and calcium ion channels has a steep relationship with voltage. In response to changes in the transmembrane voltage, structural movements of an ion channel that precede channel opening generate a capacitative gating current. The net gating charge displacement due to membrane depolarization is an index of the voltage sensitivity of the ion channel activation process. Understanding the molecular basis of voltage-dependent gating of ion channels requires the measurement and computation of the gating charge, Q. We derive a simple and accurate semianalytic approach to computing the voltage dependence of transient gating charge movement (Q–V relationship) of discrete Markov state models of ion channels using matrix methods. This approach allows rapid computation of Q–V curves for finite and infinite length step depolarizations and is consistent with experimentally measured transient gating charge. This computational approach was applied to Shaker potassium channel gating, including the impact of inactivating particles on potassium channel gating currents.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Increased intracellular magnesium attenuates β-adrenergic stimulation of the cardiac CaV1.2 channel

Increases in intracellular Mg²⁺ (Mg²⁺_i), as observed in transient cardiac ischemia, decrease L-type Ca²⁺ current of mammalian ventricular myocytes (VMs). However, cardiac ischemia is associated with an increase in sympathetic tone, which could stimulate L-type Ca²⁺ current. Therefore, the effect of Mg²⁺_i on L-type Ca²⁺ current in the context of increased sympathetic tone was unclear. We tested the impact of increased Mg²⁺_i on the β-adrenergic stimulation of L-type Ca²⁺ current. Exposure of acutely dissociated adult VMs to higher Mg²⁺_i concentrations decreased isoproterenol stimulation of the L-type Ca²⁺ current from 75 ± 13% with 0.8 mM Mg²⁺_i to 20 ± 8% with 2.4 mM Mg²⁺_i. We activated this signaling cascade at different steps to determine the site or sites of Mg²⁺_i action. Exposure of VMs to increased Mg²⁺_i attenuated the stimulation of L-type Ca²⁺ current induced by activation of adenylyl cyclase with forskolin, inhibition of cyclic nucleotide phosphodiesterases with isobutylmethylxanthine, and inhibition of phosphoprotein phosphatases I and IIA with calyculin A. These experiments ruled out significant effects of Mg²⁺_i on these upstream steps in the signaling cascade and suggested that Mg²⁺_i acts directly on Ca_V1.2 channels. One possible site of action is the EF-hand in the proximal C-terminal domain, just downstream in the signaling cascade from the site of regulation of Ca_V1.2 channels by protein phosphorylation on the C terminus. Consistent with this hypothesis, Mg²⁺_i had no effect on enhancement of Ca_V1.2 channel activity by the dihydropyridine agonist (S)-BayK8644, which activates Ca_V1.2 channels by binding to a site formed by the transmembrane domains of the channel. Collectively, our results suggest that, in transient ischemia, increased Mg²⁺_i reduces stimulation of L-type Ca²⁺ current by the β-adrenergic receptor by directly acting on Ca_V1.2 channels in a cell-autonomous manner, effectively decreasing the metabolic stress imposed on VMs until blood flow can be reestablished.     

ATP-sensitive potassium channels and vascular function

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K_V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K_V1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K_V1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth,¹ that is not seen in Shaker despite significant homology between it and Kv1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K_V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K⁺ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K⁺, and an IC₅₀ of 29 mM K⁺_o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K⁺, and further support the idea that outer pore motions occur rapidly after K_V1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.     

The HOOK region of β subunits controls gating of voltage-gated Ca2+ channels by electrostatically interacting with plasma membrane

Recently, we showed that the HOOK region of the β2 subunit electrostatically interacts with the plasma membrane and regulates the current inactivation and phosphatidylinositol 4,5-bisphosphate (PIP₂) sensitivity of voltage-gated Ca²⁺ (Ca_V) 2.2 channels. Here, we report that voltage-dependent gating and current density of the Ca_V2.2 channels are also regulated by the HOOK region of the β2 subunit. The HOOK region can be divided into 3 domains: S (polyserine), A (polyacidic), and B (polybasic). We found that the A domain shifted the voltage-dependent inactivation and activation of Ca_V2.2 channels to more hyperpolarized and depolarized voltages, respectively, whereas the B domain evoked these responses in the opposite directions. In addition, the A domain decreased the current density of the Ca_V2.2 channels, while the B domain increased it. Together, our data demonstrate that the flexible HOOK region of the β2 subunit plays an important role in determining the overall Ca_V channel gating properties.     

Effects of Copper on T-Type Ca<Superscript>2+</Superscript> Channels in Mouse Spermatogenic Cells

Low voltage-activated, rapidly inactivating T-type Ca²⁺ channels are found in a variety of cells, where they regulate electrical activity and Ca²⁺ entry. In whole-cell patch-clamp recordings from mouse spermatogenic cells, trace element copper (Cu²⁺) inhibited T-type Ca²⁺ current (I _T-Ca) with IC₅₀ of 12.06 μM. Inhibition of I _T-Ca by Cu²⁺ was concentration-dependent and mildly voltage-dependent. When voltage stepped to −20 mV, Cu²⁺ (10 μM) inhibited I _T-Ca by 49.6 ± 4.1%. Inhibition of I _T-Ca by Cu²⁺ was accompanied by a shift of −2.23 mV in the voltage dependence of steady-state inactivation. Cu²⁺ upshifted the current–voltage (I-V) curve. To know the change of the gating kinetics of T-type Ca²⁺ channels, we analyzed the effect of Cu²⁺ on activation, inactivation, deactivation and reactivation of T-type Ca²⁺ channels. Since T-type Ca²⁺ channels are a key component in capacitation and the acrosome reaction, our data suggest that Cu²⁺ can affect male reproductive function through T-type Ca²⁺ channels as a preconception contraceptive material.     

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg²⁺. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K⁺-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K⁺-flux is convergent with the gating of PIP₂ because, at a saturating concentration, PIP₂ greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K⁺-flux gating.     

Voltage-dependent C-type inactivation in a constitutively open K+ channel

Most voltage-gated potassium (Kv) channels undergo C-type inactivation during sustained depolarization. The voltage dependence and other mechanistic aspects of this process are debated, and difficult to elucidate because of concomitant voltage-dependent activation. Here, we demonstrate that MinK-KCNQ1 (I_Ks) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-I_Ks inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-I_Ks inactivation and recovery from inactivation were voltage-dependent with physiologic intracellular ion concentrations, and in the absence or presence of external K⁺, with an estimated gating charge, z_i, of ∼1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-I_Ks) exhibited constitutive C-type inactivation. The results suggest that F340W-I_Ks channels exhibit voltage-dependent C-type inactivation involving S4, without the necessity for voltage-dependent opening, allosteric coupling to voltage-dependent S6 transitions occurring during channel opening, or voltage-dependent changes in ion occupancy. The data also identify F340 as a critical hub for KCNQ1 gating processes and their modulation by MinK, and present a unique system for further mechanistic studies of the role of coupling of C-type inactivation to S4 movement, without contamination from voltage-dependent activation.     

Transfer of ion binding site from ether-à-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening

In ether-à-go-go (eag) K⁺ channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are ∼4 Å apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg²⁺. In I287D+F324D, Mg²⁺ significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg²⁺ binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg²⁺ shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg²⁺ binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.     

Activation of Slo2.1 channels by niflumic acid

Slo2.1 channels conduct an outwardly rectifying K⁺ current when activated by high [Na⁺]_i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na⁺. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC₅₀ = 2.1 mM) or flufenamic acid (EC₅₀ = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K⁺]_e, the conductance–voltage (G-V) relationship had a V_1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V_1/2 to more negative potentials (EC₅₀ = 2.1 mM) and increased the minimum value of G/G_max (EC₅₀ = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V_1/2 of the G-V relationship was shifted to more positive potentials when [K⁺]_e was elevated from 1 to 300 mM (EC₅₀ = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K⁺]_e dependency (EC₅₀ = 23.5 mM). Conductance was also [Na⁺]_e dependent. Outward currents were reduced when Na⁺ was replaced with choline or mannitol, but unaffected by substitution with Rb⁺ or Li⁺. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K⁺]_e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K⁺]_e and [Na⁺]_e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na⁺.     

Expression of a genomic clone encoding a brain potassium channel in mammalian cells using lipofection

A genomic clone encoding a mouse brain K⁺ channel (MBK1) was isolated, characterized and expressed in COS cells using the lipofection technique. Transfected COS cells expressed voltage-dependent K⁺ currents that activated within 20 ms at 0 mV and showed less than 10% inactivation during 250 ms depolarizing pulses at 60 mV. Expressed K⁺ currents were reversibly blocked by 4-aminopyridine and tetraethylammonium, and were moderately sensitive to dendrotoxin, but insensitive to charybdotoxin. Thus MBK1, expressed transiently in a mammalian cell line, exhibits features characteristic of non-inactivating K⁺ channels with a conspicuous insensitivity to charybdotoxin. Lipofection is, therefore, a valuable strategy for expression of channel proteins in mammalian cells.Abbreviations 4-AP 4 aminopyridine - TEA tetraethylammonium - CTX charybdotoxin - DTX dendrotoxin - V applied voltage - V_rev reversal potential - I current - G conductance - MBK1 mouse brain potassium channel 1 - TES N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid Correspondence to: M. Montal.     

Voltage sensor of ion channels and enzymes

Placed in the cell membrane (a two-dimensional environment), ion channels and enzymes are able to sense voltage. How these proteins are able to detect the difference in the voltage across membranes has attracted much attention, and at times, heated debate during the last few years. Sodium, Ca²⁺ and K⁺ voltage-dependent channels have a conserved positively charged transmembrane (S4) segment that moves in response to changes in membrane voltage. In voltage-dependent channels, S4 forms part of a domain that crystallizes as a well-defined structure consisting of the first four transmembrane (S1–S4) segments of the channel-forming protein, which is defined as the voltage sensor domain (VSD). The VSD is tied to a pore domain and VSD movements are allosterically coupled to the pore opening to various degrees, depending on the type of channel. How many charges are moved during channel activation, how much they move, and which are the molecular determinants that mediate the electromechanical coupling between the VSD and the pore domains are some of the questions that we discuss here. The VSD can function, however, as a bona fide proton channel itself, and, furthermore, the VSD can also be a functional part of a voltage-dependent phosphatase.     

Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

Summary The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances ofNecturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (V _mc) results in a marked decrease in apical membrane fractional resistance (fR _a), whereas hyperpolarization ofV _mc results in either no change infR _a or a small increase, and b) the voltage-dependent changes infR _a are essentially complete within 500 msec. Exposure of the tissue to 5mm TEA⁺ on the mucosal side caused no significant change in baselineV _mc (–69±2 mV) and yet virtually abolished the voltage dependence offR _a. A possible interpretation of these results is that two types of K⁺ channels exist in the apical membrane, with different voltage dependencies and TEA⁺ sensitivities. Acidification or Ba²⁺ addition to the mucosal solution also reduced the voltage-dependent changes infR _a. The time courses of the changes infR _a and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (±200 A/cm²). No secondary change infR _a was observed with mucosa-to-serosa currents, but a slow TEA⁺-sensitive decrease infR _a (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (<500 msec) voltage-dependent decrease infR _a is due to a fall in apical membrane resistance. The later decrease infR _a is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimatingfR _a than the current-induced effects on the lateral intercellular spaces. In principle, TEA⁺ can be used to prevent the nonlinear behavior ofR _a during measurements of the voltage divider or membrane resistance ratio.