Gating Charge Movement Research Articles

Biophysical reviews top five: voltage-dependent charge movement in nerve and muscle.

The discovery of gating currents and asymmetric charge movement in the early 1970s represented a remarkable leap forward in our understanding of the biophysical basis of voltage-dependent events that underlie electrical signalling that is vital for nerve and muscle function. Gating currents and charge movement reflect a fundamental process in which charged amino acid residues in an ion channel protein move in response to a change in the membrane electrical field and therefore activate the specific voltage-dependent response of that protein. The detection of gating currents and asymmetric charge movement over the past 50 years has been pivotal in unraveling the multiple molecular and intra-molecular processes which lead to action potentials in excitable tissues and excitation-contraction (EC) coupling in skeletal muscle. The recording of gating currents and asymmetric charge movement remains an essential component of investigations into the basic molecular mechanisms of neuronal conduction and muscle contraction.

Structural basis for severe pain caused by mutations in the voltage sensors of sodium channel NaV1.7.

Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.

An activator of voltage-gated K+ channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.

Structural basis for severe pain caused by mutations in voltage gated sodium channels Nav1.7.

Gain-of-function mutations in voltage-gated sodium channel NaV1.7 cause severe inherited pain syndromes, including Inherited Erythromelalgia (IEM). We introduced IEM mutations into the bacterial sodium channel NaVAb and confirmed the negative shift in the voltage dependence of activation that recapitulates the gain-of-function of these mutants. Unexpectedly, our structures of NaVAb IEM mutations reveal two complementary pathogenic mechanisms at the atomic level. Mutations in the voltage sensor domain facilitate the outward movement of S4 gating charges by widening the pathway for gating charge movement and modifying hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids. In contrast, IEM mutations in the S4-S5 linker stabilize the activated state by forming new hydrogen bonds with residues in the pore domain, including a conserved asparagine near the activation gate. These results provide key structural insights into the dual mechanisms by which these IEM mutations cause hyperexcitability and severe pain in this debilitating disease.

Tuning the temperature dependence of Shaker potassium channel with mutations at S4-S5 linker.

We investigated the effects of temperature on mutations that uncouple the voltage sensor (VSD) from the pore (PD) in the Shaker-IR potassium channel using temperature step (Tstep). We utilized a setup that achieves fast temperature changes (millisecond) and allows the recording of the cell membrane temperature with sub-millisecond resolution (https://doi.org/10.1101/2022.07.11.499616). We used the mutants V369I, I372L, S376T (ILT), in the N-terminus of the S4-S5, that produces a split in the components of the gating charge movement, and I384N, near the S4-S5 C-terminus, that uncouples the VSD charge movement from the PD. From a bath temperature of 17 ºC, we used a Tstep of around ∼9 ºC with rising phase of 1.5 ms. In ILT, applying a Tstep, after the gating currents have subsided, promotes a right shift in the first component of gating charge and a left shift of the second component (the last concerted step associated with PD opening). Consistently, Tstep applied after the onset of a depolarizing voltage pulse produces a ∼5 mV left shift in the conductance-voltage curve (G-V). The entropy, calculated from G-V, changes from 24 for WT to −42 J/K for ILT. In I384N, applying a Tstep reduces the ionic current indicating the closure of the pore. We hypothesize that the temperature dependence of the ILT G-V arises from the changes in the last step of the charge movement. On the other hand, since the VSD and PD are loosely coupled in I384N, the increase in temperature is likely to further decrease the VSD-PD coupling, thus decreasing the measured conductance. (Support: NIH: GM030376, Fondecyt: 1190203)

The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle.

Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+ is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Simultaneous imaging of Ca2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca2+ transients when APs peaked at potentials more negative than -30mV. An AP property that closely correlated with failure of the Ca2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below -21mV. We hypothesize propagation of APs and generation of Ca2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width (and thus AP integral) whereas Ca2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.

Tracking the movement of discrete gating charges in a voltage-gated potassium channel.

Positively charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. We optically tracked the movement of the two most extracellular charged residues (R1 and R2) in the Shaker potassium channel voltage sensor using a fluorescent positively charged bimane derivative (qBBr) that is strongly quenched by tryptophan. By individually mutating residues to tryptophan within the putative pathway of gating charges, we observed that the charge motion during activation is a rotation and a tilted translation that differs between R1 and R2. Tryptophan-induced quenching of qBBr also indicates that a crucial residue of the hydrophobic plug is linked to the Cole-Moore shift through its interaction with R1. Finally, we show that this approach extends to additional voltage-sensing membrane proteins using the Ciona intestinalis voltage-sensitive phosphatase (CiVSP).

Gating current noise produced by Brownian models of a voltage sensor

The activation of voltage-dependent ion channels is associated with the movement of gating charges, which give rise to gating currents. Although gating currents from a single channel are too small to be detected, analysis of the fluctuations of macroscopic gating currents from a population of channels allows a good guess of their magnitude. The analysis of experimental gating current fluctuations, when interpreted in terms of a rate model of channel activation and assuming sufficiently high bandwidth, is in accordance with the presence of a main step along the activation pathway carrying a charge of 2.3–2.4 e0. To give a physical interpretation to these results and to relate them to the known atomic structure of the voltage sensor domain, we used a Brownian model of voltage-dependent gating based on atomic detail structure, that follows the laws of electrodynamics. The model predicts gating currents and gating current fluctuations essentially similar to those experimentally observed. The detailed study of the model output, also performed by making several simplifications aimed at understanding the basic dependencies of the gating current fluctuations, suggests that in real channels the voltage sensor moves along a sequence of intermediate states separated by relatively low (<5 kT) energy barriers. As a consequence, crossings of successive gating charges through the gating pore become very frequent, and the corresponding current shots are often seen to overlap because of the relatively high filtering. Notably, this limited bandwidth effect is at the origin of the relatively high single-step charge experimentally detected.

Multiscale modeling shows that dielectric differences make NaV channels faster than KV channels.

The generation of action potentials in excitable cells requires different activation kinetics of voltage-gated Na (NaV) and K (KV) channels. NaV channels activate much faster and allow the initial Na+ influx that generates the depolarizing phase and propagates the signal. Recent experimental results suggest that the molecular basis for this kinetic difference is an amino acid side chain located in the gating pore of the voltage sensor domain, which is a highly conserved isoleucine in KV channels but an equally highly conserved threonine in NaV channels. Mutagenesis suggests that the hydrophobicity of this side chain in Shaker KV channels regulates the energetic barrier that gating charges cross as they move through the gating pore and control the rate of channel opening. We use a multiscale modeling approach to test this hypothesis. We use high-resolution molecular dynamics to study the effect of the mutation on polarization charge within the gating pore. We then incorporate these results in a lower-resolution model of voltage gating to predict the effect of the mutation on the movement of gating charges. The predictions of our hierarchical model are fully consistent with the tested hypothesis, thus suggesting that the faster activation kinetics of NaV channels comes from a stronger dielectric polarization by threonine (NaV channel) produced as the first gating charge enters the gating pore compared with isoleucine (KV channel).

α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav 1.2.

The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.

Equivalent L-type channel (CaV1.1) function in adult female and male mouse skeletal muscle fibers

Loss of total muscle force during aging has both atrophic and non-atrophic components. The former deficit is a direct consequence of reduced muscle mass while the latter has been attributed to a depression of excitation-contraction (EC) coupling. It is well established that age-onset reductions in sex hormone production regulate the atrophic component in both males and females. However, it is unknown whether the non-atrophic component is influenced by sex hormones. Since the non-atrophic component has been linked mechanistically to reduced expression of the skeletal muscle L-type Ca2+ channel (CaV1.1), we recorded L-type Ca2+ currents, gating charge movements and depolarization-induced changes in myoplasmic Ca2+ from flexor digitorum brevis (FDB) fibers of naïve and gonadectomized mice of both sexes. Our first set of experiments sought to identify any basal differences in EC coupling or L-type Ca2+ flux between the sexes; no detectable differences in any of the aforementioned parameters were observed between FDB harvested from either naïve males or females. In the latter segments of the study, ovariectomy (OVX) and orchiectomy (ORX) models were used to assess the possible influence of sex hormones on EC coupling and/or L-type Ca2+ flux. In these experiments, FDB fibers harvested from OVX and ORX mice both showed no differences in L-type Ca2+ current, gating charge movement or depolarization-induced changes in Ca2+ release from the sarcoplasmic reticulum. Taken together, our results indicate L-type Ca2+ channel function and EC coupling are: 1) equivalent between the sexes, and 2) not significantly regulated by sex hormones. Since recent NIH review guidelines mandate the consideration of sex differences as a criterion for review, our work indicates the suitability of either sex for the study of the fundamental mechanisms of EC coupling. Thus, our findings may accelerate the research process by conserving animals, labor and financial resources.

Voltage-Sensing Residues in the Voltage Sensor of the BK Channel

Voltage- and Ca2+ -activated K+ (BK) channels are modular proteins with allosteric gating. BK channels damp excitatory stimuli mediated by voltage-dependent Ca2+ channels, and they are involved in modulating physiological processes such as muscle contraction and neuronal excitability. To identify the voltage-sensing residues that contribute to the gating charge movement is crucial to understand the mechanism of BK channel voltage-dependent activation at the molecular level. Here, to determine which charged amino acids in transmembrane segments S1-S4 contribute to the voltage sensitivity of the BK channel, we have measured gating currents in BK channels containing neutralization mutations. Only neutralization of basic residues contained in S4 can decrease the voltage dependence of the BK channel voltage sensor. Neutralization of other charged residues in the voltage sensor domain changed the equilibrium of the active-resting equilibrium, revealing a modulating role in the free energy necessary to activate the voltage sensor. The results reveal that in BK channels the S4 segment is solely responsible for the gating charge movement. Supported by 1150273 to R.L. The Centro Interdisciplinario de Neurociencia de Valparaíso is a Millennium Institute (P029-022-F).

Structural Basis of Nav1.7 Inhibition by the Tarantula Toxin Protoxin-II

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Neurotoxins have provided important insights into Nav channels, but the structural basis for toxin modulation of channel gating is not well-defined. Protoxin-II (ProTx2) is an inhibitor cystine-knot peptide and a selective antagonist of the human Nav1.7 channel. Here, using X-ray crystallography and cryo-electron microscopy, we have captured ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct structural features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule of VSD2 to antagonize S4 gating charge movement through an electrostatic mechanism. ProTx2 engages a small interaction surface and few direct contacts on VSD2, allowing the S4 helix to be visualized in both activated and deactivated states. Our results illuminate pharmacological concepts employed by membrane-partitioning peptide toxins, clarify the structural basis of electromechanical coupling in voltage-gated ion channels, and provide new templates to design selective Nav channel antagonists.

Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.

Modulation of ClC-3 gating and proton/anion exchange by internal and external protons and the anion selectivity filter.

The ClC-3 2Cl- /1H+ exchanger modulates endosome pH and Cl- concentration. We investigated the relationships between ClC-3-mediated ion transport (steady-state transport current, ISS ), gating charge (Q) and cytoplasmic alkalization. ClC-3 transport is functionally unidirectional. ClC-5 and ClC-3 display indistinguishable exchange ratios, but ClC-3 cycling is less "efficient", as reflected by a large Q/ISS . An M531A mutation predicted to increase water-wire stability and cytoplasmic proton supply improves efficiency. Protonation (pH 5.0) of the outer glutamate gate (Gluext ; E224) reduces Q, inhibits transport, and weakens coupling. Removal of the central tyrosine anion gate (Y572S) greatly increases uncoupled anion current. Tyrosine -OH removal (Y572F) alters anion selectivity and impairs coupling. E224 and Y572 act as anion barriers, and contribute to gating. The Y572 side chain and -OH regulate Q movement kinetics and voltage dependence. E224 and Y572 interact to create a "closed" inner gate conformation that maintains coupling during cycling. We utilized plasma membrane-localized ClC-3 to investigate relationships between steady-state transport current (ISS ), gating charge (Q) movement, and cytoplasmic alkalization rate. ClC-3 exhibited lower transport efficiency than ClC-5, as reflected by a larger Q/ISS ratio, but an indistinguishable Cl- /H+ coupling ratio. External SCN- reduced H+ transport rate and uncoupled anion/H+ exchange by 80-90%. Removal of the external gating glutamate ("Gluext ") (E224A mutation) reduced Q and abolished H+ transport. We hypothesized that Methionine 531 (M531) impedes "water wire" H+ transfer from the cytoplasm to E224. Accordingly, an M531A mutation decreased the Q/ISS ratio by 50% and enhanced H+ transport. External protons (pH5.0) inhibited ISS and markedly reduced Q while shifting the Q-voltage (V) relationship positively. The Cl- /H+ coupling ratio at pH 5.0 was significantly increased, consistent with externally protonated Gluext adopting an outward/open position. Internal "anion gate" removal (Y572S) dramatically increased ISS and impaired coupling, without slowing H+ transport rate. Loss of both gates (Y572S/E224A) resulted in a large "open pore" conductance. Y572F (removing only the phenolic hydroxide) and Y572S shortened Q duration similarly, resulting in faster Q kinetics at all voltages. These data reveal a complex relationship between Q and ion transport. Q/ISS must be assessed together with coupling ratio to properly interpret efficiency. Coupling and transport rate are influenced by the anion, internal proton supply and external protons. Y572 regulates H+ coupling as well as anion selectivity, and interacts directly with E224. Disruption of this "closed gate" conformation by internal protons may represent a critical step in the ClC-3 transport cycle.

Structural basis for gating pore current in periodic paralysis.

Potassium-sensitive Hypokalemic and Normokalemic Periodic Paralysis (HypoPP, NormoPP) are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness1,2. They are caused by mutations in one gating charge in an S4 transmembrane segment in the voltage sensor (VS) of voltage-gated sodium channel Nav1.4 or calcium channel Cav1.11,2. Mutations of the outermost arginine gating charges (R1 and R2) cause HypoPP1,2 by creating a pathogenic gating pore in the VS through which cations leak in the resting state3,4. Mutations of the third arginine gating charge (R3) cause NormoPP5 owing to cationic leak in activated/inactivated states6. Here we present high-resolution structures of these pathogenic gating pores in the model bacterial sodium channel NaVAb7,8. Mutation of R2 in NaVAb gives gating pore current in resting states, whereas mutation of R3 gives gating pore current in activated/inactivated states. Mutations R2G and R3G have no effect on backbone structures of VS, but create aqueous space near the hydrophobic constriction site (HCS) that controls gating charge movement through VS. The R3G mutation extends the extracellular aqueous cleft completely through the activated VS. Although the R2G mutation does not create a continuous aqueous pathway in the activated state, molecular modeling of the resting state reveals a complete water-accessible pathway. Crystal structures of NaVAb/R2G in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na+ permeation through the mutant gating pore in concert with conformational fluctuations of gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from these episodic diseases.

Properties of the Voltage-Gated Proton Channel Gating Currents

The voltage-gated proton channel (Hv1) is a dimer of subunits containing four transmembrane domains (S1-S4) which play the double role of harboring the voltage-sensing charges and forming the proton conduction pathway. Some biophysical properties of Hv1, as the coupling between voltage-sensing and permeation pathway opening, the pH dependence of gating, the cooperativity between each subunit in the dimer, and the steps of charge movement during activation and deactivation can be better understood by measuring and characterizing the Hv1 gating currents. However, to measure gating currents, ionic currents needs to be eliminated or greatly reduced, a feat that is particularly challenging for Hv1 for which there is no effective blocker and proton concentration cannot be reduced more than 10 fold. Here, we expressed the low-conducting N264R Hv1 mutant from Ciona intestinalis in Xenopus laevis oocytes and succeeded in recording gating currents membrane patches with both, the monomeric and dimeric channels. In agreement with previous fluorescence measurements (Qiu et al., Neuron 77:288, 2013), the analysis of the gating currents kinetics and charge movement revealed that the ON component has a double exponential decay. Additionally, changes in pH modulate Hv1 gating currents kinetics and Q-V curves. This suggests that the pH sensors in Hv1 are located in the voltage-sensing domain. Finally, in our experimental conditions we observed charge immobilization after activation, which could be an intrinsic characteristic of Hv1 voltage-sensing domain. Supported by CONICYT-PFCHA/Doctorado Nacional/2017-21170395 to E.C., Fondecyt grants ACT 1104, Fondecyt 1160261 to C.G. and 1150273 to R.L.

Biophysical Characterization of Genetically Encoded Voltage Sensor ASAP1: Dynamic Range Improvement

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1. Gating charge movement and fluorescence kinetics track closely with each other, although ASAP1 gating currents are significantly faster than those of Ciona intestinalis voltage-sensitive phosphatase. Altering the residue before the first gating charge removes a split in the ASAP1 QV curve, but preserves the accelerated kinetics that allow for the faithful tracking of action potentials in neurons.

The Fast Component of hERG Gating Charge: An Interaction between D411 in the S1 and S4 Residues

Kv11.1 (hERG) is a voltage-gated potassium channel that shows very slow ionic current activation kinetics, and an unusual underlying biphasic gating charge movement with fast and slow components that differ greatly in time course. The structural basis and role of the fast component of gating charge (Qfast) is unclear, and its relationship to the slow activation of hERG channels is not understood. In this study we have used the cut-open oocyte voltage-clamp technique to investigate the relationship of fast gating charge movement-to-residue interactions between D411 at the bottom of the S1, and lower S4 domain charged and uncharged residues. Neutralization of D411 or K538 and V535A prevented Qfast and greatly accelerated overall charge movement. Voltage-clamp fluorometry showed a loss of a fast component of S4 fluorescence in D411N, V535A, and K538Q upon depolarization, whereas [2-(trimethyl ammonium) ethyl] methanethiosulfonate chloride modification of I521C in the outer S4 was enhanced at more negative potentials and at earlier times in these same mutants. A functional interaction between these regions during activation was suggested by ΔΔGo values >4.2 kJ/mol obtained from double mutant cycle analysis. The data indicate that interactions of S1 residue D411 with lower S4 residues stabilizes early closed states of the channel, and that disruption of these interactions results in both faster rates of activation gating and an elimination of the fast component of gating charge movement and of fluorescence. We propose that the Qfast charge movement during activation accompanies transitions through early closed states of the hERG activation pathway, and that the weak voltage dependence of these transitions limits the overall activation rate of hERG channels. Disruption of the D411-S4 interactions destabilizes these early closed states, leaving hERG channels able to activate at a rate similar to conventional potassium channels.

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation.

E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation.