Fast and slow voltage sensor movements in HERG potassium channels

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH₂-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.     

Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels

Alpha-scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.     

Coupling interactions between voltage sensors of the sodium channel as revealed by site-specific measurements

The voltage-sensing S4 segments in the sodium channel undergo conformational rearrangements in response to changes in the electric field. However, it remains unclear whether these structures move independently or in a coordinated manner. Previously, site-directed fluorescence measurements were shown to track S4 transitions in each of the four domains. Here, using a similar technique, we provide direct evidence of coupling interactions between voltage sensors in the sodium channel. Pairwise interactions between S4s were evaluated by comparing site-specific conformational changes in the presence and absence of a gating perturbation in a distal domain. Reciprocity of effect, a fundamental property of thermodynamically coupled systems, was measured by generating converse mutants. The magnitude of a local gating perturbation induced by a remote S4 mutation depends on the coupling strength and the relative equilibrium positions of the two voltage sensors. In general, our data indicates that the movement of all four voltage sensors in the sodium channel are coupled to a varying extent. Moreover, a gating perturbation in S4-DI has the largest effect on the activation of S4-DIV and vice versa, demonstrating an energetic linkage between S4-DI and S4-DIV. This result suggests a physical mechanism by which the activation and inactivation process may be coupled in voltage-gated sodium channels. In addition, we propose that cooperative interactions between voltage sensors may be the mechanistic basis for the fast activation kinetics of the sodium channel.     

Removal of the outermost arginine in IVS4 segment of the Ca(V)3.1 channel affects amplitude but not voltage dependence of gating current

Positively charged amino acids in S4 segments of voltage-dependent Ca(V)3.1 channel form putative voltage sensor. Previously we have shown that exchange of uppermost positively charged arginine in IVS4 segment for cysteine (mutation R1717C) affected deactivation and inactivation, but not activation of macroscopic current. Now we compared gating currents from both channels. Maximal amplitude of charge movement in R1717C channel decreased but voltage-dependent characteristics of charge movement were not significantly altered. We concluded that mutation of R1717C affects the coupling between S4 activation and pore opening, but not the S4 activation itself.     

Reduced voltage dependence of inactivation in the SCN5A sodium channel mutation delF1617

Deletion of a phenylalanine at position 1617 (delF1617) in the extracellular linker between segments S3 and S4 in domain IV of the human heart Na(+) channel (hH1a) has been tentatively associated with long QT syndrome type 3 (LQT3). In a mammalian cell expression system, we compared whole cell, gating, and single-channel currents of delF1617 with those of wild-type hH1a. The half points of the peak activation-voltage curve for the two channels were similar, as were the deactivation time constants at hyperpolarized test potentials. However, delF1617 demonstrated a significant negative shift of -7 mV in the half point of the voltage-dependent Na(+) channel availability curve compared with wild type. In addition, both the time course of decay of Na(+) current (I(Na)) and two-pulse development of inactivation of delF1617 were faster at negative test potentials, whereas they tended to be slower at positive potentials compared with wild type. Mean channel open times for delF1617 were shorter at potentials <0 mV, whereas they were longer at potentials >0 mV compared with wild type. Using anthopleurin-A, a site-3 toxin that inhibits movement of segment S4 in domain IV (S4-DIV), we found that gating charge contributed by the S4-DIV in delF1617 was reduced 37% compared with wild type. We conclude that deletion of a single amino acid in the S3-S4 linker of domain IV alters the voltage dependence of fast inactivation via a reduction in the gating charge contributed by S4-DIV and can cause either a gain or loss of I(Na), depending on membrane potential.     

Gating of voltage-dependent potassium channels

Activation of voltage-dependent ion channels is primarily controlled by the applied potential difference across the membrane. For potassium channels the Drosophila Shaker channel has served as an archetype of all other potassium channels in studies of activation mechanisms. In the Shaker potassium channel much of the voltage sensitivity is conferred by the S4 transmembrane helix, which contains seven positively charged residues. During gating, the movement of these charges produces gating currents. Mutagenic and fluorescence studies indicate that four of these residues are particularly important and contribute to the majority of gating charge, R362, R365, R368 and R371. The channel is thought to dwell in several closed states prior to opening. Ionic-charge pairing with negatively charged residues in the S2 and S3 helices is thought to be important in regulating these closed states and detailed kinetic models have attempted to define the kinetics and charge of the transitions between these states. Neutral residues throughout the S4 and S5 helices are thought to control late steps in channel opening and may have important roles in modulating the stability of the open state and late closed states. In response to depolarization, the S4 helix is thought to undergo a rotational translation and this movement is also important in studies of the movement of the pore helices, S5 and S6, during opening. This review will examine residues that are important during activation as well as kinetic models that have attempted to quantitatively define the activation pathway of voltage-dependent potassium channels.     

Movement and crevices around a sodium channel S3 segment

Voltage sensing is due mainly to the movement of positively charged S4 segments through the membrane electric field during changes of membrane potential. The roles of other transmembrane segments are under study. The S3 segment of domain 4 (D4/S3) in the sodium channel Na(v)1.4 carries two negatively charged residues and has been implicated in voltage-dependent gating. We substituted cysteines into nine putative "high impact" sites along the complete length of D4/S3 and evaluated their accessibilities to extracellular sulfhydryl reagents. Only the four outermost substituted cysteines (L1433C, L1431C, G1430C, and S1427C) are accessible to extracellular sulfhydryl reagents. We measured the voltage-dependent modification rates of the two cysteines situated at the extreme ends of this accessible region, L1433C and S1427C. Independent of the charge on the sulfhydryl reagents, depolarization increases the reactivity of both of these residues. Thus, the direction of the voltage dependence is opposite to that expected for a negatively charged voltage sensor, namely an inward translational movement in response to depolarization. Intrinsic electrostatic potentials were probed by charged sulfhydryl reagents and were either negative or positive, respectively, near L1433C and S1427C. The magnitude of the electrostatic potential near S1427C decreases with depolarization, suggesting that the extracellular crevice next to it widens during depolarization. S1427C experiences 44% of the electric field, as probed by charged cysteine reagents. To further explore movements around D4/S3, we labeled cysteines with the photoactivatable cross-linking reagent benzophenone-4-carboxamidocysteine methanethiosulfonate and examined the effects of UV irradiation on channel gating. After labeling with this reagent, all accessible cysteine mutants show altered gating upon brief UV irradiation. In each case, the apparent insertion efficiency of the photoactivated benzophenone increases with depolarization, indicating voltage-dependent movement near the extracellular end of D4/S3.     

Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.     

Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac

Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1–R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.     

The N-terminus of the K channel KAT1 controls its voltage-dependent gating by altering the membrane electric field.

Functional roles of different domains (pore region, S4 segment, N-terminus) of the KAT1 potassium channel in its voltage-dependent gating were electrophysiologically studied in Xenopus oocytes. The KAT1 properties did not depend on the extracellular K+ concentration or on residue H267, equivalent to one of the residues known to be important in C-type inactivation in Shaker channels, indicating that the hyperpolarization-induced KAT1 inward currents are related to the channel activation rather than to recovery from inactivation. Neutralization of a positively charged amino acid in the S4 domain (R176S) reduced the gating charge movement, suggesting that it acts as a voltage-sensing residue in KAT1. N-terminal deletions alone (e.g., delta20-34) did not affect the gating charge movement. However, the deletions paradoxically increased the voltage sensitivity of the R176S mutant channel, but not that of the wild-type channel. We propose a simple model in which the N-terminus determines the KAT1 voltage sensitivity by contributing to the electric field sensed by the voltage sensor.     

S1–S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1–S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1–S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNa_V1.4. These show that side chains of putative countercharges in hNa_V1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I–III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.     

Extracellular Mg(2+) modulates slow gating transitions and the opening of Drosophila ether-à-Go-Go potassium channels

We have characterized the effects of prepulse hyperpolarization and extracellular Mg(2+) on the ionic and gating currents of the Drosophila ether-à-go-go K(+) channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg(2+) dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg(2+) modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg(2+) slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg(2+) modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg(2+). We have also identified mutations in the S3-S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg(2+), indicating an important role for this region in the voltage-dependent activation of eag.     

Incremental reductions of positive charge within the S4 region of a voltage-gated K+ channel result in corresponding decreases in gating charge.

The S4 region of voltage-dependent ion channels is involved in the voltage-sensing mechanism of channel activation. Previous studies in fast inactivating channels have used non-steady-state measurements and thus have not allowed the quantitative assessment of activation parameters. Using site-directed mutagenesis and voltage-clamp recordings in a noninactivating channel (RCK1), we demonstrate that stepwise reductions of positive charge within the S4 region correlate with a progressive decrease in the channel's overall gating valence. In addition to testing for electrostatic behavior of individual charged residues, our study was designed to probe nonelectrostatic influences on charge movement. We provide evidence that individual charged residues behave differentially in response to the electric field, so that purely electrostatic influences cannot fully account for the gating movement of certain charges.     

Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.     

Molecular action of lidocaine on the voltage sensors of sodium channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Q(max)) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel-gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Q(max) of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near -100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.     

Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na⁺ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K⁺ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.     

Gating currents from Kv7 channels carrying neuronal hyperexcitability mutations in the voltage-sensing domain

Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases.     

Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.     

Gating Properties of a Sodium Channel with Three Arginines Substituted by Histidines in the Central Part of Voltage Sensor S4D4

In voltage-dependent sodium channels there is some functional specialization of the four different S4 voltage sensors with regard to the gating process. Whereas the voltage sensors of domains 1 to 3 control activation gating, the movement of the voltage sensor of domain 4 (S4D4) is known to be tightly coupled to sodium channel inactivation, and there is some experimental evidence that S4D4 also participates in activation gating. To further explore its putative multifunctional role in the gating process, we changed the central part of S4D4 in rat brain IIA (rBIIA) sodium channels by the simultaneous replacement of the third (R1632), fourth (R1635) and fifth (R1638) arginine by histidine (mutation R3/4/5H). As a result, the time course of current decay observed in R3/4/5H was about three times slower, if compared to wild type (WT). On the other hand, the recovery, as well as the voltage dependence of fast inactivation, remained largely unaffected by the mutation. This suggests that at physiological pH (7.5) the effective charge of the voltage sensor was not significantly changed by the amino-acid substitutions. The well-known impact of site-3 toxin (ATX-II) on the inactivation was drastically reduced in R3/4/5H, without changing the toxin affinity of the channel. The activation kinetics of WT and R3/4/5H studied at low temperature (8 degrees C) were indistinguishable, while the inactivation time course of R3/4/5H was then clearly more slowed than in WT. These data suggest that the replacement of arginines by histidines in the central part of S4D4 clearly affects the movement of S4D4 without changing the activation kinetics.     

Ion permeation through a voltage- sensitive gating pore in brain sodium channels having voltage sensor mutations

Voltage-gated sodium channels activate in response to depolarization, but it is unknown whether the voltage-sensing arginines in their S4 segments pivot across the lipid bilayer as voltage sensor paddles or move through the protein in a gating pore. Here we report that mutation of pairs of arginine gating charges to glutamine induces cation permeation through a gating pore in domain II of the Na(V)1.2a channel. Mutation of R850 and R853 induces a K(+)-selective inward cationic current in the resting state that is blocked by activation. Remarkably, mutation of R853 and R856 causes an outward cationic current with the opposite gating polarity. These results support a model in which the IIS4 gating charges move through a narrow constriction in a gating pore in the sodium channel protein during gating. Paired substitutions of glutamine allow cation movement through the constriction when appropriately positioned by the gating movements of the S4 segment.