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 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.     

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.     

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.     

Effects of bound Ts3 on voltage dependence of sodium channel transitions to and from inactivation and energetics of its unbinding

Recently, we proposed a quantitative model to explain the molecular mechanism of action of the Tityus serrulatus Ts3 α-toxin on sodium channels. In this model, the toxin acts as a stop that prevents the segment S4 of domain IV from reaching its outermost position, thus impairing the normal fast inactivation without affecting activation. In the present work, we analyze the predictions of the proposed model with regard to the voltage-dependent transitions to and from inactivation. Our results show that the recovery from inactivation was significantly faster in Ts3-bound channels and that there was no significant voltage dependence. The transition to inactivated state from open state in Ts3-modified channels presented a small but significant voltage dependence, which may derive from an intrinsic voltage dependence of inactivation or by a short movement of IVS4 in the presence of bound Ts3. We also studied the thermodynamic parameters of the voltage-dependent displacement of Ts3 from its binding site. We have observed that the activation energy to remove the toxin is 27 kJ/mol, part of which derives from the imposed depolarizing potential and the movement of an equivalent electrical charge of 0.54 c ₀. These results support the proposed model.     

Common molecular determinants of tarantula huwentoxin-IV inhibition of Na+ channel voltage sensors in domains II and IV

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.     

Modulation of human Nav1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs

Nine different voltage-gated sodium channel isoforms are responsible for inducing and propagating action potentials in the mammalian nervous system. The Na_v1.7 channel isoform plays an important role in conducting nociceptive signals. Specific mutations of this isoform may impair gating behavior of the channel resulting in several pain syndromes. In addition to channel mutations, similar or opposite changes in gating may be produced by spider and scorpion toxins binding to different parts of the voltage-gated sodium channel. In the present study, we analyzed the effects of the α-scorpion toxin OD1 and 2 synthetic toxin analogs on the gating properties of the Na_v1.7 sodium channel. All toxins potently inhibited channel inactivation, however, both toxin analogs showed substantially increased potency by more than one order of magnitude when compared with that of wild-type OD1. The decay phase of the whole-cell Na⁺ current was substantially slower in the presence of toxins than in their absence. Single-channel recordings in the presence of the toxins revealed that Na⁺ current inactivation slowed due to prolonged flickering of the channel between open and closed states. Our findings support the voltage-sensor trapping model of α-scorpion toxin action, in which the toxin prevents a conformational change in the domain IV voltage sensor that normally leads to fast channel inactivation.     

beta-Scorpion toxin modifies gating transitions in all four voltage sensors of the sodium channel

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the beta-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge-voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge-voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of beta-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.     

Immobilizing the moving parts of voltage-gated ion channels

Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation. Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.     

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.     

The role of the putative inactivation lid in sodium channel gating current immobilization

We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III-IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge-voltage relationships were not different between wild-type hH1a and ICM-hH1a(MTSET). The time constant of the development of charge immobilization assayed at -180 mV after depolarization to 0 mV was similar to the time constant of inactivation of I(Na) at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly; i.e., became immobilized. In ICM-hH1a(MTSET), immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a(MTSET) with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a(MTSET) modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1a(MTSET) in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.     

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.     

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.     

The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.     

Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization.

The highly charged transmembrane segments in each of the four homologous domains (S4D1-S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.     

Differential phospholipid binding by site 3 and site 4 toxins. Implications for structural variability between voltage-sensitive sodium channel domains

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV.     

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.     

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.     

Role of domain 4 in sodium channel slow inactivation

Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2-20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.