Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

K_V11.1 voltage-gated K⁺ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.     

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.     

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.     

Gating currents in Shaker K+ channels. Implications for activation and inactivation models.

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.     

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.     

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.     

Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels I: Wild-type skeletal muscle NaV1.4

The number of voltage-gated sodium (Na_V) channels available to generate action potentials in muscles and nerves is adjusted over seconds to minutes by prior electrical activity, a process called slow inactivation (SI). The basis for SI is uncertain. Na_V channels have four domains (DI–DIV), each with a voltage sensor that moves in response to depolarizing stimulation over milliseconds to activate the channels. Here, SI of the skeletal muscle channel Na_V1.4 is induced by repetitive stimulation and is studied by recording of sodium currents, gating currents, and changes in the fluorescence of probes on each voltage sensor to assess their movements. The magnitude, voltage dependence, and time course of the onset and recovery of SI are observed to correlate with voltage-sensor movements 10,000-fold slower than those associated with activation. The behavior of each voltage sensor is unique. Development of SI over 1–160 s correlates best with slow immobilization of the sensors in DI and DII; DIII tracks the onset of SI with less fidelity. Showing linkage to the sodium conduction pathway, pore block by tetrodotoxin affects both SI and immobilization of all the sensors, with DI and DII significantly suppressed. Recovery from SI correlates best with slow restoration of mobility of the sensor in DIII. The findings suggest that voltage-sensor movements determine SI and thereby mediate Na_V channel availability.     

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.     

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.     

Cooperative interactions between R531 and acidic residues in the voltage sensing module of hERG1 channels.

HERG1 K(+) channels are critical for modulating the duration of the cardiac action potential. The role of hERG1 channels in maintaining electrical stability in the heart derives from their unusual gating properties: slow activation and fast inactivation. HERG1 channel inactivation is intrinsically voltage sensitive and is not coupled to activation in the same way as in the Shaker family of K(+) channels. We recently proposed that the S4 transmembrane domain functions as the primary voltage sensor for hERG1 activation and inactivation and that distinct regions of S4 contribute to each gating process. In this study, we tested the hypothesis that S4 rearrangements underlying activation and inactivation gating may be associated with distinct cooperative interactions between a key residue in the S4 domain (R531) and acidic residues in neighboring regions (S1 - S3 domains) of the voltage sensing module. Using double-mutant cycle analysis, we found that R531 was energetically coupled to all acidic residues in S1-S3 during activation, but was coupled only to acidic residues near the extracellular portion of S2 and S3 (D456, D460 and D509) during inactivation. We propose that hERG1 activation involves a cooperative conformational change involving the entire voltage sensing module, while inactivation may involve a more limited interaction between R531 and D456, D460 and D509.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

Interactions among DIV voltage-sensor movement,fast inactivation,and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the Na_Vβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed Na_V1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of Na_Vβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.     

Gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains

G-protein-coupled inwardly rectifying potassium channels (GIRK / Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K⁺ ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein βγ subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast gating. This result suggests that there is a cooperative movement of both of these domains to allow fast and efficient gating of GIRK channels.     

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.     

ATP-sensitive potassium channels and vascular function

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

The 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.     

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.     

Interaction of scorpion alpha-toxins with cardiac sodium channels: binding properties and enhancement of slow inactivation

The effects of the scorpion alpha-toxins Lqh II, Lqh III, and LqhalphaIT on human cardiac sodium channels (hH1), which were expressed in human embryonic kidney (HEK) 293 cells, were investigated. The toxins removed fast inactivation with EC(50) values of <2.5 nM (Lqh III), 12 nM (Lqh II), and 33 nM (LqhalphaIT). Association and dissociation rates of Lqh III were much slower than those of Lqh II and LqhalphaIT, such that Lqh III would not dissociate from the channel during a cardiac activation potential. The voltage dependence of toxin dissociation from hH1 channels was nearly the same for all toxins tested, but it was different from that found for skeletal muscle sodium channels (muI; Chen et al. 2000). These results indicate that the voltage dependence of toxin binding is a property of the channel protein. Toxin dissociation remained voltage dependent even at high voltages where activation and fast inactivation is saturated, indicating that the voltage dependence originates from other sources. Slow inactivation of hH1 and muI channels was significantly enhanced by Lqh II and Lqh III. The half-maximal voltage of steady-state slow inactivation was shifted to negative values, the voltage dependence was increased, and, in particular for hH1, slow inactivation at high voltages became more complete. This effect exceeded an expected augmentation of slow inactivation owing to the loss of fast inactivation and, therefore, shows that slow sodium channel inactivation may be directly modulated by scorpion alpha-toxins.     

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.     

Regional specificity of human ether-a'-go-go-related gene channel activation and inactivation gating

Slow activation and rapid C-type inactivation produce inward rectification of the current-voltage relationship for human ether-a'-go-go-related gene (hERG) channels. To characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the 32 amino acids (Gly(514)-Tyr(545)) that comprise the S4 domain and the flanking S3-S4 and S4-S5 linkers. Gating and ionic currents of wild-type and mutant channels were measured using cut-open oocyte Vaseline gap and two microelectrode voltage clamp techniques to determine the voltage dependence of charge movement, activation, and inactivation. Mapping the position of the charge-perturbing mutations (defined as |DeltaDeltaG| > 1.0 kcal/mol) on a three-dimensional S4 homology model revealed a spiral pattern. As expected, mutation of these residues also altered activation. However, mutation of residues in the S3-S4 and S4-S5 linkers and the C-terminal end of S4 perturbed activation (|DeltaDeltaG| > 1.0 kcal/mol) without altering charge movement, suggesting that the native residues in these regions couple S4 movement to the opening of the activation gate or stabilize the open or closed state of the channel. Finally, mutation of a distinct set of residues impacted inactivation and mapped to a single face of the S4 helix that was devoid of activation-perturbing residues. These results define regions on the S4 voltage sensor that contribute differentially to hERG activation and inactivation gating.