Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel

KcsA is a proton-activated K⁺ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K⁺, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K⁺ selectivity, and permitted Na⁺ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.     

Structures of Gating Intermediates in a K+ channel

Regulation of ion conduction through the pore of a K⁺ channel takes place through the coordinated action of the activation gate at the bundle crossing of the inner helices and the inactivation gate located at the selectivity filter. The mechanism of allosteric coupling of these gates is of key interest. Here we report new insights into this allosteric coupling mechanism from studies on a W67F mutant of the KcsA channel. W67 is in the pore helix and is highly conserved in K⁺ channels. The KcsA W67F channel shows severely reduced inactivation and an enhanced rate of activation. We use continuous wave EPR spectroscopy to establish that the KcsA W67F channel shows an altered pH dependence of activation. Structural studies on the W67F channel provide the structures of two intermediate states: a pre- open state and a pre-inactivated state of the KcsA channel. These structures highlight key nodes in the allosteric pathway. The structure of the KcsA W67F channel with the activation gate open shows altered ion occupancy at the second ion binding site (S2) in the selectivity filter. This finding in combination with previous studies strongly support a requirement for ion occupancy at the S2 site for the channel to inactivate.     

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K⁺ channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K⁺ gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells

Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K⁺ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K⁺ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K⁺ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K⁺ (K_in) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K⁺ currents. However, KZM2 can interact with KZM3 forming heteromeric K_in channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K⁺ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the K_in channels in maize guard cells. KZM2 and KZM3 may form heteromeric K_in channel and control stomatal opening in maize.     

Protein Rearrangements Underlying Slow Inactivation of the Shaker K+ Channel

Voltage-dependent ion channels transduce changes in the membrane electric field into protein rearrangements that gate their transmembrane ion permeation pathways. While certain molecular elements of the voltage sensor and gates have been identified, little is known about either the nature of their conformational rearrangements or about how the voltage sensor is coupled to the gates. We used voltage clamp fluorometry to examine the voltage sensor (S4) and pore region (P-region) protein motions that underlie the slow inactivation of the Shaker K⁺ channel. Fluorescent probes in both the P-region and S4 changed emission intensity in parallel with the onset and recovery of slow inactivation, indicative of local protein rearrangements in this gating process. Two sequential rearrangements were observed, with channels first entering the P-type, and then the C-type inactivated state. These forms of inactivation appear to be mediated by a single gate, with P-type inactivation closing the gate and C-type inactivation stabilizing the gate''s closed conformation. Such a stabilization was due, at least in part, to a slow rearrangement around S4 that stabilizes S4 in its activated transmembrane position. The fluorescence reports of S4 and P-region fluorophore are consistent with an increased interaction of the voltage sensor and inactivation gate upon gate closure, offering insight into how the voltage-sensing apparatus is coupled to a channel gate.     

Complex interactions among residues within pore region determine the K+ dependence of a KAT1‐type potassium channel AmKAT1

KAT1‐type channels mediate K⁺ influx into guard cells that enables stomatal opening. In this study, a KAT1‐type channel AmKAT1 was cloned from the xerophyte Ammopiptanthus mongolicus. In contrast to most KAT1‐type channels, its activation is strongly dependent on external K⁺ concentration, so it can be used as a model to explore the mechanism for the K⁺‐dependent gating of KAT1‐type channels. Domain swapping between AmKAT1 and KAT1 reveals that the S5–pore–S6 region controls the K⁺ dependence of AmKAT1, and residue substitutions show that multiple residues within the S5–Pore linker and Pore are involved in its K⁺‐dependent gating. Importantly, complex interactions occur among these residues, and it is these interactions that determine its K⁺ dependence. Finally, we analyzed the potential mechanism for the K⁺ dependence of AmKAT1, which could originate from the requirement of K⁺ occupancy in the selectivity filter to maintain its conductive conformation. These results provide new insights into the molecular basis of the K⁺‐dependent gating of KAT1‐type channels.     

Calcium Channels: Unanswered Questions

Despite decades of intensive research, many questions remain unresolved regarding the structure and function of voltage-dependent calcium channels. This review considers some of those questions: Where is the activation gate? Where are the inactivation gates? How are the voltage sensors coupled to the gates? Are bacterial K⁺ channels good models for the Ca²⁺ channel pore? Are protein–protein interactions fundamental to Ca²⁺ channel function? Do voltage-dependent Ca²⁺ channels regulate basal intracellular Ca²⁺? Are N and P/Q channels specialized for fast neurotransmitter release?     

Functional role of the N-terminus of Na,K-ATPase α-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na⁺,K⁺-ATPase α-subunit shows some homology to that of Shaker-B K⁺ channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na⁺,K⁺-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the α-subunit appears to act like an inactivation gate and performs a critical step in the Na⁺,K⁺-ATPase pumping function.     

Molecular Analysis of Potential Hinge Residues in the Inactivation Gate of Brain Type IIA Na+ Channels

During inactivation of Na⁺ channels, the intracellular loop connecting domains III and IV is thought to fold into the channel protein and occlude the pore through interaction of the hydrophobic motif isoleucine-phenylalanine-methionine (IFM) with a receptor site. We have searched for amino acid residues flanking the IFM motif which may contribute to formation of molecular hinges that allow this motion of the inactivation gate. Site-directed mutagenesis of proline and glycine residues, which often are components of molecular hinges in proteins, revealed that G1484, G1485, P1512, P1514, and P1516 are required for normal fast inactivation. Mutations of these residues slow the time course of macroscopic inactivation. Single channel analysis of mutations G1484A, G1485A, and P1512A showed that the slowing of macroscopic inactivation is produced by increases in open duration and latency to first opening. These mutant channels also show a higher probability of entering a slow gating mode in which their inactivation is further impaired. The effects on gating transitions in the pathway to open Na⁺ channels indicate conformational coupling of activation to transitions in the inactivation gate. The results are consistent with the hypothesis that these glycine and proline residues contribute to hinge regions which allow movement of the inactivation gate during the inactivation process of Na⁺ channels.     

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.     

Biophysical mechanism of spike threshold dependence on the rate of rise of the membrane potential by sodium channel inactivation or subthreshold axonal potassium current

Spike threshold filters incoming inputs and thus gates activity flow through neuronal networks. Threshold is variable, and in many types of neurons there is a relationship between the threshold voltage and the rate of rise of the membrane potential (dVm/dt) leading to the spike. In primary sensory cortex this relationship enhances the sensitivity of neurons to a particular stimulus feature. While Na⁺ channel inactivation may contribute to this relationship, recent evidence indicates that K⁺ currents located in the spike initiation zone are crucial. Here we used a simple Hodgkin-Huxley biophysical model to systematically investigate the role of K⁺ and Na⁺ current parameters (activation voltages and kinetics) in regulating spike threshold as a function of dVm/dt. Threshold was determined empirically and not estimated from the shape of the Vm prior to a spike. This allowed us to investigate intrinsic currents and values of gating variables at the precise voltage threshold. We found that Na⁺ inactivation is sufficient to produce the relationship provided it occurs at hyperpolarized voltages combined with slow kinetics. Alternatively, hyperpolarization of the K⁺ current activation voltage, even in the absence of Na⁺ inactivation, is also sufficient to produce the relationship. This hyperpolarized shift of K⁺ activation allows an outward current prior to spike initiation to antagonize the Na⁺ inward current such that it becomes self-sustaining at a more depolarized voltage. Our simulations demonstrate parameter constraints on Na⁺ inactivation and the biophysical mechanism by which an outward current regulates spike threshold as a function of dVm/dt.     

Isolation and characterization of SsmTx‐I,a Specific Kv2.1 blocker from the venom of the centipede Scolopendra Subspinipes Mutilans L. Koch

Scolopendra subspinipes mutilans, also known as Chinese red‐headed centipede, is a venomous centipede from East Asia and Australasia. Venom from this animal has not been researched as thoroughly as venom from snakes, snails, scorpions, and spiders. In this study, we isolated and characterized SsmTx‐I, a novel neurotoxin from the venom of S. subspinipes mutilans. SsmTx‐I contains 36 residues with four cysteines forming two disulfide bonds. It had low sequence similarity (<10%) with other identified peptide toxins. By whole‐cell recording, SsmTx‐I significantly blocked voltage‐gated K⁺ channels in dorsal root ganglion neurons with an IC₅₀ value of 200 nM, but it had no effect on voltage‐gated Na⁺ channels. Among the nine K⁺ channel subtypes expressed in human embryonic kidney 293 cells, SsmTx‐I selectively blocked the Kv2.1 current with an IC₅₀ value of 41.7 nM, but it had little effect on currents mediated by other K⁺ channel subtypes. Blockage of Kv2.1 by SsmTx‐I was not associated with significant alteration of steady‐state activation, suggesting that SsmTx‐I might act as a simple inhibitor or channel blocker rather than a gating modifier. Our study reported a specific Kv2.1‐blocker from centipede venom and provided a basis for future investigations of SsmTx‐I, for example on structure–function relationships, mechanism of action, and pharmacological potential. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K⁺ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K⁺ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.     

Destruction of Sodium Conductance Inactivation in Squid Axons Perfused with Pronase

We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage-clamped squid axons. Internal perfusion of the axons with Pronase rather selectively destroys inactivation of the Na conductance (g_Na). At the level of a single channel, Pronase probably acts in an all-or-none manner: each channel inactivates normally until its inactivation gate is destroyed, and then it no longer inactivates. Pronase reduces _Na, possibly by destroying some of the channels, but after removal of its inactivation gate a Na channel seems no longer vulnerable to Pronase. The turn-off kinetics and the voltage dependence of the Na channel activation gates are not affected by Pronase, and it is probable that the enzyme does not affect these gates in any way. Neither the K channels nor their activation gates are affected in a specific way by Pronase. Tetrodotoxin does not protect the inactivation gates from Pronase, nor does maintained inactivation of the Na channels during exposure to Pronase. Our results suggest that the inactivation gate is a readily accessible protein attached to the inner end of each Na channel. It is shown clearly that activation and inactivation of Na channels are separable processes, and that Na channels are distinct from K channels.     

Effects of millimeter waves on ionic currents of Lymnaea neurons

The effects of mm‐waves 60.22–62.22 GHz and 75 GHz on A‐type K⁺ currents and the effects of 61.22 GHz on Ca²⁺ currents of Lymnaea neurons were investigated using a whole‐cell voltage‐clamp technique. The open end of a rectangular waveguide covered with a thin Teflon film served as a radiator. Specific absorption rates at the waveguide outlet, inserted into physiological solution, were in the range of 0–2400 W/kg. Millimeter wave irradiation increased the peak amplitudes, activation rates, and inactivation rates of both ion currents. The changes in A‐type K⁺ current were not dependent on the irradiation frequency. It was shown that the changes in the amplitudes and kinetics of both currents resulted from the temperature rise produced by irradiation. No additional effects of irradiation on A‐type K⁺ current other than thermal were found when tested at the phase transition temperature or in the presence of ethanol. Ethanol reduced the thermal effect of irradiation. Millimeter waves had no effect on the steady‐state activation and inactivation curves, suggesting that the membrane surface charge and binding of calcium ions to the membrane in the area of channel locations did not change. Bioelectromagnetics 20:24–33, 1999. © 1999 Wiley‐Liss, Inc.     

Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH

Extracellular acidification and reduction of extracellular K⁺ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K⁺]_o at pH 7.4, it is very sensitive to [K⁺]_o at pH 6.4, and in the mutant, H463G, the removal of K⁺ _o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K⁺ _o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na⁺ permeation through inactivated channels. Although the H463G currents were abolished in zero [K⁺]_o, robust Na⁺ tail currents through inactivated channels were observed. The appearnnce of H463G Na⁺ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K⁺ was replaced by NMG⁺ and the inward Na⁺ current was recorded, addition of 1 mM K⁺ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K⁺]_o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).     

Molecular Analysis of the Putative Inactivation Particle in the Inactivation Gate of Brain Type IIA Na+ Channels

Fast Na⁺ channel inactivation is thought to involve binding of phenylalanine 1489 in the hydrophobic cluster IFM in L_III-IV of the rat brain type IIA Na⁺ channel. We have analyzed macroscopic and single channel currents from Na⁺ channels with mutations within and adjacent to hydrophobic clusters in L_III-IV. Substitution of F1489 by a series of amino acids disrupted inactivation to different extents. The degree of disruption was closely correlated with the hydrophilicity of the amino acid at position 1489. These mutations dramatically destabilized the inactivated state and also significantly slowed the entry into the inactivated state, consistent with the idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation process. Substitution of a phe residue at position 1488 or 1490 in mutants lacking F1489 did not restore normal inactivation, indicating that precise location of F1489 is critical for its function. Mutations of T1491 disrupted inactivation substantially, with large effects on the stability of the inactivated state and smaller effects on the rate of entry into the inactivated state. Mutations of several other hydrophobic residues did not destabilize the inactivated state at depolarized potentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactivation at all potentials and accelerated recovery from inactivation at negative membrane potentials. Some of these mutations in L_III-IV also affected the latency to first opening, indicating coupling between L_III-IV and channel activation. Our results show that the amino acid residues of the IFM hydrophobic cluster and the adjacent T1491 are unique in contributing to the stability of the inactivated state, consistent with the designation of these residues as components of the inactivation particle responsible for fast inactivation of Na⁺ channels.     

Conformational changes in the selectivity filter of the open-state KcsA channel: an energy minimization study

Potassium channels switch between closed and open conformations and selectively conduct K⁺ ions. There are at least two gates. The TM2 bundle at the intracellular site is the primary gate of KcsA, and rearrangements at the selectivity filter (SF) act as the second gate. The SF blocks ion flow via an inactivation process similar to C-type inactivation of voltage-gated K⁺ channels. We recently generated the open-state conformation of the KcsA channel. We found no major, possibly inactivating, structural changes in the SF associated with this massive inner-pore rearrangement, which suggests that the gates might act independently. Here we energy-minimize the open state of wild-type and mutant KcsA, validating in silico structures of energy-minimized SFs by comparison with crystallographic structures, and use these data to gain insight into how mutation, ion depletion, and K⁺ to Na⁺ substitution influence SF conformation. Both E71 or D80 protonations/mutations and the presence/absence of protein-buried water molecule(s) modify the H-bonding network stabilizing the P-loops, spawning numerous SF conformations. We find that the inactivated state corresponds to conformations with a partially unoccupied or an entirely empty SF. These structures, involving modifications in all four P-loops, are stabilized by H-bonds between amide H and carbonyl O atoms from adjacent P-loops, which block ion passage. The inner portions of the P-loops are more rigid than the outer parts. Changes are localized to the outer binding sites, with innermost site S4 persisting in the inactivated state. Strong binding by Na⁺ locally contracts the SF around Na⁺, releasing ligands that do not participate in Na⁺ coordination, and occluding the permeation pathway. K⁺ selectivity primarily appears to arise from the inability of the SF to completely dehydrate Na⁺ ions due to basic structural differences between liquid water and the “quasi-liquid” SF matrix.     

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.