A Kv channel with an altered activation gate sequence displays both "fast" and "slow" activation kinetics

The Kv1–4 families of K⁺ channels contain a tandem proline motif (PXP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial "fast" mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered PXP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the PXP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the PXP motif may serve as a "hinge" and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them "reluctant" to open subsequently. voltage-gated potassium channel     

Conformational dynamics of helix S6 from Shaker potassium channel: simulation studies

Prolines in transmembrane (TM) alpha-helices are believed to play an important structural and/or functional role in membrane proteins. At a structural level a proline residue distorts alpha-helical structure due to the loss of at least one stabilizing backbone hydrogen bond, and introduces flexibility in the helix that may result in substantial kink and swivel motions about the effective "hinge." At a functional level, for example in Kv channels, it is believed that proline-induced molecular hinges may have a direct role in gating, i.e., the conformational change linked to opening/closing the channel to movement of ions. In this article we study the conformational dynamics of the S6 TM helix from of the Kv channel Shaker, which possesses the motif PVP--a motif that is conserved in Kv channels. We perform multiple molecular dynamics simulations of single S6 helices in a membrane-mimetic environment in order to effectively map the kink-swivel conformational space of the protein, exploiting the ability of multiple simulations to achieve greater sampling. We show that the presence of proline locally perturbs the helix, disrupting local dihedral angles and producing local twist and unwinding in the region of the hinge--an effect that is relaxed with distance from the PVP motif. We furthermore show that motions about the hinge are highly anisotropic, reflecting a preferred region of kink-swivel conformation space that may have implications for the gating process.     

Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels

In voltage-activated potassium (Kv) channels, basic residues in S4 enable the voltage-sensing domain to move in response to membrane depolarization and thereby trigger the activation gate to open. In the X-ray structure of the KvAP channel, the S4 helix is located near the intracellular boundary of the membrane where it forms a "voltage-sensor paddle" motif with the S3b helix. It has been proposed that the paddle is lipid-exposed and that it translocates through the membrane as it activates. We studied the interaction of externally applied Hanatoxin with the voltage-sensor paddle in Kv channels and show that the toxin binds tightly even at negative voltages where the paddle is resting and the channel is closed. Moreover, measurements of gating charge movement suggest that Hanatoxin interacts with and stabilizes the resting paddle. These findings point to an extracellular location for the resting conformation of the voltage-sensor paddle and constrain its transmembrane movements during activation.     

Gating of shaker-type channels requires the flexibility of S6 caused by prolines

The recent crystallization of a voltage-gated K+ channel has given insight into the structure of these channels but has not resolved the issues of the location and the operation of the gate. The conserved PXP motif in the S6 segment of Shaker channels has been proposed to contribute to the intracellular gating structure. To investigate the role of this motif in the destabilization of the alpha-helix, both prolines were replaced to promote an alpha-helix (alanine) or to allow a flexible configuration (glycine). These substitutions were nonfunctional or resulted in drastically altered channel gating, highlighting an important role of these prolines. Combining these mutations with a proline substitution scan demonstrated that proline residues in the midsection of S6 are required for functionality, but not necessarily at the positions conserved throughout evolution. These results indicate that the destabilization or bending of the S6 alpha-helix caused by the PXP motif apparently creates a flexible "hinge" that allows movement of the lower S6 segment during channel gating and opening.     

Closing in on the resting state of the Shaker K(+) channel

Membrane depolarization causes voltage-gated ion channels to transition from a resting/closed conformation to an activated/open conformation. We used voltage-clamp fluorometry to measure protein motion at specific regions of the Shaker Kv channel. This enabled us to construct new structural models of the resting/closed and activated/open states based on the Kv1.2 crystal structure using the Rosetta-Membrane method and molecular dynamics simulations. Our models account for the measured gating charge displacement and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by approximately 180 degrees as they move "outward" by 6-8 A. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel's S4-S5 linkers and S6 helices, allowing ion conduction. Our models are compatible with a wide body of data and resolve apparent contradictions that previously led to several distinct models of voltage sensing.     

Solution structure of Phrixotoxin 1, a specific peptide inhibitor of Kv4 potassium channels from the venom of the theraphosid spider Phrixotrichus auratus

Animal toxins block voltage-dependent potassium channels (Kv) either by occluding the conduction pore (pore blockers) or by modifying the channel gating properties (gating modifiers). Gating modifiers of Kv channels bind to four equivalent extracellular sites near the S3 and S4 segments, close to the voltage sensor. Phrixotoxins are gating modifiers that bind preferentially to the closed state of the channel and fold into the Inhibitory Cystine Knot structural motif. We have solved the solution structure of Phrixotoxin 1, a gating modifier of Kv4 potassium channels. Analysis of the molecular surface and the electrostatic anisotropy of Phrixotoxin 1 and of other toxins acting on voltage-dependent potassium channels allowed us to propose a toxin interacting surface that encompasses both the surface from which the dipole moment emerges and a neighboring hydrophobic surface rich in aromatic residues.     

Structural determinants of the regulation of the voltage-gated potassium channel Kv2.1 by the modulatory α-subunit Kv9.3

Voltage-gated potassium (Kv) channels containing alpha-subunits of the Kv2 subfamily mediate delayed rectifier currents in excitable cells. Channels formed by Kv2.1 alpha-subunits inactivate from open- and closed states with both forms of inactivation serving different physiological functions. Here we show that open- and closed-state inactivation of Kv2.1 can be distinguished by the sensitivity to intracellular tetraethylammonium and extracellular potassium and lead to the same inactivated conformation. The functional properties of Kv2.1 are regulated by its association with modulatory alpha-subunits (Kv5, Kv6, Kv8, and Kv9). For instance, Kv9.3 changes the state preference of Kv2.1 inactivation by accelerating closed-state inactivation and inhibiting open-state inactivation. An N-terminal regulatory domain (NRD) has been suggested to determine the function of the modulatory alpha-subunit Kv8.1. However, when we tested the NRD of Kv9.3, we found that the functional properties of chimeric Kv2.1 channels containing the NRD of Kv9.3 (Kv2.1(NRD)) did not resemble those of Kv2.1/Kv9.3 heteromers, thus questioning the role of the NRD in Kv9 subunits. A further region of interest is a PXP motif in the sixth transmembrane segment. This motif is conserved among all alpha-subunits of the Kv1, Kv2, Kv3, and Kv4 subfamilies, whereas the second proline is not conserved in any modulatory alpha-subunit. Exchanging this proline in Kv2.1 for the corresponding residue of Kv9.3 resulted in channels (Kv2.1-P410T) that show all hallmarks of the regulation of Kv2.1 by Kv9.3. The effect prevailed in heteromeric channels following co-expression of Kv2.1-P410T with Kv2.1. These data suggest that the alteration of the PXP motif is an important determinant of the regulatory function of modulatory alpha-subunits.     

Proline-induced hinges in transmembrane helices: possible roles in ion channel gating

A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.     

Activation gating of hERG potassium channels: S6 glycines are not required as gating hinges

The opening of ion channels is proposed to arise from bending of the pore inner helices that enables them to pivot away from the central axis creating a cytosolic opening for ion diffusion. The flexibility of the inner helices is suggested to occur either at a conserved glycine located adjacent to the selectivity filter (glycine gating hinge) and/or at a second site occupied by glycine or proline containing motifs. Sequence alignment with other K+ channels shows that hERG possesses glycine residues (Gly648 and Gly657) at each of these putative hinge sites. In apparent contrast to the hinge hypotheses, substitution of both glycine residues for alanine causes little effect on either the voltage-dependence or kinetics of channel activation, and open state block by intracellular blockers. Substitution of the glycines with larger hydrophobic residues causes a greater propensity for the channel to open. We propose that in contrast to Shaker the pore of hERG is intrinsically more stable in the open than the closed conformation and that substitution at Gly648 or Gly657 further shifts the gating equilibrium to favor the open state. Molecular dynamics simulations indicate the S6 helices of hERG are inherently flexible, even in the absence of the glycine residues. Thus hERG activation gating exhibits important differences to other Kv channels. Our findings indicate that the hERG inner helix glycine residues are required for the tight packing of the channel helices and that the flexibility afforded by glycine or proline residues is not universally required for activation gating.     

Loss of shaker K channel conductance in 0 K+ solutions: role of the voltage sensor.

In potassium-free solutions some types of K channels enter a long-lasting nonconducting or "defunct" state. It is known that Shaker K channels must open in K+-free solutions to become defunct. Gating current studies presented here indicate an abnormal conformation in the defunct state that restricts S4 movement and alters its kinetics. Thus an abnormality initiated in the P region spreads to the gating apparatus. We find that channels most readily become defunct on repolarization to an intermediate voltage, thus prolonging occupancy of one of the several intermediate closed states. The state dependence of becoming defunct was further dissected by using the gating mutant L382A. Simply closing this channel at 0 mV (reversing the last activation step) does not make the mutant channel defunct. Instead, it is necessary to move further left (more fully closed) in the activation sequence. This was confirmed with ShIR experiments showing that channels become defunct only if there is inward gating charge movement. Rapid transit through the intermediate states, achieved at very negative voltage, is relatively ineffective at making channels defunct. Several mutations that removed C-type inactivation also made the channels resistant to becoming defunct. Our results show that normal gating current cannot be stably recorded in the absence of K+.     

The intrinsic flexibility of the Kv voltage sensor and its implications for channel gating

Analysis of the crystal structures of the intact voltage-sensitive potassium channel KvAP (from Aeropyrum pernix) and Kv1.2 (from rat brain), along with the isolated voltage sensor (VS) domain from KvAP, raises the question of the exact nature of the voltage-sensing conformational change that triggers activation of Kv and related voltage-gated channels. Molecular dynamics simulations of the isolated VS of KvAP in a detergent micelle environment at two different temperatures (300 K and 368 K) have been used to probe the intrinsic flexibility of this domain on a tens-of-nanoseconds timescale. The VS contains a positively charged (S4) helix which is packed against a more hydrophobic S3 helix. The simulations at elevated temperature reveal an intrinsic flexibility/conformational instability of the S3a region (i.e., the C-terminus of the S3 helix). It is also evident that the S4 helix undergoes hinge bending and swiveling about its central I130 residue. The conformational instability of the S3a region facilitates the motion of the N-terminal segment of S4 (i.e., S4a). These simulations thus support a gating model in which, in response to depolarization, an S3b-S4a "paddle" may move relative to the rest of the VS domain. The flexible S3a region may in turn act to help restore the paddle to its initial conformation upon repolarization.     

KCNE1 constrains the voltage sensor of Kv7.1 K+ channels

Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K(+) channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K(+) channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions.     

A Gating Model for the Archeal Voltage-Dependent K Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K⁺ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.     

The S4-S5 linker directly couples voltage sensor movement to the activation gate in the human ether-a'-go-go-related gene (hERG) K+ channel

A key unresolved question regarding the basic function of voltage-gated ion channels is how movement of the voltage sensor is coupled to channel opening. We previously proposed that the S4-S5 linker couples voltage sensor movement to the S6 domain in the human ether-a'-go-go-related gene (hERG) K+ channel. The recently solved crystal structure of the voltage-gated Kv1.2 channel reveals that the S4-S5 linker is the structural link between the voltage sensing and pore domains. In this study, we used chimeras constructed from hERG and ether-a'-go-go (EAG) channels to identify interactions between residues in the S4-S5 linker and S6 domain that were critical for stabilizing the channel in a closed state. To verify the spatial proximity of these regions, we introduced cysteines in the S4-S5 linker and at the C-terminal end of the S6 domain and then probed for the effect of oxidation. The D540C-L666C channel current decreased in an oxidizing environment in a state-dependent manner consistent with formation of a disulfide bond that locked the channel in a closed state. Disulfide bond formation also restricted movement of the voltage sensor, as measured by gating currents. Taken together, these data confirm that the S4-S5 linker directly couples voltage sensor movement to the activation gate. Moreover, rather than functioning simply as a mechanical lever, these findings imply that specific interactions between the S4-S5 linker and the activation gate stabilize the closed channel conformation.     

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG⁺) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs⁺, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K⁺, suggesting that K⁺ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG⁺ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K⁺, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.     

Ion channel gating: insights via molecular simulations

Ion channels are gated, i.e. they can switch conformation between a closed and an open state. Molecular dynamics simulations may be used to study the conformational dynamics of ion channels and of simple channel models. Simulations on model nanopores reveal that a narrow (<4 A) hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small (approximately 1 A) increase in pore radius or an increase in polarity. Modelling and simulation studies confirm the importance of hydrophobic gating in K channels, and support a model in which hinge-bending of the pore-lining M2 (or S6 in Kv channels) helices underlies channel gating. Simulations of a simple outer membrane protein, OmpA, indicate that a gate may also be formed by interactions of charged side chains within a pore, as is also the case in ClC channels.     

The ladder-shaped polyether toxin gambierol anchors the gating machinery of Kv3.1 channels in the resting state

Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K⁺ permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel’s gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure–function relationship studies in Kv channels and in drug design to modulate channel function.     

Structural models for the KCNQ1 voltage-gated potassium channel

Mutations in the human voltage-gated potassium channel KCNQ1 are associated with predisposition to deafness and various cardiac arrhythmia syndromes including congenital long QT syndrome, familial atrial fibrillation, and sudden infant death syndrome. In this work 3-D structural models were developed for both the open and closed states of human KCNQ1 to facilitate structurally based hypotheses regarding mutation-phenotype relationships. The KCNQ1 open state was modeled using Rosetta in conjunction with Molecular Operating Environment software, and is based primarily on the recently determined open state structure of rat Kv1.2 (Long, S. B., et al. (2005) Science 309, 897-903). The closed state model for KCNQ1 was developed based on the crystal structures of bacterial potassium channels and the closed state model for Kv1.2 of Yarov-Yarovoy et al. ((2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7292-7207). Using the new models for KCNQ1, we generated a database for the location and predicted residue-residue interactions for more than 85 disease-linked sites in both open and closed states. These data can be used to generate structure-based hypotheses for disease phenotypes associated with each mutation. The potential utility of these models and the database is exemplified by the surprising observation that four of the five known mutations in KCNQ1 that are associated with gain-of-function KCNQ1 defects are predicted to share a common interface in the open state structure between the S1 segment of the voltage sensor in one subunit and both the S5 segment and top of the pore helix from another subunit. This interface evidently plays an important role in channel gating.     

The role of a conserved proline residue in mediating conformational changes associated with voltage gating of Cx32 gap junctions.

We have explored the role of a proline residue located at position 87 in the second transmembrane segment (TM2) of gap junctions in the mechanism of voltage-dependent gating of connexin32 (Cx32). Substitution of this proline (denoted Cx32P87) with residues G, A, or V affects channel function in a progressive manner consistent with the expectation that a proline kink (PK) motif exists in the second transmembrane segment (TM2) of this connexin. Mutations of the preceding threonine residue T86 to S, A, C, V, N, or L shift the conductance-voltage relation of wild-type Cx32, such that the mutated channels close at smaller transjunctional voltages. The observed shift in voltage dependence is consistent with a reduction in the open probability of the mutant hemichannels at a transjunctional voltage (Vj) of 0 mV. In both cases in which kinetics were examined, the time constants for reaching steady state were faster for T86N and T86A than for wild type at comparable voltages, suggesting that the T86 mutations cause the energetic destabilization of the open state relative to the other states of the channel protein. The structural underpinnings of the observed effects were explored with Monte Carlo simulations. The conformational space of TM2 helices was found to differ for the T86A, V, N, and L mutants, which produce a less bent helix ( approximately 20 degrees bend angle) compared to the wild type, which has a approximately 37 degrees bend angle. The greater bend angle of the wild-type helix reflects the propensity of the T86 residue to hydrogen bond with the backbone carbonyl of amino acid residue I82. The relative differences in propensity for hydrogen bonding of the mutants relative to the wild-type threonine residue in the constructs we studied (T86A, V, N, L, S, and C) correlate with the shift in the conductance-voltage relation observed for T86 mutations. The data are consistent with a structural model in which the open conformation of the Cx32 channel corresponds to a more bent TM2 helix, and the closed conformation corresponds to a less bent helix. We propose that the modulation of the hydrogen-bonding potential of the T86 residue alters the bend angle of the PK motif and mediates conformational changes between open and closed channel states.     

Environment of the gating charges in the Kv1.2 Shaker potassium channel

Recently, the structure of the Shaker channel Kv1.2 has been determined at a 2.9-angstroms resolution. This opens new possibilities in deciphering the mechanism underlying the function of voltage-gated potassium (Kv) channels. Molecular dynamics simulations of the channel, embedded in a membrane environment show that the channel is in its open state and that the gating charges carried by S4 are exposed to the solvent. The hydrated environment of S4 favors a local collapse of the electrostatic potential, which generates high electric-field gradients around the arginine gating charges. Comparison to experiments suggests furthermore that activation of the channel requires mainly a lateral displacement of S4. Overall, the results agree with the transporter model devised for Kv channels from electrophysiology experiments, and provide a possible pathway for the mechanistic response to membrane depolarization.