The response of a membrane-bound Kv1.2 ion channel to an applied transmembrane potential has been studied using molecular dynamics simulations. Channel deactivation is shown to involve three intermediate states of the voltage sensor domain (VSD), and concomitant movement of helix S4 charges 10-15 Å along the bilayer normal; the latter being enabled by zipper-like sequential pairing of S4 basic residues with neighboring VSD acidic residues and membrane-lipid head groups. During the observed sequential transitions S4 basic residues pass through the recently discovered charge transfer center with its conserved phenylalanine residue, F 233 . Analysis indicates that the local electric field within the VSD is focused near the F 233 residue and that it remains essentially unaltered during the entire process. Overall, the present computations provide an atomistic description of VSD response to hyperpolarization, add support to the sliding helix model, and capture essential features inferred from a variety of recent experiments.gating charge | S4 helix | voltage-gated channel V oltage sensor domains (VSDs) are membrane-embedded constructs, which work as electrical devices responding to changes in the transmembrane (TM) voltage. They are ubiquitous to voltage-gated channels (VGCs) in which four of these units are attached to the main pore (1). During channel activation, the displacements of the charges tethered to the VSD give rise to transient "gating" currents, the time integral of which is the "gating charge" (GQR) translocated across the membrane capacitance. Phenomenological kinetic models devised to describe the time course of such currents are very diverse but all indicate that during VGC activation, the VSD undergoes a complex conformational change that encompasses many transitions (2-5).Three main models have been proposed to rationalize the transfer of a large GQR across the low dielectric membrane in VGCs (6, 7). All are associated with the motion of S4, the conserved highly positively charged helix of the VSDs (8). In the sliding helix model (9, 10), the positively charged (basic) residues of the S4 segment form sequential ion pairs with acidic residues on neighboring TM segments and move a large distance perpendicular to the membrane plane. The transporter model derives from measurements of a focused electrical field within the membrane and suggests that during activation, the latter is reshaped. Accordingly, it is posited that S4 does not move its charges physically very far across the membrane (8). A third model was introduced following publication of the KvAP structure (11). Here, the position of the S3-S4 helical hairpin with respect to the pore domain suggested a gating mechanism in which the hairpin moves through the membrane in a paddle-like motion translocating S4 basic residues across the membrane, and reaching a TM position only in the activated state. Crystal structures of the Kv1.2 channel (12) and the Kv1.2-Kv2.1 paddle chimera (13) indicated later that the KvAP structure likely represented a non...

The X-ray structure of the bacterial voltage-gated sodium channel NavAb has been reported in a conformation with a closed conduction pore. Comparison between this structure and the activatedopen and resting-closed structures of the voltage-gated Kv1.2 potassium channel suggests that the voltage-sensor domains (VSDs) of the reported structure are not fully activated. Using the aforementioned structures of Kv1.2 as templates, molecular dynamics simulations are used to identify analogous functional conformations of NavAb. Specifically, starting from the NavAb crystal structure, conformations of the membrane-bound channel are sampled along likely pathways for activation of the VSD and opening of the pore domain. Gating charge computations suggest that a structural rearrangement comparable to that occurring between activated-open and resting-closed states is required to explain experimental values of the gating charge, thereby confirming that the reported VSD structure is likely an intermediate along the channel activation pathway. Our observation that the X-ray structure exhibits a low pore domain-opening propensity further supports this notion. The present molecular dynamics study also identifies conformations of NavAb that are seemingly related to the resting-closed and activated-open states. Our findings are consistent with recent structural and functional studies of the orthologous channels NavRh, NaChBac, and NavMs and offer possible structures for the functionally relevant conformations of NavAb.voltage-gated cation channels | ion channel activation mechanism V oltage-gated cation channels (VGCCs) are membrane-embedded protein pores that allow the selective flow of specific ions across the cell membrane in response to an external voltage stimulus. This remarkable property, which relies on a pore-gating mechanism, enables these channels to perform as critical devices in a number of electrically mediated biological processes including cellular secretion, hormone regulation, electric signaling in neurons, and contraction in excitable muscle cells (1). The superfamily of VGCCs includes the Na + , Ca 2+ , K + , and nonselective cationic channels, denoted Nav, Cav, Kv, and HCN channels, respectively. VGCCs are either tetrameric or pseudotetrameric with each subunit consisting of six transmembrane (TM) helices, conventionally denoted S1-S6. The helix bundle formed by segments S1-S4 constitutes the voltage-sensor domain (VSD) that triggers the closed-open transition of the conduction pore in response to TM voltage variations. The pore domain (PD) is composed of helices S5 and S6. Charge displacement across the membrane entailed by the conformational transition of the VSD, known as the gating charge (Q), results essentially from the motion of the S4 helix, which is positively charged and contains four to seven basic amino acids, mostly arginines. Gating charges have been measured for a variety of channels, as discussed extensively in recent reviews (2, 3).In 2005, the MacKinnon group reported an X-ray crystal structure...

Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamics (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na+ ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.

A dynamic transmembrane voltage field has been suggested as an intrinsic element in voltage sensor (VS) domains. Here, the dynamic field contribution to the VS energetics was analyzed via electrostatic calculations applied to a number of atomistic structures made available recently. We find that the field is largely static along with the molecular motions of the domain, and more importantly, it is minimally modified across VS variants. This finding implies that sensor domains transfer approximately the same amount of gating charges when moving the electrically charged S4 helix between fixed microscopic configurations. Remarkably, the result means that the observed operational diversity of the domain, including the extension, rate, and voltage dependence of the S4 motion, as dictated by the free energy landscape theory, must be rationalized in terms of dominant variations of its chemical free energy.voltage sensor | ion channel | free energy | molecular dynamics | electrostatics V oltage sensor (VS) domains are electrically charged membrane proteins made of four packed helices (1). The fourth segment (S4) contains four highly conserved positively charged amino acids, R 1 through R 4 . By interchanging its conformation between two main states (resting and activated) in response to voltage variations, VS domains displace the S4 charges across the membrane capacitance, giving rise to ΔQ, the so-called gating charge (2). As a result of their function of converting voltage variations into molecular motions, VS domains are ubiquitous in a number of electrically mediated processes, either as domain components of phosphatases (2) or proton (3) and ion channels (4-8).Despite the conservation of S4 sequences, nature has designed a variety of constructs (2) that present a wide range of voltage dependence and absolute rates of activation. For instance, the VS kinetics is markedly distinct between voltage-gated Na + and K + channels (9), a feature that complies with their respective role in the fast and slow phases of the action potential. Drastic kinetic shifts can even be observed in VS differing by point mutations (10,11). In all of these constructs, the S4 operation results from the fine balance between the chemical and the electrical components of the relative free energy of the segment. Whereas the former depends on the S4 energy in the absence of an electrical driving force, the latter arises essentially from ϕðrÞ (12), a dimensionless scalar field that reports the fraction of the membrane voltage coupled to every S4 charge q i . As mostly embodied in the transporter model (13), the reshaping of ϕðrÞ along with S4 displacements appears as one potential mechanism impacting the sensing process. Facing the VS diversity, it has been unknown to which extent the field reshaping may impact the S4 operation in distinct constructs and account for its energetic differences.Here, by benefiting from an increasing number of atomistic structures of VS-containing channels or enzymes made available recently, we use all-atom molecular ...