Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations Lucie Delemottea, Mounir Tareka,1, Michael L. Kleinb,1, Cristiano Amaralc, and Werner Treptowc,1 aEquipe de Chimie et Biochimie Théoriques, Unité Mixte de Recherche Structure et Réactivité des Systèmes Moléculaires Complexes, Centre National de la Recherche Scientifique, University of Nancy, 54506 Nancy, France; bInstitute of Computational and Molecular Science, Temple University, Philadelphia, PA 19122; and cLaboratório de Biologia Teórica e Computacional, Departamento de Biologia Celular, Universidade de Brasília DF, Brasilia City, 70910-900 Brazil Contributed by Michael L. Klein, February 18, 2011 (sent for review December 21, 2010) The response of a membrane-bound Kv1.2 ion channel to an ap- models of the VSD response, most of which have converged plied transmembrane potential has been studied using molecular toward the sliding helix model with S4 motion of 5–10 Å (15). dynamics simulations. Channel deactivation is shown to involve Although the latter is much larger than proposed in the transpor- three intermediate states of the voltage sensor domain (VSD), ter model, this S4 displacement is below the estimate from avidin and concomitant movement of helix S4 charges 10–15 Å along binding experiments, which suggested that KvAP S4 amino acids the bilayer normal; the latter being enabled by zipper-like sequen- move as much as 15–20 Å across the membrane (16). Recently, tial pairing of S4 basic residues with neighboring VSD acidic charge reversal mutagenesis (17) and disulfide locking (18) were residues and membrane-lipid head groups. During the observed used to probe pair interactions within the VSD of different sequential transitions S4 basic residues pass through the recently VGCs. Also, mutations with natural and unnatural amino acids, discovered charge transfer center with its conserved phenylalanine electrophysiological recordings, and X-ray crystallography were residue, F233. Analysis indicates that the local electric field within combined to identify a charge transfer center that facilitates the the VSD is focused near the F233 residue and that it remains essen- tially unaltered during the entire process. Overall, the present movement of positively charged amino acids across the mem- computations provide an atomistic description of VSD response to brane field (19). This important work enabled a dissection of hyperpolarization, add support to the sliding helix model, and VSD movements and their relation to ion channel opening. BIOPHYSICS AND capture essential features inferred from a variety of recent experi- These experiments demonstrated the existence of the sequential COMPUTATIONAL BIOLOGY ments. ion pair formation involving the S4 basic residues, an essential feature of the sliding helix model. gating charge ∣ S4 helix ∣ voltage-gated channel Here, we employ molecular dynamics (MD) simulations in atomic detail to investigate the structure of the VSD transition oltage sensor domains (VSDs) are membrane-embedded states of the Kv1.2 channel embedded in a lipid bilayer subjected Vconstructs, which work as electrical devices responding to to a hyperpolarized potential. The structure of the channel in changes in the transmembrane (TM) voltage. They are ubiquitous its open conformation (VSD up state, α) has been thoroughly to voltage-gated channels (VGCs) in which four of these units examined in previous MD simulations performed in the absence are attached to the main pore (1). During channel activation, of a depolarized TM potential (ΔV ¼ 0 mV) (20). Here, an the displacements of the charges tethered to the VSD give rise to unconstrained MD simulation has been carried out on a Kv1.2 transient “gating” currents, the time integral of which is the “gat- channel embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phos- ing charge” (GQR) translocated across the membrane capaci- phocholine (POPC) bilayer while being subjected to a hyperpo- tance. Phenomenological kinetic models devised to describe the larized TM voltage, ΔV, applied via a charge imbalance protocol time course of such currents are very diverse but all indicate that (21, 22) (Fig. S1). This 2.2-μs MD trajectory starting from the up during VGC activation, the VSD undergoes a complex conforma- α – state, , has uncovered the initial steps of the VSD response to tional change that encompasses many transitions (2 5). ΔV, which involves two intermediate states (β, γ). Specifically, the Three main models have been proposed to rationalize the VSD is observed to undergo transitions that involve a zipper-like transfer of a large GQR across the low dielectric membrane in motion of the six S4 basic residues (R1, R2, R3, R4, K5 and R6), VGCs (6, 7). All are associated with the motion of S4, the conserved highly positively charged helix of the VSDs (8). In the in a sequential ion pairing with nearby VSD acidic residues and sliding helix model (9, 10), the positively charged (basic) residues the membrane-lipid head groups. The final stages of the VSD of the S4 segment form sequential ion pairs with acidic residues response were uncovered by using biased-MD simulations, in on neighboring TM segments and move a large distance perpen- which the S4 basic residues were constrained to move along dicular to the membrane plane. The transporter model derives the spontaneously initiated pathway until reaching the down state from measurements of a focused electrical field within the mem- (ϵ) of the VSD. The combined MD simulations unveil and char- brane and suggests that during activation, the latter is reshaped. acterize five distinct states of the VSD that are involved in the Accordingly, it is posited that S4 does not move its charges deactivation process: the initial up state, α; three intermediate physically very far across the membrane (8). A third model was states, β, γ, δ; and the down state, ϵ. introduced following publication of the KvAP structure (11). Here, the position of the S3-S4 helical hairpin with respect to Author contributions: M.T., M.L.K., and W.T. designed research; L.D., C.A., and W.T. the pore domain suggested a gating mechanism in which the hair- performed research; L.D., C.A., and W.T. analyzed data; and L.D., M.T., M.L.K., and W.T. pin moves through the membrane in a paddle-like motion trans- wrote the paper. locating S4 basic residues across the membrane, and reaching a The authors declare no conflict of interest . TM position only in the activated state. Crystal structures of Freely available online through the PNAS open access option. the Kv1.2 channel (12) and the Kv1.2-Kv2.1 paddle chimera 1To whom correspondence may be addressed. E-mail: [email protected], (13) indicated later that the KvAP structure likely represented [email protected], or [email protected]. a nonnative state of the channel and its VSD (14). The newer This article contains supporting information online at www.pnas.org/lookup/suppl/ structures also provided the opportunity to develop molecular doi:10.1073/pnas.1102724108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1102724108 PNAS Early Edition ∣ 1of6 Downloaded by guest on October 1, 2021 Results mediate kinetic state previously identified in independent work Electrical Response. The electrical response of the system to a (22–24). In the subsequent QðtÞ drop (II), the VSD of subunit hyperpolarized potential ΔV was characterized by monitoring 1 underwent further salt-bridge rearrangements, which led to QðtÞ, the total GQR associated with the displacement of all yet another state, called here γ, lasting for over 0.5 μs. Then, charges in the system with respect to the membrane capacitor. in the third QðtÞ drop (III), the same VSD underwent yet another To do so, and as in electrophysiology experiments, the ionic conformational change. current through the main alpha pore was inhibited throughout The sequential conformational changes, α → β → γ, of the the MD simulations by imposing harmonic constraints on the VSDs, and especially that of subunit 1, provide a reaction path- selectivity filter backbone residues. Fig. 1 reports the variation way for the first transition events occurring in the voltage-sensing of the GQR and indicates a substantial channel electrical activity: process. In the latter, each conformation α, β, and γ is stabilized QðtÞ undergoes three major drops (I, II, and III) occurring by a maximum number of salt bridges between the S4 basic − approximately at t ¼ 0.2 μs, 1.6 μs, and 2.0 μs, respectively, with residues and negative residues of the VSD or PO4 moieties associated gating charges QðtÞ approximately − 1.4 e, −1.3 e, and of the lipids (Fig. 1C and Fig. S3). To uncover the complete −1.0 e(Æ0.3 e), respectively. VSD response from the up state (α) to the down state (ϵ), biased MD was used to simultaneously drag all the charged moieties VSD Conformational States. Throughout the MD simulation the of the basic residues from a given binding site to the next along Kv1.2 pore domain remained very stable, but the VSD undergoes the downstream path (see Materials and Methods). Along the substantial conformational changes involving zipper-like motion biased-MD trajectory between the γ and ϵ states, a fourth VSD of the salt pairing interactions. The modifications accounting conformation was identified, called here the δ-state, which is for the largest QðtÞ variations (drops I to III in Fig. 1A) involved also characterized by a specific network of salt bridges (Fig. 1 B salt-bridge rearrangements within the VSDs as a result of the S4 and C). Equilibration MD runs, each spanning approximately basic residues moving from external to internal binding sites 15 ns, confirmed the structural stability of these additional VSD along the domain. These binding sites are specifically the acidic structures, for which the distance matrix rmsd profile converged amino acids of segments S1 through S3 (E183,E226,D259, and to a value <2.4 Å(Fig.