Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations

Abstract

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.

Fig. 1.

Intermediate states of the VSD. (A) Evolution of the Kv1.2 channel gating charge, Q(t). along the 2.2-μs unbiased MD trajectory. Major Q(t) transitions are shown as I, II, and III. The standard error on each estimation of the TM voltage amounts to ± 50 mV. (B) Representative conformations (α, β, γ, δ, and ϵ) of the VSDs revealed during the unbiased and subsequent biased-MD simulations were characterized by monitoring the distances (cf. SI Text) between the S4 basic residues (numbered 1 to 6 for R1, R2, R3, R4, K5, and R6, respectively) and their binding sites (numbered 1 to 6 for top , E¹⁸³, E²²⁶, D²⁵⁹, E²³⁶, and bottom , respectively). The distances were calculated between the geometrical centers of the side chain atoms H₂N = C_ζ(NH₂)-N_εH-C_δH₂ (Arg), H₃N_ζ-C_εH₂ (Lys), HOOC_γ - C_βH₂ (Asp), HOOC_δ - C_γH₂ (Glu), and the lipid phosphate group from a representative conformation and averaged over all four subunits of the channel. (Bottom) The closest interacting pairs are shown. Note the zipper-like motion in which, for successive transitions, the pairs involving S4 basic residues are formed with lower countercharges. (C) Molecular views of the VSDs in the five key conformations highlighting the position of the S4 basic residues (blue sticks) and the salt bridges they form with the acidic residues (red sticks) of the other VSD segments or with the lipid head group moieties (yellow). The highly conserved residue F²³³ of S2 is shown as cyan spheres.

Fig. 2.

Extent of S4 motion. (A and B) Positions of the S4 basic residues R1 (black) through R6 (orange) with respect to the membrane center (z = 0). (A) Positions of the geometrical center of the charged moieties. (B) Positions of the main-chain C_α atoms. (C to G) Rigid-body movements of the S4 segment along the α → ε transition. Only the backbone atoms of the S4 residues R1 through R4 were included in the calculation. (C) Position of the segment center of mass with respect to the membrane center (z = 0). (D) Tilt Δϕ with respect to z, the bilayer normal. (E) Precession Δα around z, and (F) rotation Δω about the helical main axis Z^′ calculated as , Δα_i = α_i - α_α, and Δω_i = ω_i - ω_α, where i = {α,β,γ,δ,ε}. The S4 motions were determined after elimination of overall rotation and translation of the VSD structures, by fitting the gating charge binding sites (E¹⁸³, E²²⁶, D²⁵⁹, and E²³⁶) over the structures. (G) Coordinate systems and definition of various angles. (H) TM position (z) of the geometrical center of side chains of the charge transfer center residues (D²⁵⁹, E²³⁶, and F²³³) in each of the VSD conformational states. All the data were computed from the representative conformations considered in Fig. 1 and averaged over the four VSD subunits, with the error computed as the standard deviation from this average value.

Fig. 3.

Electric field maps under a hyperpolarized TM potential in representative VSD conformations α and δ. (A) A representative Kv1.2 (α) VSD is located in the center of the panel and for clarity, only its backbone atoms (white ribbons), the conserved F²³³ residues (cyan spheres) and the basic (blue sticks) and acidic (red sticks) residues of its TM domain are shown. The local electric field direction is shown as black arrows. Closeup view of the charge transfer center region in the α (B) and δ (C) states highlighting the electric field pointing downward and rationalizing the downward movement of the basic residues of S4.

Fig. 4.

Electrical properties of the VSD (A) Electrical distances, , for each TM residue in the α (black) and ϵ (orange) conformations and net charge per residue (green) along the Kv channel sequence (excluding the T1 domain). The position of the TM segments S1 to S6 is indicated by arrows. was normalized assuming and 0 for residues positioned, respectively, above 25 Å and below -25 Å from the bilayer center. The data were averaged over the four subunits of the channel. (B) Corresponding cumulative (orange line) and per-residue (bars) gating charges for the α → ϵ transition (basic residues in blue and acidic ones in red). (C) Contributions of each S4 basic residue to the normalized GQR associated with each transition, enabling the identification of the residue(s) transporting most of the gating charge (red arrow). Error bars correspond to the standard deviation from the average value over the four subunits. (D) Electrical distance through the VSD in each conformation as a function of z, the normal to the bilayer. (E) Gating charge (Q) and TM position (z) for the S4 basic residues in each of the VSD states α through ϵ. Circles represent the GQRs that were obtained with regular MD simulations, whereas triangles stand for the configurations that were obtained with biased MD. (F) Activation master curve, describing the dependence of Q with z.