Ion channel voltage sensors: structure, function, and pathophysiology

Abstract

Voltage-gated ion channels generate electrical signals in species from bacteria to man. Their voltage-sensing modules are responsible for initiation of action potentials and graded membrane potential changes in response to synaptic input and other physiological stimuli. Extensive structure-function studies, structure determination, and molecular modeling are now converging on a sliding-helix mechanism for electromechanical coupling in which outward movement of gating charges in the S4 transmembrane segments catalyzed by sequential formation of ion pairs pulls the S4-S5 linker, bends the S6 segment, and opens the pore. Impairment of voltage-sensor function by mutations in Na+ channels contributes to several ion channelopathies, and gating pore current conducted by mutant voltage sensors in Na(V)1.4 channels is the primary pathophysiological mechanism in hypokalemic periodic paralysis. The emerging structural model for voltage sensor function opens the way to development of a new generation of ion-channel drugs that act on voltage sensors rather than blocking the pore.

Figure 1. Na⁺ channels, conserved gating charges, and gating models

(A) Left. Drawing from a low-resolution electron microsopic image of a purified Na⁺ channel, as seen from the extracellular and intramembrane perspectives (Sato et al., 2001). Right. Transmembrane folding diagram of a single domain of a Na_V channel or a single subunit of a K_V channel. (B) Amino acid sequences of S2 and S4 segments illustrating conserved gating charges (R1-R4, bold), interacting negative charges (An1 and An2, bold) and a conserved phenylalanine residue (underline). (C) Sliding helix model of gating (Catterall, 1986a, b). Left. The S4 segment in domain IV of Na_V channels drawn as an alpha helix. Note that this S4 segment is exceptional in having 7 potential gating charges. Right. The S4 segment drawn as a cylinder with a ribbon of positive charge around it conferred by the arginine gating charges, which are neutralized by negative charges from surrounding transmembrane segments. Changes in membrane voltage cause the S4 segment to move out or in along a spiral path so that gating charges exchange ion pair partners. (D) Sliding helix model with a focused field (Catterall, 2000). The S4 segment is depicted within a voltage sensor that has aqueous vestibules on the extracellular and intracellular sides separated by a tightly fitting narrow waist that seals the voltage sensor and generates a focused electrical field along its length.

Figure 2. Evidence supporting the sliding-helix model of gating

(A) Gating charge measurements from gating current recordings on Shaker K⁺ channels (Seoh et al., 1996). The potential gating charges indicated were neutralized by mutation, and the reduction in gating current was measured and plotted in terms of equivalent gating charge. (B) Drawing of the receptor site for α-scorpion toxin binding to the resting state of Na_V channels as determined from site-directed mutagenesis and antibody mapping (Rogers et al., 1996). (C) Real-time recordings of gating movements of S4 segments from fluorescent probes covalently attached to cysteine residues that were substituted for amino acid residues at the extracellular end of the S4 segment of Shaker K⁺ channels (Glauner et al., 1999). The two traces represent fluorescence change for donor alone versus donor plus acceptor. The difference represents fluorescence resonance energy transfer (FRET), a measure of the relative distance between sites of incorporation of fluorescence probes. Fluorescent probes were incorporated at positions 356 or 359 as indicated, six or three residues upstream (and therefore extracellular) of the R1 gating charge (R362), respectively. (D) Rates of disulfide locking for interaction of R3 and An1 (blue), R4 and An2 (green), and R4 and An1 (red) (DeCaen et al., 2009; DeCaen et al., 2008). The abscissa is normalized time in units of τ_activation.

Figure 3. Gating pore current

(A) Normalized gating pore current, measured in the presence of tetrodotoxin to block the central pore, as a function of holding potential for wild-type Na_V1.2 channels (black), Na_V1.2/R1Q,R2Q (red), and Na_V1.2/R2Q,R3Q (blue) (Sokolov et al., 2005). (B) Normalized gating pore current as in panel A for HypoPP mutants of R1 and R2 (red) or NormoPP mutants of R3 (blue) ((Sokolov et al., 2007, 2008b).

Figure 4. Structural models of K_V1.2 channels in activated and resting states

The structure of the K_V1.2 channel in an activated state was determined by x-ray crystallography, and its structure in a closed state was modeled using the ROSETTA Membrane method. (A) Structure of the of Kv1.2 channel determined by x-ray crystallography (Long et al., 2005b). The voltage-sensing and pore-forming modules of two subunits are indicated in a crossection through the structure normal to the plane of the membrane. Note the labels indicating that the voltage-sensing module of subunit 4 interacts with the pore-forming module of subunit 1 (left) while the voltage-sensing module of subunit 2 interacts with the pore-forming module of subunit 3 (right). Transmembrane segments are colored: S1, dark blue; S2, light blue-green; S3, light green; S4, dark green; S5, yellow-green; S6, orange. The S4-S5 linker covalently connecting pore-forming and voltage-sensing modules are highlighted in magenta. Positions of the Cβ atoms of gating charge-carrying arginines in S4 (labeled as R1 through R4 and colored in purple) and negatively charged residues in S2 (labeled E1 (for An1) and E2 (for An2) and colored in brown) are shown in sphere representation. (B) Rosetta-Membrane resting state model of K_V1.2 channel (Pathak et al., 2007; Yarov-Yarovoy et al., 2006a). The model is colored and labeled as in panel A.

Figure 5. Stepwise movement of the S4 segment through the gating pore during activation of the bacterial Na⁺ channel NaChBac

(A) Molecular models of the series of ion pair interactions in the voltage sensor during activation. For each interaction confirmed by disulfide-locking experiments, the relevant gating charge (R1, R2, R3, R4) was constrained to form an ion pair with An1 and the voltage sensor was modeled using the Rosetta Membrane algorithm (DeCaen et al., 2009; DeCaen et al., 2008). Positions of the Cβ atoms of gating-charge-carrying arginines in S4 (labeled as R1 through R4 and colored blue) and negatively charged residues in S2 (labeled D1 and E2 and colored red) are shown in sphere representation. (B) Expanded view of gating pore structure with S2 in alpha-helical conformation and S4 in 3₁₀ helical conformation as predicted by Rosetta modeling. (C) Gating pore structure as in panel B with the mutations R1Q and R2Q. (D) Gating pore structure as in panel B with the HypoPP R2G mutation.