Sequential formation of ion pairs during activation of a sodium channel voltage sensor

Abstract

Electrical signaling in biology depends upon a unique electromechanical transduction process mediated by the S4 segments of voltage-gated ion channels. These transmembrane segments are driven outward by the force of the electric field on positively charged amino acid residues termed ''gating charges,'' which are positioned at three-residue intervals in the S4 transmembrane segment, and this movement is coupled to opening of the pore. Here, we use the disulfide-locking method to demonstrate sequential ion pair formation between the fourth gating charge in the S4 segment (R4) and two acidic residues in the S2 segment during activation. R4 interacts first with E70 at the intracellular end of the S2 segment and then with D60 near the extracellular end. Analysis with the Rosetta Membrane method reveals the 3-D structures of the gating pore as these ion pairs are formed sequentially to catalyze the S4 transmembrane movement required for voltagedependent activation. Our results directly demonstrate sequential ion pair formation that is an essential feature of the sliding helix model of voltage sensor function but is not compatible with the other widely discussed gating models.electrical excitability ͉ gating E lectrical signaling in biology depends upon a unique electromechanical transduction process that couples small changes in the electrical potential across the cell membrane to conformational changes that open and close the pores of voltage-gated ion channels. The high sensitivity of voltage-gated ion channels to small changes in membrane potential depends on gating charges within their transmembrane structure, which are driven outward across the membrane by changes in the electric field and trigger a series of conformational changes that result in opening the pore (1-4). Approximately 12-16 positive charges move across the membrane during activation of the voltage sensors of voltage-gated sodium or potassium channels (5-10). Two major thermodynamic obstacles that the voltage-sensing process must overcome are stabilization of amino acid residues that serve as gating charges in the transmembrane environment of the protein and catalysis of their outward transmembrane movement.Voltage-gated ion channels are composed of four subunits or domains that each contain six transmembrane segments (11). The S1-S4 segments serve as the voltage sensing module, and the S5 and S6 segment and their connecting P loop serve as the pore-forming module (2-4). The primary gating charges reside in the S4 segment in four to seven repeated motifs of a positively charged amino acid residue (usually arginine) followed by two hydrophobic residues (2-4). The crux of the problem of understanding the electromechanical coupling in voltage-gated ion channels is determining the structural and mechanistic basis for movement of these gating charges across the membrane and for coupling of this movement to pore opening. The sliding helix or helical screw models posit that sequential formation of ion pairs with negatively charged amino acid residues in t...

Functional Extension of Amino Acid Triads from the Fourth Transmembrane Segment (S4) into Its External Linker in Shaker K+ Channels

“…Both functional and structural findings agree that two conserved acidic residues (Glu-283 and Glu-293 in Shaker K ϩ channels) in S2 may, respectively, act as countercharges of the third and the fourth arginines (Arg-3 and Arg-4) in S4 in the activated state, and the former acidic residue may also interact with the outmost arginine (Arg-1) in S4 in the resting state (11,(17)(18)(19)(20)(21)(22). The existence of these countercharge interactions, however, cannot directly argue for or against either model.…”

Free-energy landscape of ion-channel voltage-sensor–domain activation

“…(26), disulfide locking (27,28), cadmium binding (29), and X-ray crystallography (6). Additionally, independent long timescale molecular dynamics (MD) simulations suggested the existence of metastable intermediate states compatible with this large body of experimental data (30)(31)(32).…”

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

“…Although the latter is much larger than proposed in the transporter model, this S4 displacement is below the estimate from avidin binding experiments, which suggested that KvAP S4 amino acids move as much as 15-20 Å across the membrane (16). Recently, charge reversal mutagenesis (17) and disulfide locking (18) were used to probe pair interactions within the VSD of different VGCs. Also, mutations with natural and unnatural amino acids, electrophysiological recordings, and X-ray crystallography were combined to identify a charge transfer center that facilitates the movement of positively charged amino acids across the membrane field (19).…”