Transmembrane Movement of the Shaker K+ Channel S4

Abstract

We have probed internal and external accessibility of S4 residues to the membrane-impermeant thiol reagent methanethiosulfonate-ethyltrimethlammonium (MTSET) in both open and closed, cysteine-substituted Shaker K+ channels. Our results indicate that S4 traverses the membrane with no more than 5 amino acids in the closed state, and that the distribution of buried residues changes when channels open. This change argues for a displacement of S4 through the plane of the membrane in which an initially intracellular residue moves to within 3 amino acids of the extracellular solution. These results demonstrate that the putative voltage-sensing charges of S4 actually reside in the membrane and that they move outward when channels open. We consider constraints placed on channel structure by these results.

S4 Charges Move Close to Residues in the Pore Domain during Activation in a K Channel

“…The modification was assayed functionally in two-electrode voltage-clamped oocytes, as described previously (Larsson et al 1996; Baker et al 1998). The applications of the methanethiosulfonate (MTS) reagents were computer controlled, and the MTS reagents were applied either at −80 mV (closed state; S4 in the deactivated state) or at 0 mV or +40 mV depending on the channel type (open/inactivated state; S4 in the activated state).…”

“…Cysteine accessibility studies suggest that the positive charges of S4 are either buried in the membrane or in the cytosol when the membrane is held at a hyperpolarized potential, and, in response to a depolarizing voltage pulse, the S4 charges move outward and exposes its three most external charges into the extracellular solution (Larsson et al 1996; Yang et al 1996; Yusaf et al 1996; Baker et al 1998; for review see Keynes and Elinder 1999). S4 is suggested to undergo a large-scale movement during activation of the channels, either a 180° rotation (Papazian and Bezanilla 1997; Cha et al 1999; Glauner et al 1999) or a helical screw motion with both a 180° rotation and a translational motion of 13.5 Å (Catterall 1986; Guy and Seetharamulu 1986; Glauner et al 1999; Keynes and Elinder 1999; Gandhi et al 2000).…”

Functional diversity of potassium channel voltage-sensing domains

“…48 Replication of these results in K channels showed that accessibility of introduced cysteine residues in the Shaker S4 was a function of voltage and occurred in a manner consistent with a large outward displacement of the a helix upon depolarization. 49,50 Later measurements using spectroscopic techniques, particularly the LRET and another variation of FRET involving transfer between dyes and hydrophobic quenchers, suggested that the magnitude of movement was at most a couple of Angstroms (A ). 51,52 In contrast, a proposal based on the KvAP structure and state-dependent avidin/biotin accessibility experiments, 53 suggested that the S3-S4, therein named the paddle, might traverse most of the membrane thickness in a rigid body motion.…”

“…Although there is not a consensus, most models of voltage-dependent gating seem to suggest that the S4 segment translates several angstroms and undergoes a~120 degree counter clockwise rotation. 49,54 This protein motion can be exemplified by comparing the open state of the paddle chimera with models of the closed state. 30 As can be seen in Figure 3, the displacement of the S4 helix in the closed and active states proposed by 37 is in the order of 10 A .…”

“…This is the main evidence for the existence of outward movement of the S4 of potassium channels. 49,[57][58][59][60][61] The rest of the transmembrane domains seem to remain mostly static as the S4 moves, providing a physical and electrostatic scaffold. 62,63 Given that the rest of the VSD remains static, as the charged residues in S4 move, they traverse its central portion, which contains an aromatic residue that has been dubbed the charge transfer center.…”