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.

We have acquired structural evidence that two components evident previously in the depolarization-evoked gating currents from voltage-gated Shaker K+ channels have their origin in sequential, two-step outward movements of the S4 protein segments. A point mutation greatly destabilizes the "fully retracted" state of S4 transmembrane translocation, causing instead an intermediate state to predominate at resting potentials. This state is distinguishable topologically and fluorometrically. That a point mutation effectively excludes half the range of S4 motion from physiological voltages suggests that the diverse sensitivities among voltage-gated channels might reflect not only differences in S4 valence, but also displacement. Existence of an intermediate subunit state helps explain why modeling channel activation has required positing greater than four closed states.