Proton currents constrain structural models of voltage sensor activation

The Hv1 proton channel is evidently unique among voltage sensor domain proteins in mediating an intrinsic 'aqueous' H⁺ conductance (G_AQ). Mutation of a highly conserved 'gating charge' residue in the S4 helix (R1H) confers a resting-state H⁺ 'shuttle' conductance (G_SH) in VGCs and Ci VSP, and we now report that R1H is sufficient to reconstitute G_SH in Hv1 without abrogating G_AQ. Second-site mutations in S3 (D185A/H) and S4 (N4R) experimentally separate G_SH and G_AQ gating, which report thermodynamically distinct initial and final steps, respectively, in the Hv1 activation pathway. The effects of Hv1 mutations on G_SH and G_AQ are used to constrain the positions of key side chains in resting- and activated-state VS model structures, providing new insights into the structural basis of VS activation and H⁺ transfer mechanisms in Hv1.