Hv1 Proton Channel Opening Transition Revealed by Experimental and Computational Approaches

Abstract

The Hv1 proton channel is unique among voltage sensor (VS) domain proteins in mediating an activated-state H+-selective and voltage- and pH-gated ionic conductance. Previous studies identified residues that are required for gating and selectivity in Hv1, but the structural basis for exquisite H+ selectivity and the proton transfer mechanism remain enigmatic. A more detailed understanding of Hv1 structure and function is therefore needed to explain proton channel biophysical properties. We and others demonstrated that neutralizing mutagenesis of conserved ionizable residues which are hypothesized to be required for H+ ‘shuttling’ via explicit side chain ionization fails to abrogate H+ currents in Hv1. However, the possibility that Hv1 contains a network of acidic side chains which confers functional redundancy to the H+ transfer mechanism has not been directly tested. Here we report that double and triple Hv1 mutant channels also mediate unambiguous voltage- and pH-dependent H+ currents when expressed in HEK-293 cells. The data indicate that H+ transfer is unlikely to be mediated by H+ shuttling through pairs of nearly acidic side chains, but are consistent with the hypothesis that ‘aqueous’ or water-wire proton transfer is the primary mechanism of H+ conduction in Hv1. Different mutant combinations produce similar defects in pH-dependent gating, suggesting that interactions among networked side chains confer emergent biophysical properties in Hv1. Experimental data were used to build a refined activated-state Hv1 VS model structure (Hv1 J) that was subjected to molecular dynamics simulation. Although Hv1 J is similar overall to Hv1 B (Ramsey, et al., 2010), differences in the positions of specific side chains suggest a channel-opening conformational rearrangement that follows S4 movement, as predicted from gating currents (De La Rosa, et al., 2016).