H+ Permeation in Hv1 Voltage-gated Proton Channels

Abstract

Voltage-gated H+ channels are structurally homologous to the voltage sensor domain of Kv, Nav and Cav channels but, despite the lack an ion-selective pore domain, conduct robust H+ current without apparent need for an accessory protein. Recent evidence indicates that although Hv1 is dimeric, each subunit contains a separate H+ permeation pathway that can be abrogated by amino acid mutation and chemical modification. However, the molecular mechanism of H+ permeation in Hv1 is unknown.Previous studies suggested that H+ permeation in voltage-gated proton channels is likely to employ a Grotthus-type H+-hopping mechanism involving one or more protonatable amino acids. We hypothesized that residues which are required for H+ permeation in Hv1 should be identifiable by loss of function phenotype in a mutagenesis screen. Candidate H+-acceptor residues within the voltage sensor were selected from those conserved in Hv1 species orthologues and by examination of homology models based on Hv1 structure based on known Kv channel protein structures and refined by molecular dynamics simulations. We used site-directed mutagenesis to neutralize candidate residues by substitution to Ala or Asn. Mutated GFP-Hv1 channels were expressed in mammalian culture cells and whole-cell H+ currents at fixed pipette pH and varying bath pH were elicited by depolarizing voltage steps. Although the apparent threshold for voltage-dependent activation of Hv1 current was altered by as much as −120 mV in certain mutants, the charge-neutralizing mutations we tested were insufficient to entirely abrogate expressed H+ current. Mutagenesis data and molecular models were used to generate a molecular model of H+ permeation through Hv1.