Selectivity Filter Scanning of the Human Voltage Gated Proton Channel Hhv1

Abstract

Extraordinary is crucial to all proton conducting molecules, including the human voltage gated proton channel, hHV1, because [H+] is minuscule. Here we use selectivity filter scanning to elucidate the molecular requirements for proton specific conduction in hHV1. Asp112, in the middle of the S1 transmembrane helix, is essential to the WT channel [Musset et al., 2011. Nature 480:273-277]. We neutralized Asp112 by mutating it to Ala (D112A), then introduced Asp at each position along S1 from 108 to 118, searching for second site suppressor activity. All mutants except for D112A/V109D lacked even the anion conduction exhibited by D112A. Proton specific conduction was restored with Asp or Glu at position 116. The D112V/V116D channel resembled WT in selectivity, kinetics, and ΔpH dependent gating. Both R211H and R211H/D112V/V116D were inhibited by internally applied Zn2+ when the channel was open, indicating similar S4 accessibility. At position 109 Asp allowed anion permeation in combination with D112A, but did not rescue function in the nonconducting D112V mutant, indicating that is established external to the constriction at Phe150. The three positions (109, 112, 116) that permit conduction all line the pore in our homology model, delineating the conduction pathway. Evidently, proton selective conduction requires a carboxyl group to face the pore at a constriction in the external vestibule. Molecular dynamics studies indicate reorganization of ionic networks in response to mutations and suggest that the distribution of charged groups in the external vestibule modulates charge selectivity. That the filter functions in a new location helps define local environmental features that produce proton selective conduction.