Mapping of sites facing aqueous environment of voltage-gated proton channel at resting state: A study with PEGylation protection

摘要

Hvl (also named, voltage-sensor only protein, VSOP) lacks an authentic pore domain, and its voltage sensor domain plays both roles in voltage sensing and proton permeation. The activities of a proton channel are intrinsic to protomers of Hvl, while Hvl is dimeric in biological membranes; cooperative gating is exerted by interaction between two protomers. As the signature pattern conserved among voltage-gated channels and voltage-sensing phosphatase, Hvl has multiple arginines intervened by two hydrophobic residues on the fourth transmembrane segment, S4. S4 moves upward relative to other helices upon depolarization, causing conformational change possibly leading to the formation of a proton-selective conduction pathway. However, detailed mechanisms of proton-selectivity and gating of Hvl are unknown. Here we took an approach of PEGylation protection assay to define residues facing the aqueous environment of mouse Hvl (mHvl). Accessibilities of two maleimide molecules, N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), were examined on cysteine introduced into individual sites. Only the first arginine on S4 (Rl: R201) was inaccessible by NEM and AMS in mHvl. This is consistent with previous results of electrophysiology on the resting state channel, suggesting that the accessibility profile represents the resting state of mHvl. D108, critical for proton selectivity, was accessible by AMS and NEM, suggesting that D108 faces the vestibule. F146, a site critical for blocking by a guanidinium-reagent, was accessible by NEM, suggesting that F146 also faces the inner vestibule. These findings suggest an inner vestibule lined by several residues on S2 including F146, D108 on SI, and the C-terminal half of S4.