Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this gating pore when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hH(v)1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in H(v)1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open-dosed gating, but strikingly, at negative voltages where normal gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 mu M Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val(109), Phe(150), Val(177), and Val(178), which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg(208) in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.