Engineered high-affinity zinc binding site reveals gating configurations of a human proton channel

The voltage-gated proton channel (H(V)1) is a voltage sensor that also conducts protons. The singular ability of protons to penetrate proteins complicates distinguishing closed and open channels. When we replaced valine with histidine at position 116 in the external vestibule of hH(V)1, current was potently inhibited by externally applied Zn(2+) in a construct lacking the two His that bind Zn(2+) in WT channels. High-affinity binding with profound effects at 10 nM Zn(2+) at pH(o) 7 suggests additional groups contribute. We hypothesized that Asp(185), which faces position 116 in our closed-state model, contributes to Zn(2+) chelation. Confirming this prediction, V116H/D185N abolished Zn(2+) binding. Studied in a C-terminal truncated monomeric construct, V116H channels activated rapidly. Anomalously, Zn(2+) slowed activation, producing a time constant independent of both voltage and Zn(2+) concentration. We hypothesized that slow turn-on of H(+) current in the presence of Zn(2+) reflects the rate of Zn(2+) unbinding from the channel, analogous to drug-receptor dissociation reactions. This behavior in turn suggests that the affinity for Zn(2+) is greater in the closed state of hH(V)1. Supporting this hypothesis, pulse pairs revealed a rapid component of activation whose amplitude decreased after longer intervals at negative voltages as closed channels bound Zn(2+). The lower affinity of Zn(2+) in open channels is consistent with the idea that structural rearrangements within the transmembrane region bring Arg(205) near position 116, electrostatically expelling Zn(2+). This phenomenon provides direct evidence that Asp(185) opposes position 116 in closed channels and that Arg(205) moves between them when the channel opens.