The voltage sensor (VS) domain in Hv1 proton channels mediates a voltage-dependent and H + -selective “aqueous” conductance (G AQ ) that is potently modulated by extracellular Zn 2+ . Although two conserved His residues are required for Zn 2+ effects on G AQ gating, the atomic structure of the Zn 2+ coordination site and mechanism by which extracellular Zn 2+ stabilizes a closed-state conformation remain unknown. Here we use His mutagenesis to identify residues that increase Zn 2+ potency and are therefore likely to participate in first solvation shell interactions with Zn 2+ . Experimental Zn 2+ -mapping data were then used to constrain the structure of a new resting-state Hv1 model (Hv1 F). Molecular dynamics (MD) simulations show how protein and water atoms directly contribute to octahedral Zn 2+ coordination spheres in Zn 2+ -bound and -unbound Hv1 F models. During MD simulations, we observed correlated movements of Zn 2+ -interacting side chains and residues in a highly conserved intracellular Coulombic network (ICN) that contains highly conserved Arg “gating charges” in S4 as well as acidic “counter-charges” in S2 and S3 and is known to control VS activation, suggesting that occupancy of the extracellular Zn 2+ site is conformationally coupled to reorganization of the ICN. To test this hypothesis, we neutralized an ICN Glu residue (E153) and show that in addition to shifting G AQ activation to more negative voltages, E153A also decreases Zn 2+ potency. We speculate that extracellular gating-modifier toxins and other ligands may use a generally similar long-range conformational coupling mechanism to modulate VS activation in related ion channel proteins.

Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this work, we directly measure the proton depletion caused by activation of Hv1 proton channels using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Our results provide direct evidence of local proton depletion caused by flux through the proton-selective channel.