The H_V 1 voltage-gated proton (H_V 1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best-described physiological inhibitor of H_V 1 is Zn²⁺ . Externally applied ZnCl₂ drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn²⁺ and Cd²⁺ on H_V 1 from Nicoletia phytophila, NpH_V 1. We introduced mutations at potential Zn²⁺ coordination sites and measured Zn²⁺ inhibition in different extracellular pH, with Zn²⁺ concentrations up to 1000 μm. Zn²⁺ inhibition in NpH_V 1 was quantified by the slowing of the activation time constant and a positive shift of the conductance-voltage curve. Replacing aspartate in the S3-S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage-conductance curve, such that Zn²⁺ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn²⁺ in the S3-S4 linker. A simple Hodgkin Huxley model of NpH_V 1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high-resolution clear native gel electrophoresis (hrCNE) confirmed that NpH_V 1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development.

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