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The HV1 voltage‐gated proton (HV1) 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 HV1 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 HV1 from Nicoletia phytophila, NpHV1. 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 NpHV1 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 NpHV1 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 NpHV1 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. [ABSTRACT FROM AUTHOR]

The voltage-gated proton channel 1 ( HV1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV1 channel. In the present study, we report the existence of an HV1-type proton channel in insects. We searched insect transcriptome shotgun assembly ( TSA) sequence databases and found putative HV1 orthologues in various polyneopteran insects. To confirm that these putative HV1 orthologues were functional channels, we studied the HV1 channel of Nicoletia phytophila (Np HV1), an insect of the Zygentoma order, in more detail. Np HV1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, Np HV1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. Database Nucleotide sequence data have been deposited in the GenBank database under accession number . [ABSTRACT FROM AUTHOR]

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kHV1: it is proton-specific and activated by depolarization, its gH-V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability. [ABSTRACT FROM AUTHOR]

Abstract: Voltage gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Although these molecules are distinct entities, with no proven physical interaction, their presence and activity in many cells appears to be coordinated. We describe these interactions and discuss several types of mechanisms that might explain them. [Copyright &y& Elsevier]