1. Evaluation Summary:

    This paper will be of interest both to marine biologists and to biophysicists studying voltage-gated proton channels. It describes cloning and full biophysical characterization of the first ion channel ever identified in reef-building coral species, and develops a mechanistic model for understanding regulation of voltage-gated proton channels.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    In this paper Rangel-Yescas and colleagues identify in several Cnidarian species a gene that seems to correspond to that of the Hv1 channel in humans. Cloning and heterologous expression of the putative protein from two species of reef-building corals indeed gives rise to voltage-activated proton currents. The authors provide a careful detailed biophysical characterization of gating for the cnidarian Hv channel AmHv1. They find that coral Hv channels show much faster activation/deactivation kinetics as compared to human Hv1, but otherwise show properties similar to the latter. The science is solid. Strengths of the paper include the following: (i) AmHv1 is so far the first ion channel to be cloned from a scleractinian species. Its suggested role in coral physiology might open up novel lines of research. (ii) The authors develop a mechanistic gating model which explains all the observed gating properties, including non-linear dependence on delta-pH of current activation midpoint-voltages (V0.5). The model offers a first framework for understanding the molecular mechanisms of voltage- and proton-dependence of Hv channel gating.

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  3. Reviewer #2 (Public Review):

    The authors cloned Hv1 channels from two different coral species of the genus Acropora, A. millepora and A. palmata. They found that the proteins have high sequence homology and very similar biophysical properties, despite the fact that the two organisms live in different oceans. Compared to human Hv1, the coral channels activate much more rapidly in response to membrane depolarization. The authors characterized AmHv1 in detail and used a FRET-based approach to investigate its subunit stoichiometry. Their finding is in agreement with a dimeric assembly similar to other known Hv1s. Ion selectivity and sensitivity to extracellular zinc were also investigated and found to be comparable to those of other Hv1s.

    Proton currents from Hv1 channels are modulated by the pH gradient across the membrane (deltapH). The authors discovered that the deltapH dependence of the proton current from AmHv1 shows signs of saturation at values larger than one pH unit. Based on this finding, they propose an allosteric model of pH-dependent gating based on two proton binding sites, an intracellular excitatory site, and an inhibitory extracellular site. The two sites are assumed to modulate the opening transition through allosteric coupling factors. This mechanism of pH-dependent gating can have general implications when discussed in the context of alternative models in which the S4 transmembrane segment is the pH sensor.

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  4. Reviewer #3 (Public Review):

    This is a very integral work, which couples several experimental methodologies in order to evidence the presence of Hv1 in reef-corals. The fact that this ancient species, the reef-corals, express Hv1, with all of its molecular and functional hallmarks, is a very interesting discovery, highlighting that Hv1 seems to be expressed in a myriad of cell-types and organisms dating as far back as the corals do. The present article is very clear, easy to read, and the conclusion are evident at the light of the data given to the reader. The authors show to have a great grasp over the tools used, be it genetics, electrophysiology or bioinformatics.

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