Zinc activation of OTOP proton channels identifies structural elements of the gating apparatus

  1. Bochuan Teng
  2. Joshua P Kaplan
  3. Ziyu Liang
  4. Kevin Saejin Chyung
  5. Marcel P Goldschen-Ohm
  6. Emily R Liman

  • Curated by eLife

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    eLife assessment

    This valuable study discovers that zinc ions can activate some OTOP proton channels, identifying a pharmacological tool for research, and further establishing that OTOP channels gate. The data presented provide convincing support for the conclusions made by the authors, and the study is expected to be of considerable interest to physiologists investigating OTOP and other proton channels.

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Abstract

Otopetrin proteins (OTOPs) form proton-selective ion channels that are expressed in diverse cell types where they mediate detection of acids or regulation of pH. In vertebrates there are three family members: OTOP1 is required for formation of otoconia in the vestibular system and it forms the receptor for sour taste, while the functions of OTOP2 and OTOP3 are not yet known. Importantly, the gating mechanisms of any of the OTOP channels are not well understood. Here, we show that zinc (Zn 2+) , as well as other transition metals including copper (Cu 2+ ), potently activates murine OTOP3 (mOTOP3). Zn 2+ pre-exposure increases the magnitude of mOTOP3 currents to a subsequent acid stimulus by as much as 10-fold. In contrast, mOTOP2 currents are insensitive to activation by Zn 2+ . Swapping the extracellular tm 11–12 linker between mOTOP3 and mOTOP2 was sufficient to eliminate Zn 2+ activation of mOTOP3 and confer Zn 2+ activation on mOTOP2. Mutation to alanine of H531 and E535 within the tm 11–12 linker and H234 and E238 within the 5–6 linker reduced or eliminated activation of mOTOP3 by Zn 2+ , indicating that these residues likely contribute to the Zn 2+ activating site. Kinetic modeling of the data is consistent with Zn 2+ stabilizing the opn2+en state of the channel, competing with H + for activation of the channels. These results establish the tm 11–12 and tm 5–6 linkers as part of the gating apparatus of OTOP channels and a target for drug discovery. Zn 2+ is an essential micronutrient and its activation of OTOP channels will undoubtedly have important physiological sequelae.

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  1. Version published to 10.7554/elife.85317 on eLife
  2. eLife

    eLife assessment

    This valuable study discovers that zinc ions can activate some OTOP proton channels, identifying a pharmacological tool for research, and further establishing that OTOP channels gate. The data presented provide convincing support for the conclusions made by the authors, and the study is expected to be of considerable interest to physiologists investigating OTOP and other proton channels.

  3. eLife

    Reviewer #1 (Public Review):

    OTOP ion channels are proton-activated, proton-permeable proteins that participate in sour tasting but for which other physiological roles are just beginning to be elucidated. The authors of this manuscript noticed that the isoform OTOP3 shows activation by protons that are potentiated in the presence of Zn2+ and other divalent ions, while other isoforms are not weakly or not at all potentiated. This allowed them to apply a chimeric approach to define which regions of the protein are responsible for the Zn2+ effect. The authors found that a single extracellular loop and a single histidine residue located in it are sufficient to explain the potentiation and propose that this histidine is part of a binding site that allosterically couples to yet undefined proton binding sites(s) responsible for proton gating.

    The authors have performed very high-quality experiments and carried out a careful analysis of the data. This characterization of gating behavior of OTOP channels should be a step in elucidating physiological roles and in understanding the dynamics of these proteins. For these reasons, it should be of interest to researchers working in molecular biophysics and the physiological roles of ion channels.

  4. eLife

    Reviewer #2 (Public Review):

    OTOP channels are relatively newly discovered and their physiology is poorly understood. Zn activation appears to be a differentiating feature of OTOP function and Zn is a pharmacological tool for research. The Zn potentiation of OTOP3 is a curious phenomenon that is studied very carefully here. The language in this manuscript is appropriately nuanced in the interpretation of results and is delightfully agnostic with regards to function vs binding. The major strengths of this work are the very thorough characterization of the zinc effect and the identification of the 11-12 loop as necessary and sufficient for the zinc effect.

  5. eLife

    Reviewer #3 (Public Review):

    The authors characterized the effect of Zn2+ in potentiating OTOP1 and OTOP3 proton-activated H+ currents. They took advantage of a set of chimeras with swapped extracellular loops between OTOP3 (Zn2+-dependent potentiation) and OTOP2 (no potentiation) by neatly identifying an extracellular loop that is sufficient to confer Zn2+ potentiation. The results support the idea that within this loop resides at least part of the Zn2+ binding site, a hypothesis also confirmed by the role of a histidine residue. The authors suggested that Zn2+ potentiation of OTOP3 involves different structural elements than those required for inhibition, the conclusion that is supported by the data on the OTOP3-OTOP2 chimeras. These results shed light on a new aspect of the gating mechanism of these channels, adding an important piece to the puzzle to decipher their role in cells. This manuscript provides an important result for scientists whose research is focused on proton channels, and ion channel gating mechanisms.

    Weaknesses: Although the identification of the extracellular loop represents an important result to define the structural element that confers Zn2+ potentiation to OTOP3, there are several aspects of the gating mechanism that would require a deeper analysis. The mutagenesis of the OTOP3 tm11-12 linker is very limited and does not include mutagenesis experiments in OTOP2 and OTOP1 that would further support the conclusion proposed by the authors and extend the importance of the tm11-12 linker to all the three OTOP channels (as stated in the manuscript title).
    Moreover, only one residue has been identified as important for Zn2+ binding. Given the three-dimensional structures of OTOP channels available to this date, particularly the chicken OTOP3 structure (PDB:6NF6), a structural analysis would certainly provide a set of putative partners for the histidine identified as the key residue for Zn2+ potentiation. Even if it is hard to understand what conformational state is represented in the structure, this analysis will provide a valid starting point to investigate the functional relevance of these residues.

  6. Version published to 10.1101/2022.12.15.520539v1 on bioRxiv