Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels
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Evaluation Summary:
The manuscript shows that OTOP proton channels are proton-gated with distinct pH sensitivities, and identifies regions on the proteins that alter pH-dependent gating. The main claims are well supported by the data. These findings are likely to be of interest to researchers studying acid/base physiology, sensory physiology, or ion channel biophysics.
(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 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Otopetrin (OTOP) channels are proton-selective ion channels conserved among vertebrates and invertebrates, with no structural similarity to other ion channels. There are three vertebrate OTOP channels (OTOP1, OTOP2, and OTOP3), of which one (OTOP1) functions as a sour taste receptor. Whether extracellular protons gate OTOP channels, in addition to permeating them, was not known. Here, we compare the functional properties of the three murine OTOP channels using patch-clamp recording and cytosolic pH microfluorimetry. We find that OTOP1 and OTOP3 are both steeply activated by extracellular protons, with thresholds of pH o <6.0 and 5.5, respectively, and kinetics that are pH-dependent. In contrast, OTOP2 channels are broadly active over a large pH range (pH 5 pH 10) and carry outward currents in response to extracellular alkalinization (>pH 9.0). Strikingly, we could change the pH-sensitive gating of OTOP2 and OTOP3 channels by swapping extracellular linkers that connect transmembrane domains. Swaps of extracellular linkers in the N domain, comprising transmembrane domains 1–6, tended to change the relative conductance at alkaline pH of chimeric channels, while swaps within the C domain, containing transmembrane domains 7–12, tended to change the rates of OTOP3 current activation. We conclude that members of the OTOP channel family are proton-gated (acid-sensitive) proton channels and that the gating apparatus is distributed across multiple extracellular regions within both the N and C domains of the channels. In addition to the taste system, OTOP channels are expressed in the vertebrate vestibular and digestive systems. The distinct gating properties we describe may allow them to subserve varying cell-type specific functions in these and other biological systems.
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Evaluation Summary:
The manuscript shows that OTOP proton channels are proton-gated with distinct pH sensitivities, and identifies regions on the proteins that alter pH-dependent gating. The main claims are well supported by the data. These findings are likely to be of interest to researchers studying acid/base physiology, sensory physiology, or ion channel biophysics.
(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 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The strengths of this study are the careful dissection of gating (opening and closing of channels) versus conductance of open channels. This study systematically characterizes the H+-response properties of the OTOP1, OTOP2, and OTOP3 channels and finds that their H+ conductance is gated distinctly in each. OTOP2 is constitutively open at all pH, its conductance is diminished by more acidic solutions. In contrast, OTOP3 opens exclusively to acidic pH, and OTOP1 opens to acidic or basic pH. Regions of each otopetrin protein sequence are identified that alter pH activation of H+ conductance. Weaknesses are the limited discussion of H+ current decay during pH stimulation and calibration of solution exchange kinetics. The interpretations throughout the study are grounded by experimental results. The overall …
Reviewer #1 (Public Review):
The strengths of this study are the careful dissection of gating (opening and closing of channels) versus conductance of open channels. This study systematically characterizes the H+-response properties of the OTOP1, OTOP2, and OTOP3 channels and finds that their H+ conductance is gated distinctly in each. OTOP2 is constitutively open at all pH, its conductance is diminished by more acidic solutions. In contrast, OTOP3 opens exclusively to acidic pH, and OTOP1 opens to acidic or basic pH. Regions of each otopetrin protein sequence are identified that alter pH activation of H+ conductance. Weaknesses are the limited discussion of H+ current decay during pH stimulation and calibration of solution exchange kinetics. The interpretations throughout the study are grounded by experimental results. The overall conclusions that the conductances of OTOPs can be actively gated by pH and that the 3 OTOPs provide a palette of responses to acidic and basic solutions are well-supported.
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Reviewer #2 (Public Review):
This study examines the gating mechanism of murine otopetrin channels OTOP1, OTOP2, and OTOP3 and finds that hydrogen ions are both sensed and conducted by these channels, resulting in a response to extracellular pH that is subtype-specific. OTOP2 appears to be maximally active at alkaline pHo. OTOP3 becomes active only under acidic conditions and OTOP1 displays an intermediate pHo dependence. The study also identifies the extracellular loops linking transmembrane helices 3 and 4, and 5 and 6 as protein regions contributing to the difference in pHo-dependent gating between OTOP2 and OTOP3. These findings will lead to a better understanding of the roles of otopetrins in a variety of physiological processes, including biomineralization, sour taste perception, and digestion.
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Reviewer #3 (Public Review):
In this work, the authors design experiments to determine whether Otop channels are gated by changes in extracellular pH. Understanding pH-dependent conformational changes in proteins (including ion channels) is fundamentally important. For Otops, which are voltage-independent, pH changes may represent the main physiological mechanism for controlling channel activity in vivo, so the authors' line of inquiry is of high potential value to researchers who seek to understand how Otops contribute to normal physiological mechanisms of extracellular pH sensing, intracellular pH control, and membrane potential homeostasis.
The techniques used here are generally appropriate, but electrophysiology is clearly the most incisive and the resulting data represent the major strength of the work. The authors convincingly …
Reviewer #3 (Public Review):
In this work, the authors design experiments to determine whether Otop channels are gated by changes in extracellular pH. Understanding pH-dependent conformational changes in proteins (including ion channels) is fundamentally important. For Otops, which are voltage-independent, pH changes may represent the main physiological mechanism for controlling channel activity in vivo, so the authors' line of inquiry is of high potential value to researchers who seek to understand how Otops contribute to normal physiological mechanisms of extracellular pH sensing, intracellular pH control, and membrane potential homeostasis.
The techniques used here are generally appropriate, but electrophysiology is clearly the most incisive and the resulting data represent the major strength of the work. The authors convincingly demonstrate that proton currents mediated by Otops 1-3 are differentially sensitive to changes in extracellular pH, and are therefore most likely proton-gated channels. A potential weakness of the work is that individual residues which mediate pH-dependent gating are not identified, and it is therefore difficult to ascertain any details about the structural basis of gating.
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