Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels

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 pHo <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.

Zn2+potentiation of OTOP proton channels identifies structural elements of the gating apparatus

“…While none of the other chimeras containing an mOTOP3 backbone showed a reduction in Zn 2+ potentiation, several showed an increase (Fig 6C, E, and Fig 6). Interestingly, the O3/O2(L1-2) chimera which was previously observed to be non-conductive in response to changes in extracellular pH (Teng et al, 2022), was strongly activated by Zn 2+ . Thus, for this channel, the increase in fold-potentiation mostly reflects the large decrease in the acid-induced currents, rather than an increase in the magnitude of the currents after Zn 2+ exposure and suggests that the 1-2 linker plays a role in acid-activation of mOTOP3.…”

“…Indeed, even a 64 s exposure to 1mM Zn 2+ had no effect on the magnitude or activation kinetics of the currents (Supplementary Figure 1). Interestingly, the O3/O2(L11-12) chimera is activated at a higher pH and currents are more rapidly activating than currents carried by mOTOP3 (Figure 6B and (Teng et al, 2022). This suggests that the O3/O2(L11-12) channels may be partly locked in a potentiated state.…”

“…Given the striking difference between mOTOP3 and mOTOP2 in potentiating effects of Zn 2+ , we reasoned that chimeras between the two channels might allow us to identify its structural basis. As Zn 2+ is likely to bind to an extracellular domain, we tested chimeras in which each of the six external linkers between transmembrane domains were exchanged (Teng et al, 2022). A total of twelve chimeras were tested, six in which the backbone was the mOTOP2 channel and six in which the backbone was the mOTOP3 channels.…”

“…Zn 2+ potentiation of O2/O3(L11-12) showed a time-dependence similar to that of wildtype mOTOP3 channels (Figure 6F, G). mOTOP2 and O2/O3(L11-12), but not mOTOP3, carry outward currents in response to alkaline stimuli (Teng et al, 2022). Thus, we wondered if the sensitivity to potentiation by Zn 2+ would be observed for outward currents.…”

“…Mouse OTOP1, OTOP2 and OTOP3 cDNAs were in a pcDNA3.1 vector with an N-terminal fusion tag consisting of an octahistidine tag followed by eGFP, a Gly-Thr-Gly-Thr linker and 3C protease cleavage site (LEVLFQGP) were as previously described (Saotome, 2019 #186). All chimeras and mutations were generated using In-Fusion Cloning (Takara Bio) and were confirmed by Sanger sequencing (Genewiz) (see (Teng et al, 2022)).…”