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• W4289600216 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information 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. Editor's evaluation 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, and ion channel biophysics. https://doi.org/10.7554/eLife.77946.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The activity of ion channels is often tightly controlled through a change in conformation, known as gating, that opens and closes the ion permeation pathway (Hille, 2001). Gating can be in response to voltage (voltage-dependent), chemical stimuli (ligand-gated), or membrane deformation (mechanically-gated). Understanding the gating of an ion channel is critical to understanding its physiological function. Recently, a new family of ion channels that are selective for protons and with little or no structural similarity to other ion channels was identified, collectively named the Otopetrins or OTOPs (Tu et al., 2018). These ion channels mediate proton influx in response to acid stimuli, but whether protons also gate them was unknown. Notably, while the structures of vertebrate OTOP1 and OTOP3 channels were recently solved (Chen et al., 2019; Saotome et al., 2019), it is not known if these structures are in open or closed states due to the lack of information regarding the gating of the channels. OTOP1 currents were first characterized in taste receptor cells, where pH sensitivity, proton selectivity, and inhibition by Zn2+ were described (Chang et al., 2010; Bushman et al., 2015). The founding member of the OTOP family, mOTOP1, was identified as the product of a gene mutated in a murine vestibular disorder (Hurle et al., 2003; Hughes et al., 2004) and was subsequently shown to form a proton channel that functions as a receptor for sour taste in vertebrates (Tu et al., 2018; Teng et al., 2019; Zhang et al., 2019). Most vertebrate genomes encode two related proteins, OTOP2 and OTOP3, that also form proton channels (Tu et al., 2018) and are expressed in a diverse array of tissues, including in the digestive tract, where mutations in the corresponding genes have been linked to disease (Tu et al., 2018; Parikh et al., 2019; Qu et al., 2019; Yang et al., 2019a). Functional OTOP channels are conserved across species, including in invertebrates, where they play roles in acid sensing and biomineralization (Hurle et al., 2011; Tu et al., 2018; Chang et al., 2021; Ganguly et al., 2021; Mi et al., 2021). Ion channels selective for protons are rare in nature, comprising just a small subset of the hundreds of types of ion channels that have been described over the last 80 years (Hille, 2001). The two best characterized proton-selective ion channels are M2, a viral protein involved in the acidification of the influenza virus interior, and Hv1, a voltage-gated ion channel that extrudes protons during the phagocyte respiratory burst to maintain pH (Pinto et al., 1992; Ramsey et al., 2006; Sasaki et al., 2006; Morgan et al., 2009). OTOP1 channels, unlike HV1, are not gated by voltage (Bushman et al., 2015; Tu et al., 2018) but might instead be gated by protons, like Hv1 and M2 (Cherny et al., 1995; Liang et al., 2016). However, establishing that the permeant ion gates an ion channel is not trivial. For example, an increase in current magnitudes as pH is lowered could be attributed to the opening of channels or an increase in the driving force for proton entry. Here we describe the biophysical response properties of the three murine OTOP channels to varying extracellular pH. By focusing on parameters that are independent of the driving force, we show that extracellular protons gate the three channels in a subtype-specific manner and that the extracellular linker between transmembrane domains eleven and twelve plays a role in gating. Results Differential current response profiles of three murine OTOP channels to acidic and basic stimuli We previously reported that all three murine OTOP channels conduct inward proton currents in response to acid stimuli, but show differences in their current as a function of pH (I-pH) relations (Tu et al., 2018). We reasoned that these differences likely reflect differences in gating. To test this hypothesis, we performed a careful comparative analysis of the response properties of murine OTOP1, OTOP2, and OTOP3. We used patch-clamp recording from HEK-293 cells transfected with cDNAs encoding each of the three channels for these experiments. If not otherwise stated, the intracellular solution was pH 7.4 (Cs-Aspartate-based), and the holding potential was –80 mV. All three channels carried inward currents in response to lowering pHo in a Na+-free solution, as previously reported (Tu et al., 2018). The magnitude of the OTOP1 currents increased as the extracellular pH (pHo) was lowered over a range of pH 6 to pH 4.5, while the magnitude of OTOP2 currents changed very little over the same pH range (Figure 1A). OTOP3 currents increased more steeply over the same range, with little or no inward currents in response to pH 6 (Figure 1A and C). For all three channels, currents decayed in response to prolonged acid exposure, and the rate of current decay was faster as the pHo was lowered (Figure 1A), likely due in part to intracellular accumulation of protons (DeCoursey and Cherny, 1996; Bushman et al., 2015; De-la-Rosa et al., 2016; Tu et al., 2018) (discussed below). Figure 1 Download asset Open asset Three vertebrate OTOP channels vary in their I-pH response profile across a broad pH range. (A) Proton currents elicited in response to acidic stimuli (pH 6–4.5) in the absence of extracellular Na+ measured from HEK-293 cells expressing each of the three OTOP channels as labeled (Vm = -80 mV). (B) Proton currents in response to solutions that varied in pH (pH 10–5) were measured from HEK-293 cells expressing each of the three OTOP channels as labeled (Vm = -80 mV). (C) Average data showing the peak current magnitude in response to stimuli of varying pHo, measured from experiments as in (A) and (B). OTOP3 currents are only observed in response to solutions of pH <5.5, while OTOP1 currents are evoked by solutions of <pH 6 and alkaline stimuli, and OTOP2 currents are evoked in response to all stimuli. Data represent mean ± s.e.m. of biological replicates where for each data point n=5–10 for OTOP1, n=6–10 for OTOP2, and n=4–10 for OTOP3. Figure 1—source data 1 Source data for Figure 1C. https://cdn.elifesciences.org/articles/77946/elife-77946-fig1-data1-v1.xlsx Download elife-77946-fig1-data1-v1.xlsx To further investigate the I-pH response profile of the three channels, we extended the pH range of the test solutions, now including neutral and alkaline solutions (Figure 1B and C). In OTOP1-expressing cells, we observed a small outward current in response to the alkaline solutions (pH 9–10) that was not observed in untransfected cells, which we, therefore, attribute to currents through OTOP1 channels. In response to the pH 9, but not pH 10 solution, we observed a ‘tail current’ upon return to neutral pH (for pH 9, Itail = –123+/-20 pA, n=5; for pH 10, Itail = –36+/-11 pA); this may reflect cytosolic H+ depletion during the pH 9 stimulus, creating a driving force for proton entry through open OTOP1 channels that subsequently close when the pH was restored to 7.4. In response to pH 8 and 7 solutions, no measurable change in the baseline currents was observed. A very different pH-response profile was observed in OTOP2-expressing cells (Figure 1B and C). Quite surprisingly, large outward currents were evoked in response to alkaline solutions of pH 9 and pH 10, which were similar in magnitude to the inward currents evoked in response to the acidic solutions. We also observed changes in the holding current in response to solutions at near-neutral pH (pH 8 or 7), suggesting that the channels are open at the resting pHo. Overall, we observed changes in OTOP2 currents in response to changes in extracellular pH over the entire pH range tested. The magnitude and direction of the responses were generally proportional to the driving force on the proton. This relationship broke down over the more acidic pH range, where a change of 10-fold in ion concentration (e.g. pH 6 versus pH 5; Figure 1) did not lead to a substantial increase in current magnitude. Although we do not yet understand this phenomenon, we suspect that it may be due in part to the rapid decay kinetics of the currents in response to acid stimuli and the consequent attenuation of the peak currents as well as possible inhibition of the channels by intracellular acidification. OTOP3 currents were active over a narrower pH range than OTOP1 and OTOP2 currents (Figure 1B and C). They were activated steeply below pH 6, and no outward currents were observed in response to any of the alkaline stimuli. These data suggest a sharp threshold for activating OTOP1 and OTOP3 channels of ~pH 6 (OTOP1) and pH 5.5. (OTOP3). In contrast, OTOP2 channels are active over the entire pH range. They also show that both OTOP1 and OTOP2 channels can conduct outward currents, which may be physiologically relevant under some circumstances. OTOP2 is selective for protons and open at neutral pH The unusual response properties of OTOP2 raise the question of whether OTOP2 is selective for protons. OTOP1 is highly selective for H+ over Na+, by a factor of at least 105 fold (Tu et al., 2018), and currents carried by OTOP3 follow the expectations for a proton-selective current, such as a shift in the reversal potential that follows the equilibrium potential for the H+ ion (Tu et al., 2018; Chen et al., 2019). OTOP2 is known to permeate protons (Tu et al., 2018), but selectivity for protons was not previously measured. To address this question, we first performed ion substitution experiments. We observed no change in the magnitude of the currents when the concentrations of either Na+ or Cl- in the extracellular solution were changed (Figure 2A), indicating that OTOP2 is not permeable to either ion. We also measured the reversal potential of OTOP2 currents as a function of ΔpH (pHo – pHi) under conditions designed to minimize H+ ion accumulation (see methods) (Cherny and DeCoursey, 1999; Ramsey et al., 2006; Tu et al., 2018). These experiments showed that Erev changed ~59 mV/pH as expected for an H+-selective ion channel (Figure 2B). Thus, we conclude that OTOP2 is selective for protons. Figure 2 Download asset Open asset OTOP2 is a proton selective ion channel that is open at neutral pHo. (A) Representative OTOP2 current elicited in response to a pH 5.5 solution with 160 mM Na+ replacing NMDG+ (top) or 140 mM Methane sulfonate-/ 20 mM Cl- replacing 160 mM Cl- (bottom) in the extracellular solutions at times indicated. Vm was held at –80 mV. (B) The I-V relationship (top) of the Zn2+ sensitive component of OTOP2 current in response to different pHo stimuli was obtained from ramp depolarizations in the presence and absence of Zn2+. pHi was adjusted to 6.5. Bottom: Erev measured as a function of ΔpH (pHo – pHi). pHi was adjusted to 6.0, 6.5, or 7.0 as indicated. The red dotted line is the predicted equilibrium potential for H+, EH. (C) Extracellular Zn2+ inhibits resting OTOP2 currents in a dose-dependent manner. Trace (top) shows inhibition of resting current in OTOP2-expressing HEK-293 cells by 10 μM Zn2+. Vm as indicated. Fractional inhibition was fit with a Hill slope = 0.6 and IC50=5.6 µM. Data represent mean ± s.e.m of biological replicates where n=3–4 for each data point. Figure 2—source data 1 Source data for Figure 2B and C. https://cdn.elifesciences.org/articles/77946/elife-77946-fig2-data1-v1.xlsx Download elife-77946-fig2-data1-v1.xlsx The unusual response properties of OTOP2 suggested that it might be open when pHo = 7.4. Consistent with this possibility, we routinely observed a large resting current at –80 mV in OTOP2-expressing cells. We reasoned that if this resting current is due to open OTOP2 channels, it should be inhibited by Zn2+, a blocker of OTOP1 and other proton channels (Tu et al., 2018). Indeed, OTOP2 currents at neutral pH were inhibited by Zn2+ with an IC50 of 5.6 μM (Figure 2C). Note that because the inhibition of OTOP1 by Zn2+ is pH-dependent (Bushman et al., 2015; Teng et al., 2019), the sensitivity of OTOP2 currents at neutral pH to micromolar concentrations Zn2+ as compared with the requirement of millimolar concentrations of Zn2+ to inhibit OTOP1 currents evoked in response to acid stimuli is as to be expected. Thus, we concluded that OTOP2 is a proton-selective ion channel open at neutral pH. Different pH-response profiles of three murine OTOP channels measured with pH imaging Inward and outward currents carried by protons through OTOP channels are expected to cause a change in intracellular pH. Thus, to confirm the results described above, we assessed the activity of each OTOP channel by monitoring intracellular pH. For these experiments we co-transfected HEK-293 cells with a pH-sensitive fluorescent protein – pHluorin, a variant of GFP whose fluorescence emission changes as a function of the intracellular pH (Miesenböck et al., 1998). As a control to confirm the expression and activity of pHluorin, all cells were exposed at the end of the experiment to acetic acid, which crosses cell membranes and causes intracellular acidification (Figure 3A-D, dark red bar) (Wang et al., 2011). Figure 3 Download asset Open asset OTOP channels mediate the influx and efflux of protons as measured with intracellular pH imaging. (A–D) Changes in intracellular fluorescence emission upon exposure to changing extracellular pH, as indicated, were measured from HEK-293 cells co-expressing OTOP channels and the pH-sensitive indicator pHluorin (A–C) or pHluorin alone (D). Data are shown as the mean ± SEM for n=7, 7, 11, and 3 cells for (A), (B), (C), and (D), respectively. Acetic acid, which is permeable through cell membranes and acidifies cell cytosol directly, served as a positive control. Only OTOP1 and OTOP2 conducted protons out of the cell cytosol in response to alkalinization, while all three channels conducted protons into the cell cytosol in response to acidification, albeit at different rates. The lower panel shows images of a single cell in the field of view used for these experiments taken at the pH indicated (pseudo color is arbitrary units). Figure 3—source data 1 Source data for Figure 3A–D. https://cdn.elifesciences.org/articles/77946/elife-77946-fig3-data1-v1.xlsx Download elife-77946-fig3-data1-v1.xlsx Cells expressing each of the three OTOP channels responded to the acidic solutions with large changes in intracellular pH, a response not observed in control cells (Figure 3A – D). Strikingly, we also observed changes in intracellular pH in response to an alkaline solution (pHo = 8.5) in both OTOP1- and OTOP2-expressing cells but not in OTOP3-expressing cells. This data is consistent with the patch-clamp data showing that OTOP1 and OTOP2, but not OTOP3, conduct outward proton currents in response to alkaline extracellular stimuli. We also noted differences in the time course of the response to acidic solutions (pH 6 or pH 5) in cells expressing the three OTOP channels. Notably, the response of OTOP3-expressing cells appeared slower than that of OTOP1 or OTOP2-expressing cells (compare Figure 3C with 3 A and 3B). This is likely a consequence of the smaller currents that are evoked by these stimuli in OTOP3 cells and the slower kinetics of channel activation (see below). Together these data support the conclusion that OTOP1 and OTOP2 can conduct outward proton currents in response to alkaline extracellular solution solutions, while all three channels carry inward proton currents in response to acidic stimuli. Kinetics of OTOP channels provide direct evidence of pH-dependent gating In patch-clamp recordings such as those shown in Figure 1, we noted differences in the kinetics of the currents elicited in response to acidic solutions among the three channels. For example, OTOP2 currents showed faster activation than OTOP1 currents, while OTOP3 currents were considerably slower. These differences likely reflect differences in activation of the channels by protons. In particular, for a channel that is open at neutral pH (OTOP2), we expect the currents to change in response to an increase or decrease in the concentration of the permeant ion (H+) with a time course that reflects the speed of the solution exchange. In contrast, for a channel that is closed at neutral pH (OTOP1 and OTOP3), currents may increase more slowly, with kinetics determined by the rates of agonist binding and opening of the channels. The rate of activation of each channel in response to acid stimuli was measured by fitting the time course of the current upon solution exchange with a single exponential (Figure 4A), excluding the first few milliseconds where responses deviated from an exponential time course due to a lag in the solution exchange. We also measured the rate of decay of the currents after return to neutral pH in a similar manner. Figure 4 with 1 supplement see all Download asset Open asset Activation kinetics vary dramatically between OTOP channels. (A) Representative current traces (black) from cells expressing each of the three OTOP channels in response to the application and the removal of pH 5.5 solutions. The activation and the decay kinetics of the currents were fitted with a single exponential (red and blue curves, respectively). (B) Solution exchange kinetics measured with an open K+ channel KIR2.1 are similar to the kinetics of OTOP2 currents. (C) Summary data for τon (left panel) and toff (right panel) of the three OTOP channels (OTOP1: green, OTOP2: blue, OTOP3: red) and KIR2.1 (black) in response to the application and removal of pH 5.5 extracellular solution. n=6, 5–6, 6 for OTOP1, OTOP2 and OTOP3 respectively. (D) Time constants for activation (τon, red) and deactivation (τoff, blue) of OTOP1, OTOP2, and OTOP3 currents in response to acidic stimuli (pH 6.0, 5.5, and 5.0) measured from experiments as in A. Note that the data at pH 5.5. is also shown in Panel C. n=6,5–6, and 6 for OTOP1, OTOP2, and OTOP3, respectively. Figure 4—source data 1 Source data for Figure 4C and D. https://cdn.elifesciences.org/articles/77946/elife-77946-fig4-data1-v1.xlsx Download elife-77946-fig4-data1-v1.xlsx In response to the pH 5.5 stimulus, the activation kinetics of the three OTOP channels varied over nearly two orders of magnitude (Figure 4A and C). The time constant for activation of OTOP2 was 12.0±1.6ms (n=10) which was slightly faster than the rate of solution exchange as measured using cells expressing an inward rectifier K+ channel and solutions that varied in potassium concentration (Figure 4B and C; τ=26.1 ± 2.9ms). Time constants for activation of OTOP1 and OTOP3 were 142.7±13.5ms (n=6) and 1098.0±83.0ms (n=6), respectively, considerably slower than the solution exchange suggesting that both are gated by extracellular protons. In contrast, the time constant for deactivation of OTOP1, OTOP2, and OTOP3 currents were similar to the rate of the solution exchange (τ=18.6 ± 4.4ms, n=6, 19.3±2.8ms, n=9, and 20.9±3.0ms, n=13, respectively; Figure 4A and C). The off-rates of the OTOP currents likely reflect the rate of removal of the permeant ion and not the closing rates of the channels (which could be slower). This also confirms that the differences in on-rates were not artifacts of varying solution exchange times between groups of cells. We also measured the kinetics of the currents carried by all three channels in response to solutions that varied in pHo. The activation rate of OTOP1 and OTOP3 currents increased as the pHo was lowered (Figure 4D and Figure 4—figure supplement 1). This is as expected if protons gate the channels. In contrast, the activation rate of the OTOP2 currents was insensitive to the extracellular pH over this range, consistent with an interpretation that the channels are open at neutral pH, and lowering the pH does not further increase channel open probability (Figure 4D and Figure 4—figure supplement 1). The decay rate of the currents upon return to neutral pH did not vary as a function of the pH of the stimulus for any of the three channels (Figure 4D). Together, the slow, pH-dependent kinetics of currents carried by OTOP1 and OTOP3 channels provides strong evidence that they are gated by extracellular protons. Effect of extracellular pH on slope conductance To provide further evidence that extracellular protons gate OTOP channels, we measured the slope conductance as a function of pHo for all three channels. The slope conductance (ΔI/ΔV) was measured from responses to ramp depolarizations at or near the peak of the response to the stimulus (pHo = 10–5) (Figure 5A and B) using a range of Vm from –80 mV to 0 mV, where the currents were generally linear and not contaminated by endogenous currents. Slope conductance (G) is related to the channel’s open probability (Po) by G=N*g*Po, where N is the number of channels and g is the single-channel conductance. Since the number of channels is constant, the slope conductance is a measure of Po*g. Importantly, this provides a measure of open probability, even when the holding potential is close to the equilibrium potential for H+ where currents are small (e.g. for stimuli at 8.0). Figure 5 with 1 supplement see all Download asset Open asset Changes in the slope conductance of OTOP channels as a function of extracellular pH. (A) Voltage and solution exchange protocol used to measure the slope conductance in response to changing extracellular pH. Vm was held at –80 mV and ramped to +80 mV (1 V/s at 1 Hz). The first ramp after the currents peaked was used for later measurements. (B) Representative I-V relationship from HEK-293 cells expressing each of the three OTOP channels in response to alkaline or acidic stimuli (pH 10–5) from experiments described in (A). The conductance was measured from the slope of the I-V curve between –80 mV and 0 mV to avoid contamination from outwardly rectifying Cl- currents. (C) Average slope conductance measured from cells expressing each of the three OTOP channels in response to different pHo stimuli from data as in (B). Data represent mean ± s.e.m. of biological replicates where for each data point n=5 for OTOP1, n=6–7 for OTOP2, and n=5–7 for OTOP3. Figure 5—source data 1 Source data for Figure 5C. https://cdn.elifesciences.org/articles/77946/elife-77946-fig5-data1-v1.xlsx Download elife-77946-fig5-data1-v1.xlsx In OTOP1-expressing cells, the slope conductance varied as a function of extracellular pH, dramatically increasing when the extracellular pH was lowered below pHo = 6.0 (Figure 5B and C). We also observed an increase in the slope conductance when the pHo was made more alkaline, consistent with data in Figure 1 showing that an alkaline pHo of 9.0 can be activating. In OTOP2-expressing cells, the slope conductance was highest in alkaline pH (pHo = 10) and decreased as the pHo was lowered (Figure 5B and C). The response of OTOP3-expressing cells was more similar to that of OTOP1, but with some clear differences (Figure 5B and C). The slope conductance remained very small when extracellular pH was between 10 and 6 and increased drastically when pHo was 5. These data support the conclusion that OTOP1 and OTOP3 are gated by extracellular protons, while OTOP2 is conductive over a broad pH range. This series of experiments also provided an opportunity to test the extent to which intracellular acidification accounts for the decay of the macroscopic currents. In particular, we noted that the decay of the currents was often accompanied by a shift in Erev (Figure 5—figure supplement 1A, B). Although in these experiments the currents were not leak-subtracted and, consequently, Erev cannot be used to precisely measure ΔpH, shifts in Erev can, nonetheless, be considered as evidence of a relative change in intracellular pH. For OTOP1, the observation that Erev shifted by –42.5+/-10.4 mV (n=5) between the first and the fourth ramp depolarization (4 seconds apart) suggests that the cytosol acidified by ~0.7 pH units (Figure 5—figure supplement 1B). A more modest shift of –27.9+/-3.4 mV (n=3) was observed for OTOP2 currents, while for slowly activating OTOP3 currents, Erev shifted slightly but significantly in the positive direction (6.82+/-2.5 mV, n=7; p=0.03, two-tailed paired Student’s T-test comparing t=0 s and t=4 s). To test whether acidification of the cytosol could account for the decay of OTOP1 and OTOP2 currents, we calculated the expected effect of the change in driving force (Vm-Erev) on the current magnitude at 1 s timepoints as the currents decayed and compared this to the observed current magnitudes at these times. As seen in Figure 5—figure supplement 1C, the shift in driving force mostly accounts for the decay of OTOP1 currents. For OTOP2, the decays in current magnitudes is much greater than can be explained by a change in driving force, suggesting that intracellular acidification may directly inhibit OTOP2 channel activity or conductance. We could not perform a similar analysis of OTOP3 currents, which in these experiments did not decay significantly during the stimulus. Extracellular linkers are key determinants for pH-sensitive gating The difference in pHo sensitivity of the three murine OTOP channels suggested that structural domains that vary among the channels contribute to the gating apparatus and that these domains could be identified using chimeric channels. Specifically, we generated chimeras between mOTOP2 and mOTOP3, which, as described above, are the most divergent functionally of the three murine OTOP channels. OTOP channels contain twelve transmembrane domains (S1-S12), with N and C termini located intracellularly. The transmembrane domains 1–6 and 7–12 respectively constitute the structurally homologous ‘N’ and ‘C’ domains. Reasoning that the residues involved in sensing the extracellular pH and gating the channels would be located on the extracellular surface of the channels, we swapped each of the six extracellular linkers that connect transmembrane helices (Figure 6—figure supplement 1). Each of the twelve chimeric channels was then tested over a range from pH 10 to pH 5. To simplify the analysis, we divided the chimeras into four categories: OTOP2 N domain (OTOP2 backbone with OTOP3 linkers), OTOP2 C domain, OTOP3 N domain, and OTOP3 C domain. Of the three OTOP2 N domain chimeras, two were functional. Strikingly, the replacement of the OTOP2 S5-S6 linker (L5-6) with that from OTOP3 nearly eliminated the outward currents in response to alkaline stimuli and reduced the response to the mildly acidic stimulus (pH 6) but had little effect on the magnitude or kinetics of the inward currents elicited in response to the pH 5 stimulus (Figure 6A–C; OTOP2/OTOP3(L5-6)). Replacement of the OTOP2 S1-2 linker with that from OTOP3 reduced current magnitudes (possibly due to effects on trafficking) but did not significantly change relative responses to the stimuli of varying pH. In contrast to the OTOP2 N domain chimeras, the two functional C domain chimeras (L7-8 and L11-12) showed a similar pH dependence as WT OTOP channels, suggesting that the mutations did not specifically affect channel gating (Figure 6A and D–E). None of the chimeras showed a significant change in kinetics (Figure 7A, B and E). Figure 6 with 1 supplement see all Download asset Open asset Chimeric channels with external l" @default.
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• W4289600216 title "Decision letter: Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels" @default.
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