Gating elements for carvacrol activation of the OTOP1 proton channel

Abstract

Otopetrin 1 (OTOP1) is a proton-activated channel crucial for animals' perception of sour taste. Despite its significance, the gating mechanism of OTOP1 remains poorly understood. Here, we demonstrate that carvacrol activates the mouse OTOP1 (mOTOP1) channel under neutral and acidic conditions. Functional analysis showed that carvacrol enhances pH fluorescence signals in OTOP1-expressing cells, with reduced efficacy at lower pH levels. Carvacrol selectively activates mOTOP1, while mOTOP2, mOTOP3, and Chelonia mydas OTOP1 (CmOTOP1) are insensitive to carvacrol activation under neutral pH. Through chimera and point mutation experiments, swapping S134 in transmembrane segment 3 (TM3) and T247 in the TM5-6 linker abolished carvacrol activation of mOTOP1 and conferred activation on CmOTOP1, suggesting these two residues are critical for carvacrol sensitivity. These findings highlight TM3 and TM5-6 linker as pivotal gating apparatus of OTOP1 channels and potential docking sites for drug design.

Fig. 1. Carvacrol dose-dependently activates the mOTOP1 channel.

a Representative traces showing mOTOP1 currents elicited in response to pH 6.0 stimulus or 1 mM carvacrol at pH 6.0 (pH_i = 7.3, membrane potential V_m = 0 mV). b Fold potentiation as a function of different concentrations of carvacrol (I_carvacrol/I_pH6), mean ± standard error of the mean (SEM), n = 6. The current amplitude refers to the stabilized current level after stimulation. c The current–voltage (I–V) relationships of currents in mOTOP1-expressing HEK293T cells were recorded using different concentrations of carvacrol at pH 7.4 extracellular solution, voltage ramps from −100 mV to +80 mV. d Whole-cell patch clamp ramp recordings of mOTOP1 currents in response to carvacrol at different concentrations, currents recorded at −100 mV (pH_i = 7.3). e Change in currents under different concentrations of carvacrol was measured as outlined in d. mean ± SEM, n = 3–6. f Hill equation fitting of dose-dependent activation of mOTOP1 channel by carvacrol with EC₅₀ of 692.27 ± 28.86 μM. Hill coefficient, 1.68. Current was measured at −100 mV, data points represent mean ± SEM, n = 6.

Fig. 2. Carvacrol-activated OTOP1 channel selectively permeates protons.

a Fluorescence intensity of pH indicator pHrodo in response to different stimuli were measured, in HEK293T cells expressing mOTOP1(n = 20) and sham transfected cells (n = 13), F/F₀ is relative value of pH fluorescence intensity, shown as mean ± SEM based on the data from traces for mOTOP1 and mock transfected cells, the initial fluorescence intensity of cells was defined as F₀. b Changes in intracellular fluorescence intensity were statistically analysed by Two-way ANOVA, Dunnett’s multiple comparisons test, ****p < 0.0001, ΔF = F − F_0, mOTOP1, n = 20, pH 5.2: ΔF = 0.549 ± 0.04, pH 5.2 + 1 mM carvacrol: ΔF = 1.791 ± 0.11 ; sham cells, n = 13, mean ± SEM. The F value is measured as marked with an arrow in Fig. 2a. c mOTOP1 currents were evoked in response to 500 μM carvacrol at pH 5.5, with Na⁺, K⁺, Li⁺, Cs⁺ (160 mM each), or Ca²⁺ (40 mM) replacing NMDG⁺ in the extracellular solution as indicated (V_m = 0 mV). d Percentage change of currents for each ion replacement respectively. Na⁺, n = 5, 0.011 ± 0.007; K⁺, n = 8, −0.010 ± 0.009; Li⁺, n = 6, 0.008 ± 0.005; Cs⁺, n = 7, −0.002 ± 0.013 and Ca²⁺, n = 11, −0.030 ± 0.008, mean ± SEM. e I–V relationships of mOTOP1 channels measured under 130 mM Li⁺ replacing NMDG⁺ in the pH 7.3 extracellular solution as indicated, NMDG⁺ or Li⁺-containing solution both dissolve 500 μM carvacrol. f Ionic permeability ratio of Na⁺, K⁺, Li⁺, and Cs⁺ to H⁺ when mOTOP1 channels were activated by 500 μM carvacrol at pH 7.3 extracellular solution.

Fig. 3. Selectivity of carvacrol in activating OTOP1 channels.

a, b I–V relationships of mOTOP2 or mOTOP3 channels in response to different concentrations of carvacrol or pH 5.0. Carvacrol at different concentrations was prepared using pH 7.4 extracellular solutions. Voltage ramps from −100 mV to +80 mV. c Plot of normalized currents (I_carvacrol/I_pH5, %) versus carvacrol in mOTOP1, mOTOP2, or mOTOP3 channels (mean ± SEM, n = 3–5 cells). currents recorded at −100 mV. d–h Representative I–V curves of five vertebrate OTOP1 channels in response to 500 μM carvacrol at pH 7.4, voltage ramps from −100 mV to +80 mV. i Statistics of activity under 500 μM carvacrol on OTOP1 channels in various vertebrates, currents recorded at −100 mV (pH_o= 7.4). Animal icons: Oryx dammah adapted from “Oryx gazella”, credited to Jan A. Venter, Herbert H. T. Prins, David A. Balfour & Rob Slotow (vectorized by T. Michael Keesey). Gallus gallus reprinted from “Gallus gallus domesticus”, credited to Soledad Miranda-Rottmann. Both “Oryx gazella” and “Gallus gallus domesticus” were retrieved from the Attribution 3.0 Unported license: https://creativecommons.org/licenses/by/3.0/. Bufo gargarizans, Chelonia mydas and Danio rerio adapted from public domain, retrieved from CC0 1.0 Universal Public Domain Dedication license: https://creativecommons.org/publicdomain/zero/1.0/.

Fig. 4. Phe⁶⁷-Thr¹⁴³ and Val²¹⁶-Arg²⁹⁴ regions are sufficient for carvacrol activation in mOTOP1.

a–e Representative currents recorded from HEK293T cells expressing either wild-type mOTOP1 channels or chimeric channels (substituting homologous fragments of CmOTOP1 for mOTOP1) in response to 500 μM carvacrol. Voltage ramps from −100 mV to +80 mV. f Current amplitude changes of mOTOP1 and its chimeras, with recorded at −100 mV. ΔI= I_carvacrol - I_{pH 7.4,} (mean ± SEM, n = 3–6 cells), Cm(451-620)/M(443-600) chimera was observed to be non-conductive. g–k Representative currents recorded from HEK293T cells expressing either wild-type CmOTOP1 channels or chimeric channels (substituting homologous fragments of mOTOP1 for CmOTOP1) in response to 500 μM carvacrol. Voltage ramps from −100 mV to +80 mV. l Current amplitude changes of CmOTOP1 and its chimeras under 500 uM carvacrol, currents recorded at −100 mV. ΔI = I_carvacrol−I_{pH 7.4} (mean ± SEM, n = 3–6 cells).

Fig. 5. S134 and T247 are essential for carvacrol activation.

Representative traces of mOTOP1_S134G (a), mOTOP1_T247A (b), and mOTOP1_S134G_T247A (c) currents in response to 500 μM carvacrol or pH 4.5 extracellular solutions. d Normalized currents for fold potentiation measured from single and double mutations of mOTOP1. mean ± SEM, n = 3–5. Statistical significance compared to wild-type was determined using one-way ANOVA followed by Bonferroni’s post hoc test, ***p < 0.001, ****p < 0.0001. Representative traces of CmOTOP1_G134S (e) and CmOTOP1_A247T (f) currents in response to 500 μM carvacrol or pH 4.5 extracellular solutions. g Normalized currents for fold potentiation measured from single mutations of CmOTOP1. mean ± SEM, n = 3. Statistical significance compared to wild-type was determined using one-way ANOVA followed by Bonferroni’s post hoc test, *p < 0.05, **p < 0.01. h Representative traces of mOTOP1 currents in response to 500 μM p-Cymene. i Images were generated using the AlphaFold predicted structure of mOTOP1. Left panel shows sideview. Each zoom-in highlights residues abrogates the activatory effect of carvacrol.