An evolutionarily conserved gene family encodes proton-selective ion channels

Abstract

Ion channels form the basis for cellular electrical signaling. Despite the scores of genetically identified ion channels selective for other monatomic ions, only one type of proton-selective ion channel has been found in eukaryotic cells. By comparative transcriptome analysis of mouse taste receptor cells, we identified Otopetrin1 (OTOP1), a protein required for development of gravity-sensing otoconia in the vestibular system, as forming a proton-selective ion channel. We found that murine OTOP1 is enriched in acid-detecting taste receptor cells and is required for their zinc-sensitive proton conductance. Two related murine genes, Otop2 and Otop3, and a Drosophila ortholog also encode proton channels. Evolutionary conservation of the gene family and its widespread tissue distribution suggest a broad role for proton channels in physiology and pathophysiology.

Fig. 1

Expression analysis of taste-cell enriched genes identifies OTOP1 as a novel proton channel. (A) Transcriptome profiling of PKD2L1 and TRPM5 taste receptor cells (Each data point represents the average of 5 replicates). Genes tested by electrophysiology are highlighted in magenta or red (OTOP1). RPM, reads per million. (B) Magnitude of currents evoked in response to pH 4.5 Na⁺-free solution in Xenopus oocytes expressing the genes indicated (V_m = −80mV; data are mean ± SEM, n = 3 – 37; for OTOP1, n = 5). ****, P < 0.0001 compared to uninjected oocytes (n = 3). One-way ANOVA with Bonferroni correction. Inset: currents evoked in an OTOP1-expressing oocyte to the acid stimulus at V_m = −80mV (left) and the current-voltage relationship before (gray), during acid application (green) and during Zn²⁺ application (black). (C) Current measured by two-electrode voltage clamp in a Xenopus oocyte expressing OTOP1 in response to Na⁺-free extracellular solutions with pH_o as indicated (V_m = −80 mV). (D) I-V relation of the current in (A) from voltage ramps (1V/s). (E) Evoked current (ΔI; mean ± SEM) as a function of pH in Xenopus oocytes expressing OTOP1 (blue circle; n = 4) and uninjected oocytes (grey circles; n = 4). (F) Currents measured by whole-cell patch clamp recording in a HEK-293 cell expressing OTOP1 in Na⁺-free extracellular solutions (pH_i = 7.3, V_m = −80 mV). (G) I-V relation of currents in OTOP1-expressing HEK-293 cell from experiments as in (G) with voltage ramps (1V/s). (H) Evoked currents (ΔI; mean ± SEM) as a function of pH in HEK-293 cells expressing OTOP1 (blue squares; n = 5) and untransfected cells (grey squares; n = 3).

Fig 2

Selectivity of OTOP1 for protons. (A) Fluorescence emission of the pH indicator pHrodo Red in HEK-293 cells expressing OTOP1 (n = 9) and sham-transfected cells (n = 11; mean ± SEM) in response to the stimuli indicated. Similar results were obtained in 3 replicates. (B) Average data (mean ± SEM; n = 28-29 cells) were analyzed by 2-tailed t-test. ****: P < 0.0001. HOAc, acetic acid, which shuttles protons across membranes (4) served as a positive control. (C) OTOP1 currents in HEK-293 cells were evoked in response to a pH 5.5 solution with Na⁺, Li⁺ or Cs⁺ (160 mM each) or Ca²⁺ (40 mM) replacing NMDG⁺ in the extracellular solution as indicated (V_m = −80 mV). Percentage change in currents was 0.4 ± 0.7, n = 8, 2.7 ± 0.7, n = 8, 2.4 ± 0.5, n = 8, 3.6 ± 1.7, n = 7 for each ion replacement respectively. (D) Isolated OTOP1 currents in response to voltage ramps (1 V/s) at varying extracellular pH (pH_i = 6.0; Zn²⁺-sensitive component is shown; see Fig. S5 and methods). (E) E_rev as a function of ΔpH (pH_i-pH_o) from experiments as in (D). The red line is E_H. The data were fit by linear regression with a slope of 53 mV/ΔpH and a Y intercept of 3.6 mV (R² = 0.99).

Fig 3

An evolutionarily conserved family of genes, expressed in diverse tissues and encoding proton channels. (A) Maximum-likelihood phylogenetic tree from the multi-sequence alignment of 13 otopetrin domain proteins. Scale bar is amino acid substitutions per site. (B) Distribution of Otops in selected murine tissues from microarray data (16). Scale represents expression level in arbitrary units (mean ± SEM, n = 2). (C, F, I) Representative traces (V_m = −80 mV) showing currents evoked in Xenopus oocytes expressing OTOP2, OTOP3 or dmOTOPLc in response to varying pH_o of the Na+-free extracellular solution. (D, G, J) I-V relationship (from voltage ramps at 1 V/s) from experiments as in (C, F, I). (E, H, K) The average current induced at V_m = −80 mV (ΔI) as a function of pH for oocytes expressing each of the channels (black circles; mean ± SEM, n = 3–7) and for uninjected oocytes (gray triangles, mean ± SEM, n = 3).

Fig 4

Requirement of OTOP1 for the proton current in taste receptor cells. (A) Read counts per million (RPM) for the genes indicated from RNA-seq data obtained from single PKD2L1 (n = 19) or TRPM5 taste cells (n = 5). 0 RPM was adjusted to 0.01 RPM. (B) Confocal images showing taste buds in the circumvallate papillae from a mouse in which Pkd2l1 drives expression of YFP immunostained with antibodies against YFP (green), OTOP1 (magenta) and TRPM5 (cyan). Scale bar is 10 μM. Arrow indicates taste pore. (C) Current in response to pH 5.0 stimulus in isolated PKD2L1 TRCs from tlt mutant or wildtype mice in NMDG⁺-based solution (V_m = −80 mV). (D) Average data from experiments as in (C) (****: P < 0.0001 by two tailed t-test, n = 8 cells/genotype). (E) Response of PKD2L1 TRCs to NMDG⁺-based extracellular solution of varying pH (V_m = −80 mV). (F) Average data from experiments as in (E). (G) Voltage-gated Na⁺ currents in TRCs from tlt and wildtype mice were indistinguishable (P > 0.05, two-tailed t-test).