An evolutionarily conserved gene family encodes proton-selective ion channels

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Science  02 Mar 2018:
Vol. 359, Issue 6379, pp. 1047-1050
DOI: 10.1126/science.aao3264
  • Fig. 1 Expression analysis of taste-cell–enriched genes identifies OTOP1 as a previously unknown proton channel.

    (A) Transcriptome profiling of PKD2L1 and TRPM5 taste receptor cells (each data point represents the average of five 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 (Vm = –80 mV; data are mean ± SEM, n = 3 to 37 cells; for OTOP1, n = 5). ****P < 0.0001 compared to uninjected oocytes (n = 3). One-way analysis of variance with Bonferroni correction. (Inset) Currents evoked in an OTOP1-expressing oocyte in response to the acid stimulus at Vm = –80mV (left) and the current-voltage (I-V) relationship before application (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 pHo as indicated (Vm = –80 mV). (D) I-V relation of the current in (A) from voltage ramps (1 V/s). (E) Evoked current (ΔI; mean ± SEM) as a function of pH in Xenopus oocytes expressing OTOP1 (blue circle; n = 4) and uninjected oocytes (gray circles; n = 4). (F) Currents measured by whole-cell patch clamp recording in a HEK-293 cell expressing OTOP1 in Na+-free extracellular solutions (pHi = 7.3, Vm = –80 mV). (G) I-V relation of currents in an OTOP1-expressing HEK-293 cell from experiments as in (G) with voltage ramps (1 V/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 (gray 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 three replicates. (B) Average data (mean ± SEM; n = 28 or 29 cells) were analyzed by two-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 (Vm = –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), and 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 (pHi = 6.0; Zn2+-sensitive component is shown; see fig. S5 and methods). (E) Erev as a function of ΔpH (pHi – pHo) from experiments as in (D). The red line is the equilibrium potential for H+, EH. The data were fit by linear regression with a slope of 53 mV/ΔpH and a y intercept of 3.6 mV (correlation coefficient 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 multisequence alignment of 13 otopetrin domain proteins. Scale bar indicates amino acid substitutions per site. dm, Drosophila melanogaster; ce, Caenorhabditis elegans. (B) Distribution of Otop genes in selected murine tissues from microarray data (16). Scale represents expression level in arbitrary units (mean ± SEM, n = 2). (C, F, I) Representative traces (Vm = –80 mV) showing currents evoked in Xenopus oocytes expressing OTOP2, OTOP3, or dmOTOPLc in response to varying pHo 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), and (I). (E, H, K) The average current induced at Vm = –80 mV (ΔI) as a function of pH for oocytes expressing each of the channels (black circles; mean ± SEM, n = 3 to 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-sequencing 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, 10 μM. Arrow indicates taste pore. (C) Current in response to a pH 5.0 stimulus in isolated PKD2L1 TRCs from tlt mutant or wild-type (WT) mice in NMDG+-based solution (Vm = –80 mV). (D) Average data from experiments as in (C) (****P < 0.0001 by two tailed t test, n = 8 cells per genotype). (E) Response of PKD2L1 TRCs to NMDG+-based extracellular solution of varying pH (Vm = –80 mV). (F) Average data from experiments as in (E). (G) Voltage-gated Na+ currents in TRCs from tlt and wild-type mice were indistinguishable (P > 0.05, two-tailed t test).

Supplementary Materials

  • An evolutionarily conserved gene family encodes proton-selective ion channels

    Yu-Hsiang Tu, Alexander J. Cooper, Bochuan Teng, Rui B. Chang, Daniel J. Artiga, Heather N. Turner, Eric M. Mulhall, Wenlei Ye, Andrew D. Smith, Emily R. Liman

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S11
    • Table S1
    • References

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