Fig. 1: The extracellular S5–S6 loop and the S11–S12 loop are essential for Otop1 function.

a The putative transmembrane topology of the human Otop1 channel, showing the N (transmembrane segments S1–S6) and C (S7–S12) domains, the loops connecting the TM segments, and the intracellular termini. Loops studied in this work are indicated. b The top (left) and side (right) views of the zebrafish Otop1 channel (PDB 6NF4)²³. The numbers of the transmembrane segments are indicated on one of the subunits on the left. The three putative pores formed within the N and C domains and the intrasubunit interface between the two domains are indicated by dotted red circles. c Representative recording showing the currents of un-injected and human Otop1-injected oocytes at indicated pHs. Oocytes were clamped at −60 mV. d Representative current–voltage relationship (I–V curve) of human Otop1 channel at indicated pHs. e, g Left: the representative I–V curves of the indicated WT and mutant Otop1 channels. Right: the scatter plot and bar graph showing the currents of the indicated channels recorded at −100 mV at the indicated pHs. L5–6∆: S5–S6 loop deletion mutant; L6–7∆: S6–S7 loop deletion mutant; L10–11∆: S10–S11 loop deletion mutant; L5–6/3HA: the mutant with the S5–S6 loop replaced by three fused HA tags; L11–12/HA: the mutant with the S11–S12 loop replaced by an HA tag. Data in the bar graphs are presented as mean ± SD. Currents of the mutants were compared to that of the WT with Student’s t-test (***P < 0.001, n.s.: no significance). Oocyte numbers for scatter plots and bar graphs are indicated in parentheses. f, h Western blot images show the expression of the indicated Otop1 channels. Asterisks indicate the monomer bands of the WT and mutant proteins. Both L5–6/3HA and L11-12/HA gave stronger signals since they contain more than one copy of the HA tag. Mutant L5–6/3HA ran at a higher position on the gel for an unknown reason.