Fig. 4: L11–12 is structurally and functionally essential for the Otop1 channel.

a Top view of the cryo-EM structure of the zebrafish Otop1 (PDB 6NF4)²³, showing that L11–12, which is in purple, crosses the center of the C pore. b Side view of the cryo-EM structure of the zebrafish Otop1 (left) and a cartoon (right) show the gap between S7 and S12 in the top 1/3 portion of the C barrel caused by the relatively lower position of S11 in the transmembrane part of the Otop1 structure. L11–12 extends upwards and crosses the pore to reach S12. S6 is bent (indicated by dashed lines) to further leave the gap open for access. c Top view of the cryo-EM structure of the zebrafish Otop1 to show the two very conserved tight interactions (shown in the black dotted line) that anchor L11–12 in the right position. Side chains of the four residues involved in the interactions, as well as D544 (D570 in human), are shown as sticks. Residue numbers are indicated in both zebrafish and human (in parenthesis) sequences. d Left: the representative I–V curves of the indicated WT and mutant human Otop1 channels. Right: the scatter plot and bar graph showing the currents of the indicated channels recorded at −100 mV at the indicated pHs. 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 < 0.05, ***P < 0.001, n.s.: no significance). Oocyte numbers for scatter plots and bar graphs are indicated in parentheses. e Western blot showing the overall expression (in lysate samples) and surface expression of indicated WT and mutant channels. The surface proteins were purified by surface biotinylation.