Human OTOP3 requires strong acidification for activation and exhibits mixed proton-anion permeation.

The OTOPetrin (OTOP) proton channel family comprises three members: OTOP1, OTOP2, and OTOP3. While OTOP1 has been established as the primary sour taste receptor, the physiological roles of OTOP2 and OTOP3 remain largely unknown. Although OTOP channels are widely recognized as proton-selective ion channels, their functional diversity is not fully understood. Here, we show that human OTOP3 (hOTOP3) exhibits distinct gating and permeation properties compared with mouse OTOP3 (mOTOP3) and other OTOP family members. Using two-electrode voltage clamp recordings in Xenopus oocytes, we found that hOTOP3 requires stronger extracellular acidification for activation, indicating a higher threshold for proton-dependent gating. We further identify the extracellular loop between the fifth and the sixth transmembrane segments (L5-6) contributes to this difference in pH sensitivity. Notably, under strongly acidic conditions, hOTOP3 exhibits a measurable anion permeability, a property not observed in human or mouse OTOP1 or OTOP2, nor in mOTOP3. Mutational analysis reveals coordinated effects on proton and anion currents, suggesting that the two permeation processes are mechanistically coupled. Together, these findings demonstrate that hOTOP3 exhibits a higher activation threshold and condition-dependent mixed proton-anion permeation. These results expand the functional diversity of the OTOP channel family and suggest that hOTOP3 may be adapted to function in highly acidic microenvironments, where coupled proton and anion flux could help maintain electrochemical homeostasis.