Zn²⁺potentiation of OTOP proton channels identifies structural elements of the gating apparatus

Otopetrin proteins (OTOPs) form proton-selective ion channels that are expressed in diverse cell types where they may mediate detection of acids or regulation of pH. In vertebrates there are three family members: OTOP1 is required for formation of otoconia in the vesibular system and it forms the receptor for sour taste, while the functions of OTOP2, and OTOP3 are not yet known. Importantly, the gating mechanisms of any of the OTOP channels are not well-understood, and until recently, it was not even known if the channels were gated. Here we show that Zn²⁺, as well as other transition metals including copper (Cu²⁺), potently activate murine OTOP3. Zn²⁺pre-exposure increases the magnitude of OTOP3 currents to a subsequent acid stimulus by as much as 10-fold. In contrast, OTOP2 currents are insensitive to potentiation by Zn²⁺. Swapping the extracellular tm 11-12 linker between OTOP3 and OTOP2 was sufficient to eliminate Zn²⁺potentiation of OTOP3 and confer Zn²⁺potentiation on OTOP2. We also show that H531 within the tm 11-12 linker is essential for potentiation of OTOP3 by Zn²⁺, likely by forming part of its binding site. Kinetic modeling of the data is consistent with Zn²⁺stabilizing the open state of the channel, possibly competing with H⁺for activation of the channels. These results establish the tm 11-12 linker as part of the gating apparatus of OTOP channels and a target for drug discovery. Zinc is an essential micronutrient and its regulation of OTOP channels will undoubtedly have important physiological sequelae.