Zinc activation 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 mediate detection of acids or regulation of pH. In vertebrates there are three family members: OTOP1 is required for formation of otoconia in the vestibular 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. Here, we show that zinc (Zn²⁺⁾, as well as other transition metals including copper (Cu²⁺), potently activates murine OTOP3 (mOTOP3). Zn²⁺ pre-exposure increases the magnitude of mOTOP3 currents to a subsequent acid stimulus by as much as 10-fold. In contrast, mOTOP2 currents are insensitive to activation by Zn²⁺. Swapping the extracellular tm 11-12 linker between mOTOP3 and mOTOP2 was sufficient to eliminate Zn²⁺ activation of mOTOP3 and confer Zn²⁺ activation on mOTOP2. Mutation to alanine of H531 and E535 within the tm 11-12 linker and H234 and E238 within the 5-6 linker reduced or eliminated activation of mOTOP3 by Zn²⁺, indicating that these residues likely contribute to the Zn²⁺ activating site. Kinetic modeling of the data is consistent with Zn²⁺ stabilizing the opn2+en state of the channel, competing with H⁺ for activation of the channels. These results establish the tm 11-12 and tm 5-6 linkers as part of the gating apparatus of OTOP channels and a target for drug discovery. Zn²⁺ is an essential micronutrient and its activation of OTOP channels will undoubtedly have important physiological sequelae.