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. Significance Statement A family of proton-activated H+ ion channels was recently identified that includes the sour receptor OTOP1. Here we show that members of the OTOP channel family are differentially sensitive to Zn2+, which strongly activates OTOP3 but not OTOP2. By studying chimeric channels, we identify structural elements in the extracellular linker between transmembrane domains 11-12 that are necessary and sufficient for Zn2+ activation of OTOP channels. In addition to the taste system, OTOP channels play important roles in biomineralization in both vertebrate and invertebrates and are expressed in the digestive tract where their expression is a predictor of cancer prognosis. Our identification of the tm 11-12 linker as part of the gating apparatus makes it a promising target for pharmaceutical discovery.