Structures of the Otopetrin Proton Channels Otop1 and Otop3

Otopetrins (Otop1-Otop3) comprise one of only two known eukaryotic proton-selective channel families. Otop1 is required for formation of otoconia and is a candidate mammalian sour taste receptor. Here, we report cryo-EM structures of zebrafish Otop1 and chicken Otop3 in lipid nanodiscs. The structures reveal a dimeric architecture of Otopetrins with each subunit consisting of twelve transmembrane helices divided into structurally related N and C domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a cavernous central tunnel. Two hydrophilic vestibules, as well as the intrasubunit interface between N and C domains, form conduits for water entry into the membrane plane in molecular dynamics simulations, suggesting they each could provide pathways for proton conduction. We also demonstrate the functional relevance of a salt bridge in the C domain vestibule by mutagenesis. Our results provide a structural basis for understanding the function of the Otopetrin proton channel family.