Mechanically activated ion channels underlie touch, hearing, shear-stress sensing, and response to turgor pressure. OSCA/TMEM63s are a newly-identified family of eukaryotic mechanically activated ion channels opened by membrane tension. The structural underpinnings of OSCA/TMEM63 function are not explored. Here, we elucidate high resolution cryo-electron microscopy structures of OSCA1.2, revealing a dimeric architecture containing eleven transmembrane helices per subunit and surprising topological similarities to TMEM16 proteins. We locate the ion permeation pathway within each subunit by demonstrating that a conserved acidic residue is a determinant of channel conductance. Molecular dynamics simulations reveal membrane interactions, suggesting the role of lipids in OSCA1.2 gating. These results lay a foundation to decipher how the structural organization of OSCA/TMEM63 is suited for their roles as MA ion channels.

Otopetrins (Otop1–Otop3) comprise one of two known eukaryotic proton-selective channel families. Otop1 is required for otoconia formation and 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, with each subunit forming twelve transmembrane helices divided into structurally similar amino (N) and carboxy (C) domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a central tunnel. In molecular dynamics simulations, hydrophilic vestibules formed by the N and C domains and in the intrasubunit interface between N and C domains form conduits for water entry into the membrane core, suggesting three potential proton conduction pathways. By mutagenesis, we test the roles of charged residues in each putative permeation pathway. Our results provide a structural basis for understanding selective proton permeation and gating of this conserved family of proton channels.

Main 41We purified Arabidopsis thaliana OSCA1.2 expressed in HEK293F cells and determined 42 cryo-EM reconstructions in lipidic nanodiscs and LMNG detergent micelle with cholesteryl 43 hemisuccinate (CHS) ( Fig. 1a-d, Extended Data Figs. 1,2). The resulting density maps 44 allowed building of 82% of the full-length OSCA1.2 sequence (Extended Data Fig. 3, 45 Extended Data Table 1). Structures of OSCA1.2 in nanodisc and LMNG were nearly 46 identical (RMSD=0.81 Å, Extended Data Fig. 4a-c). We refer to the nanodisc structure 47