TMEM63 proteins form monomeric high-threshold mechanosensitive ion channels

OSCA/TMEM63s constitute the largest family of mechanically activated ion channels identified to date and are evolutionarily related to TMEM16/anoctamin and TMC channels. OSCAs are found in plants while TMEM63 proteins exist in animals. Recent human genetics and animal studies demonstrated that TMEM63s play important roles in the pathophysiology of central nervous system and hearing. How TMEM63 channels are gated by mechanical stimuli and contribute to mechanotransduction processes in vivo remains unclear. Here, we uncover an unanticipated monomeric configuration of TMEM63 proteins that allows these channels to specifically respond to high threshold mechanical stimuli. Using single-particle cryo-electron microscopy, we first resolved atomic structures of TMEM63A and TMEM63B in monomeric configurations, which contrasts with dimeric OSCA, TMEM16 and TMC1 channels. The monomeric states of TMEM63s were confirmed by multiple lines of biochemical and single-molecule imaging evidence. TMEM63s possess 11 transmembrane (TM) domains and a large intracellular loop (IL2) between TM2 and TM3. IL2 mediates dimerization in OSCAs and sequence alignment revealed evolutionary variations of IL2 in TMEM63s. Replacement of the IL2 of OSCA1.2 with that of TMEM63A or mutations in key variable residues in IL2 of OSCA1.2 resulted in monomeric OSCA proteins, revealing IL2 as a molecular determinant underlying the unique monomeric state of TMEM63s. Functionally, monomeric TMEM63s or OSCA1.2 derivatives evoked mechanically-activated currents with significantly higher thresholds, relative to dimeric OSCA1.2. Structural analyses uncovered substantial conformational differences in mechano-sensing domain IL2 and gating helix TM6 between monomeric TMEM63s and dimeric OSCA1.2, which render TMEM63s well-suited to specifically detect high threshold mechanical stimuli. Our studies provide a framework for better understanding of the mechanical gating of TMEM63 channels and suggest that TMEM63s act as high-threshold mechanosensitive ion channels, complementary to Piezos, TMCs and other mechanosensitive channels.