Mechanically activated (MA) ion channels convert physical forces into electrical signals, and are essential for eukaryotic physiology. Despite their importance, few bona-fide MA channels have been described in plants and animals. Here, we show that various members of the OSCA and TMEM63 family of proteins from plants, flies, and mammals confer mechanosensitivity to naïve cells. We conclusively demonstrate that OSCA1.2, one of the Arabidopsis thaliana OSCA proteins, is an inherently mechanosensitive, pore-forming ion channel. Our results suggest that OSCA/TMEM63 proteins are the largest family of MA ion channels identified, and are conserved across eukaryotes. Our findings will enable studies to gain deep insight into molecular mechanisms of MA channel gating, and will facilitate a better understanding of mechanosensory processes in vivo across plants and animals.

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.

IntroductionMechanotransduction, the conversion of mechanical cues into biochemical signals, is crucial for many biological processes in plants and animals 1,2 . In mammals, some mechanosensory processes such as touch sensation and vascular development are mediated by the PIEZO family of mechanically activated (MA) ion channels [3][4][5] . In plants, the impact of gravity or soil properties on root development, wind on stem growth, and turgor pressure on plant-cell size and shape are proposed to involve activation of MA ion channels 6,7 . Homologues of the bacterial MA channel MscS (MSLs) exist in plants, and MSL8 is shown to be involved in pollen hydration 8 ; however, the identity of the MA channels required for most mechanotransduction processes in plants have remained elusive 9 . Here, we identify various members of the 15 OSCA proteins from Arabidopsis thaliana (previously reported as hyperosmolarity sensors 10,11 ) as MA ion channels. Purification and reconstitution of OSCA1.2 in liposomes induced stretch-activated currents, suggesting that OSCAs are inherently mechanosensitive, pore-forming ion channels. This conclusion is confirmed by a high-resolution electron microscopy structure of OSCA1.2 described in a companion paper 12 . Beyond plants, we present evidence that fruit fly, mouse, and human TMEM63 family of proteins, homologues of OSCAs, induce MA currents when expressed in naïve cells. Our results suggest that OSCA/TMEM63 proteins are the largest family of MA ion channels identified, and are conserved across eukaryotes. We anticipate that further characterization of OSCA isoforms which have diverse biophysical properties, will help gain substantial insight on the molecular mechanism of MA ion channel gating and permeation. OSCA1.1 mutant plants have impaired leaf and root growth under stress, potentially linking this ion channel to a mechanosensory role 11 . We expect future studies to uncover novel roles of OSCA/TMEM63 channels in mechanosensory processes across plants and animals.3 MainIn Arabidopsis thaliana, hyperosmolarity-evoked intracellular calcium increase is dependent on the genes OSCA1.1 and OSCA1.2 10,11 . However, the activation mechanism for these proteins and whether they encode a pore-forming ion channel remains unknown. We synthesized human codon optimized versions of OSCA1.1 (At4g04340) and OSCA1.2 (At4g22120) cDNA in pIRES2-mCherry vector, heterologously expressed them in mechanicallyinsensitive PIEZO1-knockout HEK293T cells (HEK-P1KO) 13,14 , and electrophysiologically characterized hyperosmolarity-activated currents. In contrast to published reports 10,11 , we find that hyperosmolarity-evoked whole-cell currents recorded from OSCA1.1-or OSCA1.2expressing cells were only modestly larger than baseline currents (Extended Data Fig.1).We next explored the possibility that OSCA1.1 and OSCA1.2 are mechanosensitive, and that the modest hyperosmolarity-induced currents might be due to osmotic shock causing cell shrinking, and affecting membrane tension 15 . In cells, MA currents are commo...

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