Abstract

Mechanosensitive ion channels such as OSCA1.2 enable cells to sense and respond to mechanical forces by translating membrane tension into ionic flux. While lipid rearrangement in the inter-subunit cleft has been proposed as a key activation mechanism, the contributions of other domains to OSCA gating remain unresolved. Here, we combined the genetic encoding of the photoactivatable crosslinker p-benzoyl-L-phenylalanine (BzF) with functional Ca2+ imaging and molecular dynamics simulations to dissect the roles of specific residues in OSCA1.2 gating. Targeted UV-induced crosslinking at positions F22, H236, and R343 locked the channel in a non-conducting state, indicating their functional relevance. Structural analysis revealed that these residues are strategically positioned: F22 interacts with lipids near the activation gate, H236 lines the lipid-filled cavity, and R343 forms cross-subunit contacts. Together, these results support a model in which mechanical gating involves a distributed network of residues across multiple channel regions, allosterically converging on the activation gate. This study expands our understanding of mechanotransduction by revealing how distant structural elements contribute to force sensing in OSCA channels.