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.

The proton-activated chloride channel PAC/TMEM206 is broadly expressed in mammalian tissues and contributes to acid-induced cell injury in pathological settings such as ischemia, inflammation, and tumor acidosis. In addition to its established proton sensitivity, PAC is strongly modulated by temperature: heating potentiates proton-evoked currents and shifts activation toward less acidic pH yet does not open the channel at neutral pH. How proton and thermal inputs are structurally integrated remains unclear. Here, we combined site-specific incorporation of the environmentally sensitive fluorescent amino acid ANAP with automated patch-clamp electrophysiology and molecular dynamics simulations to identify structural elements underlying PAC thermal modulation. ANAP reporters introduced across the extracellular domain and vestibule–pore coupling region revealed residue-specific thermal- and proton-dependent spectral shifts, showing that heating and acidification remodel overlapping but non-identical local environments. The strongest ANAP-reported temperature-dependent changes were observed at R93, Y111, F196, R237, and F282, whereas proton-dependent changes prominently involved R93, Y111, H130, and R237. Several temperature-sensitive ANAP reporters mapped to an intersubunit region also highlighted by dynamic correlation analysis. Together, our results identify structural correlates of PAC thermal modulation consistent with a model in which protonation creates an activation-permissive landscape while heating reweights coupling across an intersubunit scaffold; this model generates testable predictions that should be addressed by targeted mutagenesis and thermodynamic characterization.