Conformational Fingerprints Underlying Thermal Modulation of PAC/TMEM206 Gating

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.