Schneiter, D. M.; Rougier, J.-S.; Abriel, H.; Stahlberg, H.; Ekundayo, B. E.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated cation channel whose pharmacology is shaped by its molecular environment. It remains poorly understood how temperature and membrane context influence inhibitor recognition. Here we combine cryo-electron microscopy of membrane-derived vesicles and detergent-solubilized TRPM4 to investigate lipid-associated architecture and binding of the potent anthranilic anilide inhibitor PBA. We find that membrane vesicles preserve a native-like paralipid environment and reveal lipid binding patterns highly similar to those observed in GDN, supporting detergent-solubilized TRPM4 as a structurally relevant system for ligand analysis. Strikingly, PBA occupies distinct binding pockets at 8 {degrees}C and 37 {degrees}C. At low temperature, PBA binds in a previously described inhibitor pocket formed by S3, S4, the S4-S5 linker and the TRP helix, whereas at physiological temperature it relocates to a distinct site within the S1-S4 domain proximal to the Ca2+ regulatory region. These findings reveal temperature-dependent plasticity in TRPM4 ligand recognition.