Temperature-dependent ligand relocation reveals plasticity of TRPM4 inhibition

Abstract 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 °C and 37 °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.