Temperature and intrinsic Ca 2+ reshape TRPM4 pharmacology

Abstract Proteins operate in dynamic environments where ions, lipids, and temperature collectively define their properties, yet most studies rely on simplified conditions that overlook these intrinsic variables. Here, we show two such factors—temperature and Ca2+—profoundly remodel the function and pharmacology of TRPM4, an ion channel implicated in cardiac conduction, immune regulation, cancer, and intestinal fluid homeostasis. At physiological temperature and Ca2+, TPPO—previously considered a selective TRPM5 inhibitor inactive toward TRPM4—potently activates TRPM4, revealing strong synergy among temperature, Ca2+, and ligand binding. In contrast, Necrocide-1, a necroptotic activator targeting the same binding pocket, defies this logic: it opens TRPM4 without Ca2+ but is antagonized by Ca2+. Meanwhile, the inhibitors NBA and CBA engage a nearby pocket, locking the channel in a non-conductive pre-open state. Our findings highlight that even rigid binding pockets can exhibit temperature-dependent ligand recognition, revealing hidden pharmacology and informing selective, environment-aware therapeutic strategies. Competing Interest Statement The authors have declared no competing interest. Footnotes This version of the manuscript has been revised to include additional mutational analyses of the agonist and antagonist binding sites within the S1-S4 domain, along with an updated Discussion.