A single allosteric site merges activation, modulation and inhibition in TRPM5

TRPM5 is a Ca²⁺-activated monovalent cation channel essential for taste perception, insulin secretion and gastrointestinal chemosensation. Canonical TRPM5 activation requires Ca²⁺ binding at two distinct sites: an agonist site within the lower vestibule of the S1-S4 pocket in the transmembrane domain (Ca_TMD) and a modulatory site in the intracellular domain (Ca_ICD) that tunes voltage dependence and agonist sensitivity. Here we characterize CBTA as a noncalcium agonist that binds to the upper vestibule of the S1-S4 pocket, directly above Ca_TMD. CBTA alone mimics the dual role of Ca_TMD and Ca_ICD, merging agonist activation with voltage modulation. CBTA also renders TRPM5 supersensitive to Ca²⁺, synergistically hyperactivating the channel even at near-resting Ca²⁺ levels. We further demonstrate that the inhibitor triphenylphosphine oxide binds the same site but stabilizes a nonconductive state. These opposing effects reveal the upper S1-S4 pocket as a multifunctional regulatory hub integrating activation, inhibition and modulation in TRPM5.