Abstract
TRPM5 is a Ca(2+)-activated monovalent cation channel essential for taste perception, insulin secretion and gastrointestinal chemosensation. Canonical TRPM5 activation requires Ca(2+) 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 Ca(2+), synergistically hyperactivating the channel even at near-resting Ca(2+) 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.