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 (CaTMD) and a modulatory site in the intracellular domain (CaICD) 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 CaTMD. CBTA alone mimics the dual role of CaTMD and CaICD, 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. TRPM5 activation requires Ca2+ binding at an S1–S4 pocket in the transmembrane domain (TMD) and a modulatory site in the intracellular domain (ICD). Structural and functional analyses of TRPM5 with Ca2+, the agonist CBTA and the inhibitor triphenylphosphine oxide—all engaging the S1–S4 pocket—reveal how modulation of channel activity is communicated across the TMD and ICD.