biostudies-literatureSchmiege PU.S. Department of Health & Human Services | NIH | National Institute of General Medical SciencesU.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)NHLBI NIH HHSU.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood InstituteG. Harold and Leila Y. Mathers FoundationWelch FoundationG. Harold and Leila Y. Mathers Foundation (G. Harold & Leila Y. Mathers Foundation)U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)NIGMS NIH HHS5325https://www.ebi.ac.uk/biostudies/studies/S-EPMC12174366biostudies-literatureUnknown16(1)TRPML2 activity is critical for endolysosomal integrity and chemokine secretion, and can be modulated by various ligands. Interestingly, two ML-SI3 isomers regulate TRPML2 oppositely. The molecular mechanism underlying this unique isomeric preference as well as the TRPML2 agonistic mechanism remains unknown. Here, we present six cryo-EM structures of human TRPML2 in distinct states revealing that the π-bulge of the S6 undergoes a π-α transition upon agonist binding, highlighting the remarkable role of the π-bulge in ion channel regulation. Moreover, we identify that PI(3,5)P₂ allosterically affects the pose of ML2-SA1, a TRPML2 specific activator, resulting in an open channel without the π-α transition. Functional and structural studies show that mutating the S5 of TRPML1 to that of TRPML2 enables the mutated TRPML1 to be activated by (+)ML-SI3 and ML2-SA1. Thus, our work elucidates the activation mechanism of TRPML channels and paves the way for the development of selective TRPML modulators.Nature communicationsTRPML2 in distinct states reveals the activation and modulation principles of the TRPML family.PMC12174366R35 GM149533MF-2302-03702P01HL160487R35GM149533I-1957P01 HL020948Hatton AJaslan DLi XSadanandan NPFine MElghobashi-Meinhardt NSchmiege PGrimm CfalseTRPML2 in distinct states reveals the activation and modulation principles of the TRPML family.TRPML2 activity is critical for endolysosomal integrity and chemokine secretion, and can be modulated by various ligands. Interestingly, two ML-SI3 isomers regulate TRPML2 oppositely. The molecular mechanism underlying this unique isomeric preference as well as the TRPML2 agonistic mechanism remains unknown. Here, we present six cryo-EM structures of human TRPML2 in distinct states revealing that the π-bulge of the S6 undergoes a π-α transition upon agonist binding, highlighting the remarkable role of the π-bulge in ion channel regulation. Moreover, we identify that PI(3,5)P₂ allosterically affects the pose of ML2-SA1, a TRPML2 specific activator, resulting in an open channel without the π-α transition. Functional and structural studies show that mutating the S5 of TRPML1 to that of TRPML2 enables the mutated TRPML1 to be activated by (+)ML-SI3 and ML2-SA1. Thus, our work elucidates the activation mechanism of TRPML channels and paves the way for the development of selective TRPML modulators.2025-01-01T00:00:00Z2025 Jun2026-06-03T07:55:22.556Z2026-04-26T03:10:41.392ZS-EPMC121743664052787310.1038/s41467-025-60710-8