Vera Moiseenkova-Bell, Ph.D., University of Pennsylvania
Advancing the Understanding of TRP Channels Structure and Function through CryoEM and CryoET
Transient receptor potential (TRP) channels integrate cellular signals across diverse tissues, yet key questions remain about their ligand modulation and native membrane organization. We combine single-particle cryoEM, cryoET, proteomics, and electrophysiology to advance both fronts. First, we define a druggable allosteric site in the TRPV subfamily using the uricosuric agent probenecid (PBC). Electrophysiology shows that PBC robustly potentiates rat TRPV2 responses to known stimuli but does not open the channel on its own. High-resolution cryoEM reveals that PBC binds within a previously unrecognized intracellular pocket and engages a conserved TRPV2-specific histidine that stabilizes the channel away from an inactivated carboxyl-terminal conformation. Substituting the corresponding residue in TRPV1 and TRPV3 from glutamine to histidine confers PBC sensitivity, indicating a transferable molecular determinant for pharmacology across TRPV paralogs. In combination with 2-aminoethoxydiphenyl borate, PBC drives TRPV2 into an intermediate, potentiated state, mapping a structural path toward therapeutic modulation.
Second, to place these molecular insights into their cellular context, we developed a cryoET workflow that visualizes membrane protein architecture directly within intact endolysosomes. We isolate native organelles by independently targeting two lysosomal membrane proteins, TRPML1 and TMEM192, yielding vesicles with the expected morphology and proteomic signature of the endolysosomal system. Sub-tomogram averaging resolves key complexes in situ, including V-ATPase, flotillin, and clathrin, and uncovers their heterogeneous distributions across organelles. Integrating proteomics with cryoET establishes a general platform for interrogating lysosomal membrane protein organization and dynamics in health and disease.
Together, these studies identify a ligandable TRPV2 pocket that tunes gating and provide an in situ framework to relate TRP channel pharmacology to native lysosomal membrane architecture, accelerating structure-guided discovery for cardiovascular, neurological, and immunometabolic indications.