Inactivation Switch for Run-up in TRPV2

The homotetrameric thermosensitive transient receptor potential vanilloid 2 (TRPV2) channel exhibits both inactivation and run-up in response to heat or 2-aminoethoxydiphenyl borate (2-APB). Although a three-state model has been proposed to explain the run-up phenomenon, the role of inactivation remains unclear. In this computational study, a highly sensitive thermoring energetic model was employed to analyze and compare three-dimensional cryo-EM structures of TRPV2 in activated and inactivated states induced by different chemical perturbations. The analysis revealed that, in response to 2-APB or a mild detergent, the weakest tertiary bridge between the pre-S1 domain and the ankyrin repeat domain, which is present in the pre-open closed state, remains intact in the activated state but is disrupted in the inactivated state. In contrast, the highly conserved swapping π bridges near the lower gate are broken in the activated state but re-established in the inactivated state. Furthermore, the greater dynamic systematic thermal instability of the pre-open inactivated state compared with the activated state may account for similar, or mirrored, run-up responses induced by either 2-APB or heat. A stable pre-open activated state was also identified, exhibiting a lower activation threshold and reduced thermosensitivity that were well matched to facilitate subsequent heat activation and thereby enhance heat-induced run-up. These findings demonstrate that thermoring energetic analysis of protein structures in distinct functional states can precisely identify the dynamic allosteric communication networks that regulate protein activity. (229 words).