Cytoplasmic inter-subunit interface modulates TRPC5 activity: Molecular mechanism behind intellectual disability-related R175C mutation

The TRPC5 (Transient Receptor Potential Canonical 5) receptor, which is highly expressed in the brain, kidney, and sensory neurons, is emerging as a pharmacological target due to its involvement in renal physiology, pain, metabolic homeostasis, and various neurodevelopmental disorders. The recently identified R175C mutation associated with intellectual disability and autism spectrum disorders has supported the critical importance of TRPC5 for central nervous system function. Compared to other neuronal TRPs, TRPC5 remains understudied, with its activation mechanisms not well understood. Here, we focused on elucidating the molecular mechanisms of TRPC5 gating that are impaired by the R175C mutation. Based on the structural analyses and bioinformatics profiling, we recognized R175 as part of an inter-subunit interface involved in various modes of TRPC5 gating. Using molecular simulations, site-directed mutagenesis and electrophysiological measurements, we identified that a unique evolutionarily conserved region (dubbed zero-tolerance) contributes to the dynamic network of hydrogen bonds at this interface. Our data show that while the stability of the closed conformation is associated with more contacts at the lower part (Q309) of the zero-tolerance region, fewer contacts here and more at the upper part (R323) apparently accompany the open conformation. Moreover, the activity of TRPC5 can be tuned from this interface by cellular processes, as it is a site of post-translational modification, including putative phosphorylation at S193. In conclusion, our results characterize the molecular mechanism underlying the functional impairment associated with the R175C mutation and provide a possible explanation for the functional importance of the zero-tolerance region in TRPC5.