Molecular Determinants of the Sensitivity to Gq/11-Phospholipase C-dependent Gating, Gd3+ Potentiation, and Ca2+ Permeability in the Transient Receptor Potential Canonical Type 5 (TRPC5) Channel

Transient receptor potential canonical type 5 (TRPC5) is a Ca2+ -permeable cation channel that is highly expressed in the brain and is implicated in motor coordination, innate fear behavior, and seizure genesis. The channel is activated by a signal downstream of the G-protein-coupled receptor (GPCR)-G(q/ 11) -phospholipase C (PLC) pathway. In this study we aimed to identify the molecular mechanisms involved in regulating TRPC5 activity. Wereport that Arg-593, a residue located in the E4 loop near the TRPC5 extracellular Gd3+ binding site, is critical for conferring the sensitivity to GPCR-G(q/11)-PLC-dependent gating on TRPC5. Indeed, guanosine 5' -O-(thiotriphosphate) and GPCR agonists only weakly activate the TRPC5(R593A) mutant, whereas the addition of Gd3+ rescues the mutant's sensitivity to GPCR-G(q/ 11)-PLC-dependent gating. Computer modeling suggests that Arg-593 may cross-bridge the E3 and E4 loops, forming the molecular fulcrum. While validating the model using site-directed mutagenesis, we found that the Tyr542 residue is critical for establishing a functional Gd3+ binding site, the Tyr-541 residue participates in fine-tuning Gd3+ -sensitivity, and that the Asn-584 residue determines Ca2+ permeability of the TRPC5 channel. This is the first report providing molecular insights into the molecular mechanisms regulating the sensitivity to GPCR-G(q/ 11)-PLC-dependent gating of a receptor- operated channel.