Chen, XingjuanLi, WennanRiley, Ashley M.Soliman, MarioChakraborty, Saikat ChakrabortStamatkin, Christopher W.Obukhov, Alexander G.2018-05-312018-05-312017-01-20Chen, X., Li, W., Riley, A. M., Soliman, M., Chakraborty, S., Stamatkin, C. W., & Obukhov, A. G. (2017). 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. The Journal of Biological Chemistry, 292(3), 898–911. http://doi.org/10.1074/jbc.M116.755470https://hdl.handle.net/1805/16310Transient 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)-Gq/11-phospholipase C (PLC) pathway. In this study we aimed to identify the molecular mechanisms involved in regulating TRPC5 activity. We report 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-Gq/11-PLC-dependent gating on TRPC5. Indeed, guanosine 5'-O-(thiotriphosphate) and GPCR agonists only weakly activate the TRPC5R593A mutant, whereas the addition of Gd3+ rescues the mutant's sensitivity to GPCR-Gq/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 Tyr-542 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-Gq/11-PLC-dependent gating of a receptor-operated channel.en-USPublisher PolicyG-protein-coupled receptor (GPCR)CalciumIon channelPatch clampTransient receptor potential channels (TRP channels)Article