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

2017 
Abstract 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)-Gq/11-phospholipase C (PLC) pathway. In this study, we aimed to identify the molecular mechanisms involved in regulating TRPC5 activity. We report that R593, a residue located in the E4 loop near the TRPC5′s extracellular Gd3+-binding site, is critical for conferring the sensitivity to GPCR-Gq/11-PLC-dependent gating on TRPC5. Indeed, GTPγS and GPCR agonists only weakly activate the TRPC5R593A mutant, whereas addition of Gd3+ rescues the mutant′s sensitivity to GPCR-Gq/11-PLC-dependent gating. Computer modeling suggests that R593 may cross-bridge the E3 and E4 loops, forming the ″molecular fulcrum.″ While validating the model using site-directed mutagenesis, we found that the Y542 residue is critical for establishing a functional Gd3+ binding site, the Y541 residue participates in fine-tuning Gd3+-sensitivity, and that the N584 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.
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