title: 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.
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 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 TRPC5R593Amutant, 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.
authors.affiliation: From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Xingjuan
authors.initials: X
authors.lastname: Chen
authors.affiliation: From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Wennan
authors.initials: W
authors.lastname: Li
authors.affiliation: From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Riley
authors.affiliation: From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Mario
authors.initials: M
authors.lastname: Soliman
authors.affiliation: From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Saikat
authors.initials: S
authors.lastname: Chakraborty
authors.affiliation: From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Stamatkin
authors.affiliation: From the Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202 aobukhov@iu.edu.
authors.forename: Alexander G
authors.identifier: 0000-0002-3862-6004
authors.initials: AG
authors.lastname: Obukhov
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D005682:Gadolinium / Q000493:pharmacokinetics*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; D050052:TRPC Cation Channels; C491780:TRPC5 protein, human; C112021:Trpc5 protein, mouse; D005682:Gadolinium; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
keywords: G-protein-coupled receptor (GPCR); calcium; ion channel; patch clamp; transient receptor potential channels (TRP channels)