title: Critical roles of Gi/o proteins and phospholipase C-δ1 in the activation of receptor-operated TRPC4 channels.
abstract: Transient Receptor Potential Canonical (TRPC) proteins form nonselective cation channels commonly known to be activated downstream from receptors that signal through phospholipase C (PLC). Although TRPC3/C6/C7 can be directly activated by diacylglycerols produced by PLC breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. We show here that TRPC4 activation requires coincident stimulation of Gi/o subgroup of G proteins and PLCδ, with a preference for PLCδ1 over PLCδ3, but not necessarily the PLCβ pathway commonly thought to be involved in receptor-operated TRPC activation. In HEK293 cells coexpressing TRPC4 and Gi/o-coupled µ opioid receptor, µ agonist elicited currents biphasically, with an initial slow phase preceding a rapidly developing phase. The currents were dependent on intracellular Ca(2+) and PIP2. Reducing PIP2 through phosphatases abolished the biphasic kinetics and increased the probability of channel activation by weak Gi/o stimulation. In both HEK293 cells heterologously expressing TRPC4 and renal carcinoma-derived A-498 cells endogenously expressing TRPC4, channel activation was inhibited by knocking down PLCδ1 levels and almost completely eliminated by a dominant-negative PLCδ1 mutant and a constitutively active RhoA mutant. Conversely, the slow phase of Gi/o-mediated TRPC4 activation was diminished by inhibiting RhoA or enhancing PLCδ function. Our data reveal an integrative mechanism of TRPC4 on detection of coincident Gi/o, Ca(2+), and PLC signaling, which is further modulated by the small GTPase RhoA. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration in brain and other systems.
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX 77030; Graduate Studies in Cell and Regulatory Biology, The University of Texas Health Science Center at Houston, Houston, TX 77030;
authors.forename: Dhananjay P
authors.initials: DP
authors.lastname: Thakur
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX 77030;
authors.forename: Jin-bin
authors.initials: JB
authors.lastname: Tian
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX 77030;
authors.forename: Jaepyo
authors.initials: J
authors.lastname: Jeon
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX 77030; Graduate Studies in Cell and Regulatory Biology, The University of Texas Health Science Center at Houston, Houston, TX 77030;
authors.forename: Jian
authors.initials: J
authors.lastname: Xiong
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX 77030; Graduate Studies in Cell and Regulatory Biology, The University of Texas Health Science Center at Houston, Houston, TX 77030;
authors.forename: Yu
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Experimental and Clinical Pharmacology and Toxicology, Saarland University, 66421 Homburg, Germany.
authors.forename: Veit
authors.initials: V
authors.lastname: Flockerzi
authors.affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX 77030; Graduate Studies in Cell and Regulatory Biology, The University of Texas Health Science Center at Houston, Houston, TX 77030; Michael.x.zhu@uth.tmc.edu.
authors.forename: Michael X
authors.initials: MX
authors.lastname: Zhu
mesh_terms: D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D054803:Phospholipase C delta / Q000502:physiology*; D015398:Signal Transduction; D050052:TRPC Cation Channels / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000502:physiology
chemical_list: D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C066737:RHOA protein, human; D002217:Carbachol; D054803:Phospholipase C delta; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020742:rhoA GTP-Binding Protein; D002118:Calcium
keywords: G proteins; TRP channels; calcium; pertussis toxin; phospholipase C