title: Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes.
abstract: The present work investigates the effect of phosphatidylinositol-4,5-bisphosphate (PIP(2)) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co-immunoprecipitation methods. Inclusion of 100 microM diC8-PIP(2) in the patch pipette and bathing solutions, respectively, inhibited angiotensin II (Ang II)-evoked whole-cell cation currents and TRPC6 channel activity by over 90%. In inside-out patches diC8-PIP(2) also inhibited TRPC6 activity induced by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) with an IC(50) of 7.6 microM. Anti-PIP(2) antibodies potentiated Ang II- and OAG-evoked TRPC6 activity by about 2-fold. Depleters of tissue PIP(2) wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP(2) scavenger poly-L-lysine. Wortmannin reduced Ang II-evoked TRPC6 activity by over 75% but increased OAG-induced TRPC6 activity by over 50-fold. Co-immunoprecipitation studies demonstrated association between PIP(2) and TRPC6 proteins in tissue lysates. Pre-treatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP(2). These results provide for the first time compelling evidence that constitutively produced PIP(2) exerts a powerful inhibitory action on native TRPC6 channels.
authors.affiliation: Ion Channels & Cell Signalling Research Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London SW17 0RE, UK. aalbert@sgul.ac.uk
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Albert
authors.forename: Sohag N
authors.initials: SN
authors.lastname: Saleh
authors.forename: William A
authors.initials: WA
authors.lastname: Large
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004594:Electrophysiology; D008638:Mesenteric Arteries / Q000166:cytology*; D009025:Morpholines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism / Q000494:pharmacology*; D011817:Rabbits; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors*; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D000906:Antibodies; D002867:Chromones; D004075:Diglycerides; D009025:Morpholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D050052:TRPC Cation Channels; D014662:Vasoconstrictor Agents; C044295:1,2-dioctanoylglycerol; D000804:Angiotensin II; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C038237:1-oleoyl-2-acetylglycerol; D000077191:Wortmannin