Obligatory role for phosphatidylinositol 4,5-bisphosphate in activation of native TRPC1 store-operated channels in vascular myocytes.
In the present study the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) was studied on a native TRPC1 store-operated channel (SOC) in freshly dispersed rabbit portal vein myocytes. Application of diC8-PIP(2), a water soluble form of PIP(2), to quiescent inside-out patches evoked single channel currents with a unitary conductance of 1.9 pS. DiC8-PIP(2)-evoked channel currents were inhibited by anti-TRPC1 antibodies and these characteristics are identical to SOCs evoked by cyclopiazonic acid (CPA) and BAPTA-AM. SOCs stimulated by CPA, BAPTA-AM and the phorbol ester phorbol 12,13-dibutyrate (PDBu) were reduced by anti-PIP(2) antibodies and by depletion of tissue PIP(2) levels by pre-treatment of preparations with wortmannin and LY294002. However, these reagents did not alter the ability of PIP(2) to activate SOCs in inside-out patches. Co-immunoprecipitation techniques demonstrated association between TRPC1 and PIP(2) at rest, which was greatly decreased by wortmannin and LY294002. Pre-treatment of cells with PDBu, which activates protein kinase C (PKC), augmented SOC activation by PIP(2) whereas the PKC inhibitor chelerythrine decreased SOC stimulation by PIP(2). Co-immunoprecipitation experiments provide evidence that PKC-dependent phosphorylation of TRPC1 occurs constitutively and was increased by CPA and PDBu but decreased by chelerythrine. These novel results show that PIP(2) can activate TRPC1 SOCs in native vascular myocytes and plays an important role in SOC activation by CPA, BAPTA-AM and PDBu. Moreover, the permissive role of PIP(2) in SOC activation requires PKC-dependent phosphorylation of TRPC1.
Saleh
Sohag N
SN
Ion Channels and Cell Signalling Research Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, UK.
Albert
Anthony P
AP
Large
William A
WA
D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000494:pharmacology; D053119:Benzophenanthridines / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007155:Immunologic Factors / Q000494:pharmacology; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D009025:Morpholines / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000037:antagonists & inhibitors / Q000502:physiology*; D011169:Portal Vein / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011817:Rabbits; D050052:TRPC Cation Channels / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D045163:Antibodies, Phospho-Specific; D053119:Benzophenanthridines; D002614:Chelating Agents; D002867:Chromones; D007155:Immunologic Factors; D007211:Indoles; D009025:Morpholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D047428:Protein Kinase Inhibitors; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004533:Egtazic Acid; C016299:chelerythrine; D019870:1-Phosphatidylinositol 4-Kinase; D011493:Protein Kinase C; C064758:Ro 31-8220; C000543:cyclopiazonic acid; D000077191:Wortmannin