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.