J Physiol 587.22 PIP3 and PIP2 activation of vascular TRPC1 5369 by PI-PLC, since 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeant DAG analogue, activates TRPC1 channel activity through a PKC-dependent mechanism in rabbit mesenteric artery, portal vein and also coronary artery (see Albert & Large, 2002; Saleh et al. 2006, 2008). Moreover, our recent ﬁndings extended this hypothesis by proposing an obligatory role for PIP2 in PKC-dependent activation of TRPC1 channels in portal vein smooth muscle cells (Saleh et al. 2009) and therefore we investigated the effects of PIP2 in coronary artery myocytes. Bath application of 10 μM diC8-PIP2 to inside-out patches induced cation channel activity which had a mean NPo of 0.34 ± 0.11 at −70 mV (n = 11) and a unitary conductance of 2.6 pS and an Er of about 0 mV (Fig. 6Aa and b). In addition, the mean NPo of diC8-PIP2-evoked channel activity was signiﬁcantly reduced by co-application of anti-TRPC1 antibodies (from 0.24 ± 0.08 to 0.02 ± 0.01, 88 ± 6% inhibition, n = 5, P < 0.01, Fig. 6B) and by 3 μM chelerythrine (from 0.31 ± 0.08 to 0.05 ± 0.03, 97 ± 6% inhibition, n = 6, P < 0.01, Fig. 6C). These data provide evidence that exogenous PIP2 activates TRPC1 channel currents via a PKC-dependent mechanism. OAG-evoked channel activity was signiﬁcantly inhibited by anti-TRPC1 antibodies (Fig. 6D, mean NPo from 0.23 ± 0.08 to 0.03 ± 0.01, 87 ± 1% inhibition, n = 4, P < 0.01) and by anti-PIP2 antibodies (Fig. 6E, mean NPo from 0.32 ± 0.06 to 0.01 ± 0.01, 97 ± 1% inhibition, n = 6, P < 0.01) in inside-out patches. Figure 6F also shows that when tissues were pre-treated with 20 μM wortmannin for 30 min to deplete tissue PIP2 levels (see Fig. 7) OAG did not evoke TRPC1 channel activity in cell-attached patches (mean NPo of 0.01 ± 0.01, n = 6). These results suggest that endogenous PIP2 has an obligatory role for OAG-evoked TRPC1 channel activation. These studies demonstrate that stimulation of ETB receptors induces TRPC1 channel activation through stimulation of PI-PLC to generate DAG which activates PKC leading to channel opening through a mechanism involving endogenous PIP2. Figure 6. Obligatory role of PIP2 in mediating OAG-induced TRPC1 channel activity via a PKC-dependent mechanism Aa and b, bath application of 10 μM diC8-PIP2 activates cation channel activity in inside-out patches at −70 mV which has a unitary conductance of 2.6 pS and a Er of about 0 mV. B and C, diC8-PIP2-evoked channel activity is inhibited by 1 : 200 anti-TRPC1 antibodies (B) and also by 3 μM chelerythrine (C). D and E, OAG-induced channel activity is inhibited by 1 : 200 anti-TRPC1 antibodies (D) and also by 1 : 200 anti-PIP2 antibodies (E) in inside-out patches held at −70 mV. F, pre-treatment with 20 μM wortmannin for 30 min prevented activation of TRPC1 activity by OAG in a cell-attached patches at −70 mV. C 2009 The Authors. Journal compilation C 2009 The Physiological Society