5370 S. N. Saleh and others J Physiol 587.22 PIP3 evokes TRPC1 channel activity independently of PIP2 The present work shows that stimulation of ETA receptors activates TRPC1 activity through a PI-3-kinase-mediated pathway involving PIP3 (see Figs 4 and 5). Moreover we demonstrate that exogenous diC8-PIP3 evokes PKC-dependent TRPC1 channel activity (see Fig. 5). In contrast, our data indicate that activation of TRPC1 channels by stimulation of ETB receptors involves a permissive role for PIP2. In a previous report we stated that PIP2 had an obligatory role for TRPC1 channel activation in rabbit portal vein myocytes (Saleh et al. 2009). Therefore we investigated whether endogenous PIP2 was necessary for activation of TRPC1 channels by PIP3 in coronary artery smooth muscle cells. Figure 7A shows that following pre-treatment of myocytes with 20 μM wortmannin for 30 min to deplete PIP2 levels (see Fig. 5Ca) bath application of 3 μM diC8-PIP3 activated TRPC1 channel activity with a mean peak NPo value of 0.31 ± 0.05 (n = 6) in inside-out patches, which is similar to control values of channel activity induced by 3 μM PIP3 in the absence of wortmannin (see above and Fig. 7Aa). Figure 7B shows that anti-PIP2 antibodies had no effect on PIP3-induced TRPC1 channel activity in inside-out patches (control mean NPo of 0.23 ± 0.06 and 0.24 ± 0.08 in the presence of anti-PIP2 antibodies, n = 6). Both of these procedures blocked OAG-evoked TRPC1 channel activity (cf. Fig. 6E and F). In other experiments the anti-PIP2 antibody reduced TRPC1 channel activation by both ETA and ETB receptor stimulation (data not shown). This is pre- dictable since PIP2 acts as a substrate for PIP3 generated by PI-3-kinase (ETA pathway) and DAG produced by PI-PLC (ETB pathway). Therefore with regard to direct TRPC1 channel activation endogenous PIP2 is not obligatory for TRPC1 channel activation by PIP3 (ETA receptor pathway) but Figure 7. PIP3 activation of TRPC1 channel activity is independent of PIP2 A, diC8-PIP3-evoked TRPC1 channel activity in inside-out patches held at −70 mV was not inhibited by pre-treatment with 20 μM wortmannin for 30 min. B, 1 : 200 anti-PIP2 antibodies did not inhibit diC8-PIP3-mediated channel activation. Ca, co-immunoprecipitation experiment showing association between PIP2 and TRPC1 proteins at rest (Con) after immunoprecipitation with anti-TRPC1 antibodies and blotting with anti-PIP2 antibodies which was reduced following pre-treatment with 20 μM wortmannin (Wort). Cb, upper panel shows co-immunoprecipitation experiment following immunoprecipitation with anti-PIP2 antibodies and blotting with anti-TRPC1 antibodies illustrating that PIP2 association with TRPC1 proteins is unaltered following pre-treatment with 100 nM ET-1 and stimulation of ETA (100 nM ET-1 + 100 nM BQ-788) or ETB (100 nM ET-1 + 100 nM BQ-123) receptors. Lower panel shows a Western blot in which following preincubation with its antigenic peptide (AgP), detection of TRPC1 protein with anti-TRPC1 antibodies was reduced. Ca and b also show that wortmannin and antigenic peptide had no effect on expression of β-actin proteins. D, dot-blot showing the presence of PIP2 but not PIP3 levels in tissue lysates at rest and the reduction of PIP2 levels and increase of PIP3 levels following stimulation of ETA receptors. C 2009 The Authors. Journal compilation C 2009 The Physiological Society