J Physiol 587.22 PIP3 and PIP2 activation of vascular TRPC1 5365 ET-1-evoked channel activity shown in Fig. 1Aa could be ﬁtted by the sum of four Gaussian curves representing one closed and three open levels of the same conductance, i.e. there were at least three channels in the patch. Figure 1B and F shows that pre-treatment with the ETA receptor antagonist 100 nM BQ-123 for 5 min had no effect on ET-1-induced channel activity (n = 10). In addition, Fig. 1C and F shows that pre-treatment with the ETB receptor antagonist 100 nM BQ-788 for 5 min also had no effect on ET-1-evoked channel activity (n = 10). Figure 1E shows that ET-1 activated the same 2.6 pS channel currents in the presence of either BQ-123 or BQ-788 and in the absence of receptor antagonists. However, Fig. 1D and F illustrates that that pre-treatment with co-application of both 100 nM BQ-123 and 100 nM BQ-788 for 5 min almost completely abolished ET-1-induced channel activity (n = 8, P < 0.001). These data showing that both ETA and ETB receptor antagonists must be present to block channel activity by ET-1 indicate that stimulation of both ETA and ETB receptors can lead to channel opening. Stimulation of ETA and ETB receptors activates TRPC1 channel currents through a PKC-dependent mechanism Previously we have shown that ET-1 and agents that deplete internal Ca2+ stores, cyclopiazonic acid (CPA) and BAPTA-AM, evoke native 2.6 pS TRPC1 channel currents in coronary artery myocytes which are inhibited by PKC inhibitors (Saleh et al. 2008; Albert et al. 2009). Therefore we investigated the role of PKC and TRPC1 subunits in mediating both ETA and ETB receptor-mediated channel activity. In these experiments we bath applied ET-1 in the presence of either BQ-788 or BQ-123 to evoke respectively ETA or ETB receptor-coupled pathways in cell-attached patches. Figure 2A shows that the mean NPo of ETA receptor-mediated channel activity, activated by 100 nM ET-1 in the presence of 100 nM BQ-788, was signiﬁcantly reduced from 0.37 ± 0.04 to 0.06 ± 0.01 (83 ± 5% inhibition, n = 6, P < 0.01) by the PKC inhibitor chelerythrine (3 μM). Figure 2B shows that the mean Figure 2. Stimulation of ETA and ETB receptors activates native TRPC1 channel currents via a PKC-dependent mechanism A and B, stimulation of ETA (100 nM BQ-788 present) or ETB receptors (100 nM BQ-123 present) with 100 nM ET-1 evoked channel activity that was inhibited by co-application of 3 μM chelerythrine in cell-attached patches held at −70 mV. C and D, stimulation of respectively ETA and ETB receptors evoked channel activity initially induced in cell-attached patches which was inhibited by application of 1 : 200 dilution of anti-TRPC1 antibodies following excision of patches into the inside-out conﬁguration (i/o). E and F, following pre-incubation of anti-TRPC1 anti- bodies (1 : 200) with its antigenic peptide (1 : 100, AgP) bath application of the complex had no effect on ETA receptor-mediated or ETB receptor-mediated channel activity. C 2009 The Authors. Journal compilation C 2009 The Physiological Society