5368 S. N. Saleh and others J Physiol 587.22 Involvement of PIP3 in ETA receptor-mediated activation of TRPC1 channel currents The above results suggest that generation of PIP3 produced from the action of PI-3-kinase on PIP2 is required for ETA receptor-mediated TRPC1 channel stimulation. Consequently we investigated if exogenous PIP3 directly activates TRPC1 channel currents in coronary artery myocytes. Figure 5A and B shows that bath application of 3 μM diC8-PIP3, a water soluble form of PIP3, to the cyto- solic surface of inside-out patches activated cation channel activity with a mean NPo of 0.32 ± 0.06 (n = 11) and a unitary conductance of 2.6 pS with an Er of about 0 mV. The threshold concentration of diC8-PIP3 was approximately 1 μM and maximum channel activation was obtained with 10–20 μM diC8-PIP3 (data not shown). Figure 5C and D also illustrates that mean NPo of diC8-PIP3-evoked channel activity in inside-out patches was signiﬁcantly inhibited from 0.21 ± 0.09 to 0.02 ± 0.02 (94 ± 4% inhibition, n = 5, P < 0.01) by co-application with 3 μM chelerythrine and from 0.28 ± 0.05 to 0.03 ± 0.02 (84 ± 7% inhibition, n = 5, P < 0.01) with anti-TRPC1 antibodies. The role of endogenous PIP3 in ETA receptor-mediated stimulation of TRPC1 channel activity was investigated using an anti-PIP3 antibody. Figure 5E shows that the mean NPo of ETA receptor-mediated TRPC1 channel stimulation, initially activated in cell-attached patches in the presence of the ETB receptor antagonist BQ-788, was signiﬁcantly reduced from 0.25 ± 0.07 to 0.01 ± 0.01 (97 ± 1% inhibition, n = 6, P < 0.01) by bath application of an anti-PIP3 antibody to the cytosolic surface of the patches. In contrast, Fig. 5F illustrates that an anti-PIP3 antibody had no effect on ETB receptor-mediated TRPC1 channel activity (control mean NPo from 0.48 ± 0.08 to 0.51 ± 0.11 in anti-PIP3 antibody, n = 6). These data clearly show that exogenous PIP3 and ET-1 activate the same PKC-dependent TRPC1 channel currents and also indicate that endogenous PIP3 mediates activation of TRPC1 channel currents by ETA receptor stimulation. Involvement of PIP2 in ETB receptor-mediated activation of TRPC1 channel currents The above results indicate that ETB receptor-mediated TRPC1 channel activity is coupled to a PI-PLC pathway and to stimulation of PKC (see Figs 2 and 3). Previous work suggests that this biochemical cascade is likely to involve generation of DAG, through hydrolysis of PIP2 Figure 5. PIP3 activates TRPC1 channel currents via a PKC-dependent mechanism A, bath application of 3 μM diC8-PIP3 activates cation channel activity in an inside-out patch at −70 mV. B, current–voltage relation of diC8-PIP3-evoked cation channel activity yielded a unitary conductance of 2.6 pS and an Er of about 0 mV. Each point was at least n = 4. C and D, respectively 3 μM chelerythrine and 1 : 200 dilution of anti-TRPC1 antibodies inhibited diC8-PIP3-induced channel activity in inside-out patches at −70 mV. E and F, co-application of 1 : 200 dilution of anti-PIP3 antibodies reduced ETA receptor-mediated TRPC1 channel activity (E) but had no effect on ETB receptor-mediated TRPC1 channel activity (F). C 2009 The Authors. Journal compilation C 2009 The Physiological Society