J Physiol 587.22 PIP3 and PIP2 activation of vascular TRPC1 5371 is necessary for OAG (DAG)-induced (ETB receptor pathway) TRPC1 channel stimulation. To further investigate the role of PIP2 and PIP3 in mediating ET-1-induced TRPC1 channel activation we carried out co-immunoprecipitation and dot-blot studies. Figure 7Ca illustrates a co-immunoprecipitation experiment which shows that at rest PIP2 is associated with TRPC1 proteins in coronary artery when tissue lysates were immunoprecipitated with anti-TRPC1 anti- bodies and then blotted with anti-PIP2 antibodies to detect a predicted band of ∼75 kDa (see Methods). In addition Fig. 7Ca shows that pre-treatment of coronary arteries with 20 μM wortmannin for 30 min reduced PIP2 association with TRPC1 proteins whereas total β-actin levels were not altered. The upper panel in Fig. 7Cb shows that stimulation of ETA or ETB receptors did not alter PIP2 association with TRPC1 proteins following immuno- precipitation with anti-PIP2 antibodies and blotting with anti-TRPC1 antibodies to detect a predicted band of ∼100 kDa (see Methods). The middle panel shows a control experiment in which pre-incubation of the anti-TRPC1 antibody with its antigenic peptide (AgP) reduced the detection of the predicted band for TRPC1 proteins on a Western blot. The lower panel shows that the antigenic peptide had no effect on the expression of β-actin. It was not possible to detect total PIP3 levels using Western blotting or association between PIP3 and TRPC1 proteins using co-immunoprecipitation at rest or after stimulation of ETA and ETB receptors. This is probably due to resting and receptor-mediated generation of PIP3 levels being too small to resolve with the limited amounts of available coronary artery tissue. Therefore we measured PIP3 and PIP2 levels using tissue lysate and dot-blot techniques with their respective antibodies. Figure 7D illustrates that at rest total cell lysates from coronary arteries contained detectable PIP2 but not PIP3 whereas upon stimulation of ETA receptors (ET-1 in the pre- sence of BQ-788) the levels of PIP2 were reduced and generation of PIP3 was detected whereas levels of β-actin were unaffected. These data provide novel evidence that PIP3 can activate TRPC1 channels independently of PIP2 in coronary artery myocytes. Discussion The present work provides the ﬁrst evidence that stimulation of ETA and ETB receptors by ET-1 activates native TRPC1 channel currents in freshly dispersed coronary artery myocytes by two distinct parallel phosphoinositide signalling pathways. Evidence is provided to show that stimulation of ETA receptors evokes TRPC1 channel currents through PI-3-kinase-mediated C 2009 The Authors. Journal compilation C 2009 The Physiological Society generation of PIP3 which leads to opening of TRPC1 channels, possibly by a direct action. In contrast ETB receptors are coupled to PI-PLC and production of DAG leading to PIP2-mediated TRPC1 channel activation. Moreover it appears that PKC is involved in activation of TRPC1 channel currents by both PIP3 and PIP2. Previously we have shown a permissive role for PIP2 in activating TRPC1 channels in rabbit portal vein myocytes (Saleh et al. 2009) but this is the ﬁrst demonstration that PIP3 also activates native TRPC1 channel currents. Furthermore, to our knowledge, this is the ﬁrst evidence that PI-3-kinase may be involved in activation of TRPC1 channel. Importantly, this pathway involving PI-3-kinase-mediated generation of PIP3 represents a novel activation mechanism of TRPC channels. ETA receptor transduction mechanism and activation of TRPC1 channel currents ETA receptor-mediated stimulation of TRPC1 channel activity is blocked by PI-3-kinase inhibitors and by an anti-PIP3 antibody which did not inhibit TRPC1 channel activation induced by ETB receptor stimulation. Moreover exogenous PIP3 applied to inside-out patches evoked cation channel currents with identical properties to those stimulated by ET-1, i.e. native TRPC1 channels. Importantly, PIP3-induced TRPC1 channel activation did not require endogenous PIP2 since PIP3 readily activated TRPC1 channel currents in tissues pre-treated with high concentrations of wortmannin, which reduced association of PIP2 with TRPC1. Moreover an anti-PIP2 antibody which blocked responses to OAG did not inhibit PIP3-evoked TRPC1 channel activity. Thus generation of PIP3 by stimulation of ETA receptors activates TRPC1 channels with PIP3 possibly being the activating ligand, which represents a novel mechanism of ion channel activation. Stimulation of ETA receptors expressed in Chinese hamster ovary cells has been shown to increase PI-3-kinase activity and PIP3 formation which was inhibited by low concentrations of wortmannin (Sugawara et al. 1996). Our data also show that ETA receptor stimulation increases PIP3 production. In vascular smooth muscle ET-1 receptor stimulation leads to activation of several signalling pathways including PI-3-kinase (see review by Boualleque et al. 2007) and this mechanism is involved in vasoconstriction (Kawanabe et al. 2004). Previously PIP3 has been shown to bind to expressed TRPC1 proteins (Kwon et al. 2007) although another study suggested that PIP3 did not activate expressed TRPC1 channels (Tseng et al. 2004). However in the same work it was shown that PIP3 produces marked stimulation of TRPC6 channel activity (Tseng et al. 2004). Previously we indicated that the 2.6 pS ET-1-induced conductance in coronary artery