5372 S. N. Saleh and others J Physiol 587.22 myocytes may be a heteromeric channel consisting of TRPC1, TRPC5 and TRPC6 subunits (Saleh et al. 2008). Therefore it is possible that the heteromeric structure of native TRPC1 channels is more sensitive to PIP3 then heterologously expressed TRPC1 proteins or that PIP3 binds to proposed TRPC5 or TRPC6 subunits of the native conductance in coronary artery myocytes. The present work does not reveal how ETA receptors are linked to PI-3-kinase in coronary arteries but in other systems it has been shown, and is generally accepted, that Gβγ subunits activate PI-3-kinase (see Clapham & Neer, 1997; Vanhaesebroeck et al. 1997). ETB receptor transduction mechanism and activation of TRPC1 channel currents The present work shows that ETB receptor-induced stimulation of TRPC1 channel activity was markedly inhibited by the PI-PLC inhibitor U73122, which did not effect ETA receptor-mediated activation of TRPC1 channel activity. In addition OAG, an analogue of DAG which is a product of PI-PLC stimulation, induced TRPC1 channel activity which was also inhibited by an anti-PIP2 antibody. Moreover OAG did not evoke TRPC1 channel activity in cells pre-treated with high concentrations of wortmannin, which depleted tissue PIP2 levels. These electrophysiological data are consistent with a pathway in which ETB receptors are coupled to PI-PLC, which generates DAG and subsequently induces PIP2-mediated activation of TRPC1 channels. Application of exogenous PIP2 evoked TRPC1 channel currents and co-immunoprecipitation studies showed that PIP2 co-associated with TRPC1 proteins in resting and ET-1-stimulated tissues. This ﬁnding is similar to a previous study in rabbit portal vein myocytes in which it was concluded that PIP2 is tethered to TRPC1 proteins at rest but PKC-mediated phosphorylation of TRPC1 proteins was necessary to cause channel opening (Saleh et al. 2009, see Large et al. 2009 for more detail). We propose that a similar mechanism may be important for ETB receptor stimulation in coronary artery myocytes. Therefore the present work shows that both PIP2 and PIP3 can activate TRPC1 channels in coronary artery myocytes and our evidence is that PIP3 is obligatory for ETA receptor-mediated stimulation of TRPC1 channels whereas PIP2 is necessary for ETB receptor-mediated activation of the same ion channel. An interesting observation is that ETA and ETB receptor-mediated TRPC1 channel activity is not additive and that antagonism of both ETA and ETB receptors is required to block ET-1-induced activation of TRPC1 channels. This suggests that both pathways were equally effective in activating TRPC1 channels with the conditions used in our experiments and may indicate a safeguard mechanism for channel activation. Moreover these data indicate how two receptor subtypes converge onto the same TRPC1 channel utilising different transduction pathways. Role of PKC in activation mechanism of native TRPC1 channels by ETA and ETB receptor stimulation Stimulation of TRPC1 activity by both ETA and ETB receptors in coronary artery myocytes was almost abolished by the PKC inhibitor chelerythrine. In addition, the responses of PIP3 and PIP2, the proposed mediators of respectively ETA and ETB receptors stimulation, were also blocked by chelerythrine. Therefore it is evident that PKC plays a central role in the activation mechanism of TRPC1 channels by ET-1. Previously we demonstrated in rabbit portal vein myocytes that TRPC1 channel activation by the sarcoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA), phorbol 12,13-dibutyrate (PDBu), a PKC stimulant, and PIP2 was associated with phosphorylation of TRPC1 proteins which was inhibited by chelerythrine (Saleh et al. 2009). Ahmmed et al. (2004) also demonstrated that PKC-evoked phosphorylation of expressed TRPC1 channels regulated store-operated Ca2+ entry in cultured endothelial cells. Importantly, the present work adds signiﬁcant support for the postulated activation mechanism of TRPC1 channels (see Large et al. 2009 for fully explanation) by showing that PIP3, another notable endogenous phospholipid, also acts as a stimulatory ligand of TRPC1 channels and requires a PKC-dependent process which is likely to involve phosphorylation of TRPC1 subunits. In future experiments it will be interesting to investigate the molecular basis of PIP3/PIP2-mediated activation mechanisms of native TRPC1 channels using expressed heterotetrameric channels involving TRPC1 subunits. It has been shown that PIP3 also activates some PKC isoforms in vitro (Nakanishi et al. 1993). Therefore on ETA receptor stimulation production of PIP3 is likely both to activate PKC and also to activate TRPC1 channels, which leads to opening of channels through a positive feedback process in which increased PKC-dependent phosphorylation of TRPC1 proteins results in greater PIP3-mediated channel activity. A similar transduction mechanism has been proposed to link expressed M2 muscarinic receptors to an endogenous chloride channel in Xenopus oocytes (Wang et al. 1999). The observation that bath application of ET-1 evoked channel activity recorded in a cell-attached patch suggests that important signalling molecule(s) outlined above translocate from receptors stimulated outside the patch to ion channels underneath the pipette tip. A characteristic of native TRPC channels is that once these signalling pathways are activated by bath applied agonists in C 2009 The Authors. Journal compilation C 2009 The Physiological Society