J Physiol 587.22 (2009) pp 5361–5375 5361 Activation of native TRPC1/C5/C6 channels by endothelin-1 is mediated by both PIP3 and PIP2 in rabbit coronary artery myocytes Sohag N. Saleh, Anthony P. Albert and William A. Large Ion Channels and Cell Signalling Research Centre, Division of Basic Medical Sciences, St George’s, University of London, Cranmer Terrace, London SW17 0RE, UK We investigate activation mechanisms of native TRPC1/C5/C6 channels (termed TRPC1 channels) by stimulation of endothelin-1 (ET-1) receptor subtypes in freshly dispersed rabbit coronary artery myocytes using single channel recording and immunoprecipitation techniques. ET-1 evoked non-selective cation channel currents with a unitary conductance of 2.6 pS which were not inhibited by either ETA or ETB receptor antagonists, respectively BQ-123 and BQ788, when administered separately. However, in the presence of both antagonists, ET-1-evoked channel activity was abolished indicating that both ETA and ETB receptor stimulation activate this conductance. Stimulation of both ETA and ETB receptors evoked channel activity which was inhibited by the protein kinase C (PKC) inhibitor chelerythrine and by anti-TRPC1 anti- bodies indicating that activation of both receptor subtypes causes TRPC1 channel activation by a PKC-dependent mechanism. ETA receptor-mediated TRPC1 channel activity was selectively inhibited by phosphoinositol-3-kinase (PI-3-kinase) inhibitors wortmannin (50 nm) and PI-828 and by antibodies raised against phosphoinositol-3,4,5-trisphosphate (PIP3), the product of PI-3-kinase-mediated phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2). Moreover, exogenous application of diC8-PIP3 stimulated PKC-dependent TRPC1 channel activity. These results indicate that stimulation of ETA receptors evokes PKC-dependent TRPC1 channel activity through activation of PI-3-kinase and generation of PIP3. In contrast, ETB receptor-mediated TRPC1 channel activity was inhibited by the PI-phospholipase C (PI-PLC) inhibitor U73122. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), an analogue of diacylglycerol (DAG), which is a product of PI-PLC, also activated PKC-dependent TRPC1 channel activity. OAG-induced TRPC1 channel activity was inhibited by anti-phosphoinositol-4,5-bisphosphate (PIP2) antibodies and high concentrations of wortmannin (20 μm) which depleted tissue PIP2 levels. In addition exogenous application of diC8-PIP2 activated PKC-dependent TRPC1 channel activity. These data indicate that stimulation of ETB receptors evokes PKC-dependent TRPC1 activity through PI-PLC-mediated generation of DAG and requires a permissive role of PIP2. In conclusion, we provide the ﬁrst evidence that stimulation of ETA and ETB receptors activate native PKC-dependent TRPC1 channels through two distinct phospholipids pathways involving a novel action of PIP3, in addition to PIP2, in rabbit coronary artery myocytes. (Received 14 August 2009; accepted after revision 16 September 2009; ﬁrst published online 21 September 2009) Corresponding author A. Albert: Ion Channels and Cell Signalling Research Centre, Division of Basic Medical Sciences, St George’s, University of London, Cranmer Terrace, London SW17 0RE, UK. Email: aalbert@sgul.ac.uk Abbreviations AgP, antigenic peptide; CPA, cyclopiazonic acid; DAG, diacylglycerol; ET-1, endothelin-1; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PI-3-kinase, phosphoinositol-3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; phosphatidylinositol-3,4,5-trisphosphate; PLC, phosphoslipase C; PKC, protein kinase C; TRPC, canonical transient receptor potential. C 2009 The Authors. Journal compilation C 2009 The Physiological Society DOI: 10.1113/jphysiol.2009.180331