5362 Introduction Endothelin-1 (ET-1) produces vasoconstriction by a direct action on vascular smooth muscle cells through stimulation of predominantly ETA receptors, although ETB receptors are involved in some vascular beds (Sumner et al. 1992; Davenport & Battistini, 2002). Moreover in the coronary circulation activation of ET-1 receptors has been linked to exaggerated constriction of human coronary artery leading to myocardial ischaemia in coronary artery disease (Schiffrin & Touyz, 1998; Kinlay et al. 2001). ET-1-induced vasoconstriction is mediated almost entirely by inﬂux of Ca2+ ions through voltage-independent ion channels (see Miwa et al. 2005). These data suggest that ET-1 contracts vascular smooth muscle by opening Ca2+-permeable non-selective cation channels. Consistent with this notion we demonstrated that ET-1 activates two distinct types of canonical transient receptor potential (TRPC) channels in freshly dispersed rabbit coronary myocytes. At low concentrations (1–10 nM) ET-1 activates a non-selective cation channel with four subconductance states of between 16 and 68 pS (Peppiatt-Wildman et al. 2007). These responses were mediated mainly by ETA receptors and were mimicked by the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) via a protein kinase C (PKC)-independent mechanism. Evidence indicated that this cation channel protein is a heteromeric structure consisting of TRPC3/TRPC7 subunits (Peppiatt-Wildman et al. 2007). In contrast at higher concentrations (100 nM) ET-1 evokes a PKC-dependent 2.6 pS Ca2+-permeable cation channel which has characteristics of a heteromeric TRPC1/TRPC5/TRPC6 structure (subsequently referred to as TRPC1 channels, Saleh et al. 2008). With this concentration of ET-1 the TRPC3/TRPC7 conductance is not observed. In the present study we have investigated the trans- duction mechanisms linking ET-1 receptors to native TRPC1 ion channels described above in coronary artery myocytes. The results demonstrate that TRPC1 channels may be activated by stimulation of either ETA or ETB receptors using two distinct phosphoinositide signalling pathways involving respectively phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2). This is the ﬁrst demonstration that PIP3, in addition to PIP2, activates native TRPC1 channels. Methods Cell isolation New Zealand White rabbits (2–3 kg) were killed using I.V. sodium pentobarbitone (120 mg kg−1, in accordance S. N. Saleh and others J Physiol 587.22 with the UK Animals (Scientiﬁc Procedures Act) 1986). Experimental methods were carried out as speciﬁed by St George’s animal welfare committee and according to the policies of The Journal of Physiology (Drummond, 2009). Right and left anterior descending coronary arteries were dissected free from fat and connective tissue in physio- logical salt solution containing (mM): NaCl (126), KCl (6), glucose (10), Hepes (11), MgCl2 (1.2) and CaCl2 (1.5), with pH adjusted to 7.2 with 10 M NaOH. An incision was made along the longitudinal axis of the blood vessels and the exposed endothelium was gently removed using a cotton bud. Enzymatic digestion and smooth muscle cell isolation were subsequently carried using methods previously described (Saleh et al. 2006). Electrophysiology Single channel currents were recorded in voltage-clamp mode using cell-attached and inside-out patch conﬁgurations (Hamill et al. 1981) with a HEKA EPC 8 patch-clamp ampliﬁer (HEKA Elektronik, Lambrecht/Pfalz, Germany) at room temperature (20–23◦C). Patch pipettes were manufactured from borosilicate glass to produce pipettes with resistances of 6–10 M for isolated patch recording when ﬁlled with patch pipette solution. To reduce ‘line’ noise the recording chamber (vol. ca 150–200 μl) was perfused using two 20 ml syringes, one ﬁlled with external solution and the other used to drain the chamber, in a ‘push and pull’ technique. The external solution could be exchanged twice within 30 s. In cell-attached patch recording, the membrane potential was set to ∼0 mV using a high KCl bathing solution (see below). In both cell-attached and inside-out patch recordings, +70 mV was applied to the patch and held at this level except for measuring current–voltage (I –V ) relationships when the applied patch voltage was manually altered between +120 mV and −50 mV. According to convention in the text membrane potential is given with respect to the internal potential and thus, the resting holding potential is referred to as −70 mV. Single channel currents were initially recorded onto digital audiotape (DAT) using a Sony PCM-R300 digital tape-recorder (BioLogic Science Instruments, Claix, France) at a bandwidth of 5 kHz (HEKA EPC 8 patch-clamp ampliﬁer) and a sample rate of 48 kHz. For off-line analysis, single channel currents were ﬁltered at 100 Hz (see below, −3 db, low pass 8-pole Bessel ﬁlter, model LP02, Frequency Devices Inc., Ottawa, IL, USA) and acquired using a Digidata 1322A and pCLAMP 9.0 at a sampling rate of 1 kHz. Data were captured with a Dell Dimension 5150 personal computer. Single channel current amplitudes were calculated from idealised traces of at least 60 s in duration using the 50% threshold method and analysed using pCLAMP C 2009 The Authors. Journal compilation C 2009 The Physiological Society