J Physiol 587.22 PIP3 and PIP2 activation of vascular TRPC1 5373 the cell-attached conﬁguration channel activity persists after excision into the inside-out conﬁguration. In this conﬁguration there is no agonist present and it is possible that processes that normally inhibit channel activity are lost (e.g. cytosolic factors) when the membrane patch is excised. Multiple transduction mechanisms and TRPC channels in vascular smooth muscle In cell lines, receptor-mediated activation of expressed TRPC channels is generally shown to be via stimulation of Gαq/11 and activation of PI-PLC (e.g. see Hardie, 2007), but in vascular smooth muscle more diverse signalling pathways are involved. Therefore α1-adrenoceptors and angiotensin II (Ang II) receptors are coupled to TRPC6 channels via PI-PLC in respectively rabbit portal vein and mesenteric artery myocytes (Helliwell & Large, 1997; Inoue et al. 2001; Saleh et al. 2006). In contrast constitutive TRPC3 channels in rabbit ear artery myocytes are coupled to Gαi/o proteins linked PC-PLD-induced production of DAG (Albert & Large, 2004; Albert et al. 2005, 2006). The present work adds yet another signalling cascade for TRPC channels in which ETA receptor stimulation causes PI-3-kinase-mediated production of PIP3 to activate TRPC1 channels. Agents that deplete intracellular Ca2+ stores also stimulate TRPC1 channel activity and therefore these channels are often termed store-operated channels (SOCs). The present results with ET-1 and previous work with noradrenaline in portal vein (Albert & Large, 2002) and Ang II in mesenteric artery (Saleh et al. 2006) indicate that membrane-delimited lipid pathways induce TRPC1 channel activity in isolated patches. Consequently TRPC1 channels behave more as receptor-operated channels than as SOCs according to their strict deﬁnition. Phospholipids and TRPC channels There is increasing evidence that phospholipids regulate transient receptor potential channels including TRPC channel subtypes in native vascular myocytes and in expression systems (Hardie 2007; Rohacs, 2007; Voets & Nilius, 2007; Nilius et al. 2008; Large et al. 2009). Endogenous PIP2 inhibits the excitatory effects of DAG on TRPC6 in mesenteric artery myocytes (Albert et al. 2008) and also inositol 1,4,5-trisphosphate potentiates the excitatory effects of DAG on both native TRPC6 and TRPC1 channels in rabbit portal vein myocytes (Albert & Large, 2003; Liu et al. 2005; Saleh et al. 2008). PIP2 has also been shown to have complex actions on expressed TRPC conductances with this phospholipid increasing TRPC3, TRPC6 and TRPC7 channel activity (Lemonnier et al. 2007), inhibiting TRPC4 whole-cell currents (Otsuguro C 2009 The Authors. Journal compilation C 2009 The Physiological Society et al. 2008) and having both excitatory and inhibitory effects on TRPC5 channel activity (Trebak et al. 2008). There is little information on the action on PIP3 on TRPC channels although this phospholipid has been shown to increase expressed TRPC6-mediated Ca2+ entry in HEK293 cells recorded with a Ca2+-sensitive dye (Tseng et al. 2004). However the present data provide the ﬁrst direct evidence that PIP3 activates native TRPC channels in any cell type. Conclusion This study demonstrates that ET-1 activates native TRPC1 channels in rabbit coronary artery myocytes using two distinct phospholipid signalling pathways. The data show that PIP3 and PIP2 mediate the responses to respectively ETA and ETB receptor stimulation and facilitate opening of native TRPC1 channels. This is the ﬁrst demonstration that PIP3 activates native TRPC1 channels in vascular smooth muscle. 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