TRPC1 Ca2+-permeable cation channels regulate contraction and remodelling of vascular myocytes; phenotypes associated with cardiovascular disease (CVD, Abramowitz & Birnbaumer, 2009). It is apparent that phosphatidylinositol 4,5-bisphosphate (PIP2) and protein kinase C (PKC) are obligatory for TRPC1 channel activation in vascular myocytes (Albert, 2011). Moreover, calmodulin (CaM) is also important for activation of TRPC1 channels (Albert et al, 2006). The present work investigates how interactions between PIP2, PKC and CaM induce TRPC1 channel opening, and reveals a novel role for MARCKS, a membrane-associated phospholipid-binding protein (McLaughlin et al, 2005). Rabbits were sacrificed using i.v. sodium pentobarbitone (120 mgkg-1), and wild-type (WT) or TRPC1-/- mice were killed by cervical dislocation; both protocols carried out in accordance with the UK Animals Scientific Procedures Act, 1986. Portal vein myocytes were enzymatically dispersed for immunocytochemical staining and patch clamp recording, and tissue lysates were used for immunoprecipiation, Western blot and dot-blot analysis (n=at least 3 replicates or n=at least 6 patches per test condition). Mean data were ± S.E.M, and statistical significance was determined using Students t-test (p<0.05). MARCKS protein was expressed, and predominantly distributed at the plasma membrane of portal vein myocytes. In rabbit and WT murine portal vein myocytes, a MARCKS inhibitory peptide (MANS) activated whole-cell and single cation channel activities; these channel activities were absence in TRPC1-/- myocytes. MANS-activated TRPC1 channel activity was prevented by lowering PIP2 levels and PKC inhibitors but was unaffected by CaM inhibitors. These results suggest that constitutive MARCKS activity prevents TRPC1 channel opening, and that TRPC1 channel activation by PIP2/PKC and CaM occur downstream and upstream of MARCKS respectively. In un-stimulated tissue lysates, TRPC1 associated with MARCKS, and PIP2 primarily interacted with MARCKS. Following stimulation with noradrenaline or MANS, TRPC1 dissociated from MARCKS, and PIP2 was released from MARCKS and interacted with TRPC1. Importantly, noradrenaline stimulated PKC-dependent phosphorylation of TRPC1 but not MARCKS. We propose that noradrenaline induces TRPC1 channel activity through PKC-dependent phosphorylation of channel proteins, which increases binding of PIP2 to TRPC1 subunits that opens the channels; these levels of PIP2 are provided by MARCKS, which acts as a PIP2 buffer. It is likely that CaM activates TRPC1 by causing MARCKS to release PIP2. Our conclusions raise the idea that MARCKS is a novel Ca2+ signalling molecule, through modulating TRPC1 and possibly other ion channels, and is a potential therapeutic target for CVD.