Interactions between DAG, IP3 and PIP2 Govern Activation of Heterotetrameric TRPC6/C7 Channel Activity in Rabbit Portal Vein Myocytes

Abstract

Previously we have shown that synergism between inositol-1,4,5-trisphoshate (IP3) and diacylglycerol (DAG) mediates activation of TRPC6-like channel activity by noradrenaline (NA, Albert & Large, 2003) in rabbit portal vein myocytes. Moreover, a recent study showed that endogenous phosphatidylinositol-4,5-bisphosphate (PIP2) produced a marked inhibitory action on TRPC6 activity in mesenteric artery myocytes (Albert et al, 2008). In the present work we investigated interactions between DAG, IP3 and PIP2 in regulating TRPC6-like activity in portal vein myocytes using patch clamp and immunoprecipitation methods. In inside-out and cell-attached patches, bath application of respectively 10 μM IP3 and the cell-permeable IP3 analogue, 10 μM 6-IP3, both potentiated OAG-induced TRPC6-like channel activity by 3-fold but had no effect when applied on their own. In inside-out patches, pre-treatment with 20 μM wortmannin, to deplete endogenous PIP2 levels, increased OAG-evoked channel activity by 75-fold compared to control patches. Moreover, anti-PIP2 antibodies activated TRPC6-like activity in quiescent inside-out patches. In wortmannin-treated inside-out patches, 10 μM diC8-PIP2 inhibited OAG evoked channel activity (IC50 = 0.74 μM) which was rescued by over 50 % by co-application of 10 μM IP3. Anti-TRPC6 and anti-TRPC7 antibodies inhibited TRPC6-like activity induced by NA by over 80%, but channel activity was unaffected by other TRPC antibodies. Co-immunoprecipitation studies showed association between TRPC6 and TRPC7 proteins and that both these channel proteins interacted with PIP2. Pretreated with 6-IP3, reduced association between PIP2 and TRPC7 but not TRPC6, whereas OAG reduced PIP2 interactions with TRPC6 but not TRPC7. These results indicate that endogenous PIP2 has a pronounced inhibitory action on TRPC6/TRPC7 heteromeric channels in portal vein myocytes. Moreover channel activation by DAG requires both this triglyceride and IP3 to remove associations between PIP2 and these channel proteins.