Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC117

Poster Communications

IP3 rescues heterotetrameric canonical transient receptor potential 6/7 (TRPC6/C7) channel activity from inhibition by phosphatidylinositol-4,5-bisphosphate (PIP2) in rabbit vascular myocytes

M. Ju1, S. N. Saleh1, A. P. Albert1, W. A. Large1

1. Division of Basic Medical Sciences, St George's University of London, London, United Kingdom.

We have shown that synergism between the diacylglycerol analogue OAG and IP3 on TRPC6-like channel activity in portal vein myocytes although the mechanism governing the increase in activity is not known (Albert & Large, 2003). In recent work we proposed that endogenous PIP2 produces a marked inhibitory action on homotetrameric TRPC6 activity in mesenteric artery myocytes (Albert et al, 2008). The aims of the present work are to investigate whether PIP2 also inhibits TRPC6-like activity in portal vein myocytes and, to study if removal of an inhibitory action of PIP2 by DAG and/or IP3 mediate opening of these channels. In freshly dispersed single portal vein myocytes, single cation channel currents were recorded at room temperature using the inside-out patch configuration of the patch clamp technique (Albert et al, 2008). Mean values are of n cells ± S.E.M. Statistical analysis was carried out using unpaired and paired Student’s t-test with the level of significance set at P<0.05. To test the effects of PIP2 on OAG-evoked activity we compared control cells with cells pre-treated with 20 µM wortmannin for 30 min to deplete PIP2 levels. Mean open probability of OAG (10 µM)-induced TRPC6-like activity was significantly increased from 0.03 ± 0.01 (n=9) in control cells to 2.53 ± 0.37 (n=48) in cells pre-treated with wortmannin at -50 mV. Application of PIP2 inhibited OAG-evoked activity with an IC50 value of 0.74 µM at -50 mV. Inhibition of OAG-induced activity by 10 µM PIP2 was significantly rescued by over 50 % by co-application of 10 µM IP3 (n=5,). In addition the rescuing action of IP3 was not affected by IP3 receptor blocker heparin (1 mgml-1, n=5). Noradrenaline-evoked channel activity was inhibited by anti-TRPC6 (88 ± 9 %, n=7) and anti-TRPC7 antibodies 85 ± 13 % (n=10) but not by other anti-TRPC antibodies. These results show that endogenous PIP2 has a pronounced inhibitory action on TRPC6-like activity in portal vein myocytes which can be removed by interactions with IP3. Moreover these data suggest that these channels are likely to be composed of TRPC6/TRPC7 heterotetramers and that TRPC7 proteins mediate interactions between PIP2 and IP3.

Where applicable, experiments conform with Society ethical requirements