Ins(1,4,5)P3 interacts with PIP2 to regulate activation of TRPC6/C7 channels by diacylglycerol in native vascular myocytes.

We investigated synergism between inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG) on TRPC6-like channel activity in rabbit portal vein myocytes using single channel recording and immunoprecipitation techniques. Ins(1,4,5)P(3) at 10 microm increased 3-fold TRPC6-like activity induced by 10 microm 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue. Ins(1,4,5)P(3) had no effect on OAG-induced TRPC6 activity in mesenteric artery myocytes. Anti-TRPC6 and anti-TRPC7 antibodies blocked channel activity in portal vein but only anti-TRPC6 inhibited activity in mesenteric artery. TRPC6 and TRPC7 proteins strongly associated in portal vein but only weakly associated in mesenteric artery tissue lysates. Therefore in portal vein the conductance consists of TRPC6/C7 subunits, while OAG activates a homomeric TRPC6 channel in mesenteric artery myocytes. Wortmannin at 20 microm reduced phosphatidylinositol 4,5-bisphosphate (PIP(2)) association with TRPC6 and TRPC7, and produced a 40-fold increase in OAG-induced TRPC6/C7 activity. Anti-PIP(2) antibodies evoked TRPC6/C7 activity, which was blocked by U73122, a phospholipase C inhibitor. DiC8-PIP(2), a water-soluble PIP(2) analogue, inhibited OAG-induced TRPC6/C7 activity with an IC(50) of 0.74 microm. Ins(1,4,5)P(3) rescued OAG-induced TRPC6/C7 activity from inhibition by diC8-PIP(2) in portal vein myocytes, and this was not prevented by the Ins(1,4,5)P(3) receptor antagonist heparin. In contrast, Ins(1,4,5)P(3) did not overcome diC8-PIP(2)-induced inhibition of TRPC6 activity in mesenteric artery myocytes. 2,3,6-Tri-O-butyryl-Ins(1,4,5)P(3)/AM (6-Ins(1,4,5)P(3)), a cell-permeant analogue of Ins(1,4,5)P(3), at 10 microm increased TRPC6/C7 activity in portal vein and reduced association between TRPC7 and PIP(2), but not TRPC6 and PIP(2). In contrast, 10 microm OAG reduced association between TRPC6 and PIP(2), but not between TRPC7 and PIP(2). The present work provides the first evidence that Ins(1,4,5)P(3) modulates native TRPC channel activity through removal of the inhibitory action of PIP(2) from TRPC7 subunits.