Expression of functional receptor-coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells

The TRPC3 channel, an intensively studied member of the widely expressed tr ansient receptor potential (TRP) family, is a Ca2+-conducting channel activ ated in response to phospholipase C-coupled receptors. Despite scrutiny, th e receptor-induced mechanism to activate TRPC3 channels remains unclear. Ev idence indicates TRPC3 channels interact directly with intracellular inosit ol 1,4,5-trisphosphate receptors (InsP(3)Rs) and that channel activation is mediated through coupling to InsP(3)Rs. TRPC3 channels were expressed in D T40 chicken B lymphocytes in which all three InsP(3)R genes were deleted (D T40InsP(3)R-k/o). Endogenous B-cell receptors (BCR) coupled through Syk kin ase to phospholipase C-gamma (PLC-gamma) activated the expressed TRPC3 chan nels in both DT40w/t and DT40InsP(3)R-k/o cells. The diacylglycerol (DAG) a nalogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also activated TRPC3 channels i ndependently of InsP(3)Rs. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor, U-73122, and also blocked by wortmannin-induced PLC substrate depletion. Neither U-73122 nor wortmannin modified either OAG-ind uced TRPC3 activation or store-operated channel activation in DT40 cells. C otransfection of cells with both G protein-coupled M5 muscarinic receptors and TRPC3 channels resulted in successful M5 coupling to open TRPC3 channel s mediated by PLC-beta. We conclude that TRPC3 channels are activated indep endently of InsP(3)Rs through DAG production resulting from receptor-mediat ed activation of either PLC-gamma or PLC-beta.