Figure 7.

IGF1 enhances G_q-coupled receptor activation of TRPC6 via WNK1. (A) Cells co-expressing TRPC6 and M3R were serum-starved for 18 h and incubated for 10 min with 100 ng/ml IGF1, 20 nM wortmannin, both or neither before ruptured whole-cell recording. Carbachol was applied to the bath where shown. (B) Peak CCh-induced current density from panel A. *, p < 0.01 between indicated groups. (C) Peak CCh-induced inward current density in cells transfected with control oligonucleotides or WNK1 siRNA and with or without IGF1 pretreatment. *, p < 0.01 between indicated groups. #, p < 0.01 vs cells transfected with control oligo and without IGF1 pretreatment. (D) Peak CCh-induced inward current density in cells transfected with WNK1 siRNA and cDNA for either wild type WNK1 or T58A mutant WNK1, and with or without IGF1 pretreatment. *, p < 0.01 between indicated. (E) Peak CCh-activated TRPC6 currents in cells transfected with control oligonucleotides or PI4KIIIα siRNA and with or without IGF1 pretreatment. *, p < 0.01 between indicated. (F) Interaction of WNK1, G_q-PLC-β and PI4KIIIα signaling pathways. Components of the pathways are shown in black. Inhibitors are color-coded per their biochemical sites of action and placed where their inhibitory effects on cellular signaling were noted in the experiments. Pathways are indicated by black arrows. The experimental results indicate that the point at which G_q and WNK1 interacts is at the level of PLC activity (marked by dark green box). (see also Figure S5).