Figure 2.
Role of Gq in the activation of TRPC6 by WNK1. (A) GDPβS (0.2 mM) inhibits WNK1 activation of TRPC6. (B) Anti-WNK1 antibody inhibits TRPC6 activated by GTPγS. TRPC6 single channel activity was recorded from inside-out patches from TRPC6-expressing HEK cells. Buffer, GTPγS, anti-WNK1 Ab and/or OAG was applied to the cytoplasmic face as indicated. Each data point is NPo (number × open probability) averaged over 30 s. Inset shows mean ± SEM of peak open probability. *, p < 0.01 vs “buffer”. **, p < 0.01 “GTPγS” vs “GTPγS+anti-WNK1 antibody”. (C) Whole-cell WNK1-activated TRPC6 currents were measured in Gαq/11-null fibroblasts transfected with wild type Gαq (Gαq/WT) or empty vector. (D) Single channel activity was measured in inside-out patches from TRPC6-expressing Gαq/11-null fibroblasts that have endogenous WNK1 depleted by siRNA. Data show responses to GTPγS-preactivated Gαq (“Gαq*”) and/or WNK1 applied to the cytoplasmic face. Holding membrane potential is −80 mV. Inset shows mean ± SEM of peak open probability. *, p < 0.01 vs “buffer”. **, p < 0.05 vs “buffer”. ***, p < 0.02 between indicated groups. (E, F) Single channel activity was recorded in inside-out patches from HEK cells expressing both TRPC6 and wild type Gαq (Gαq/WT) (panel E) or G188S-mutant Gαq (Gαq/G188S) (panel F). Buffer, WNK11–491 and/or RGS4 (0.5 µM) was applied to the cytoplasmic face as indicated. NPo values were averaged every 20 s. There was a 2 min time gap between application of buffer and WNK1. (see also Figure S2).