Figure 1.

Activation of TRPC6 by WNK1 requires PLC activity and is independent of WNK1 kinase activity. (A) Top: Domain structure of rat WNK1. Akt, its phosphorylation site; PRD, proline-rich domain; KD, kinase domain. Bottom: whole-cell patch-clamp recording of TRPC6. CCh: carbachol (100 µM); M3R: M3 muscarinic cholinergic receptor. Intracellular free [Ca²⁺] was 70 nM except as indicated in panel F. (B) Activation of TRPC6 by CCh via M3R, GTPγS or WNK1_1–491. HEK cells were transfected with TRPC6 with or without M3R. Data points (circles and error bars) are means ± SEM of inward current density (pA/pF at −100 mV; n = 5 for each). At the end of each recording, extracellular Na⁺ was replaced by the TRPC6-impermeable cation N-methyl-D-glucamine (NMDG) to gauge the background current. Inset shows current-voltage (I–V) relationships of activated TRPC6 currents. (C) Concentration-response curve for activation of TRPC6 by WNK1_1–491. (D) Effect of PLC inhibitor U73122 or inactive analog U73343 (2.5 µM each) on WNK1_1–491 activation of TRPC6. Inward current at −100 mV from a representative experiment. Similar results were observed in 6 independent experiments. (E) Cells were preincubated with 15 µM edelfosine for 1 hr and washed before recording. TRPC6 currents in response to 100 nM WNK1_1–491, 5 nM PLC-β1 or both were measured. (F) TRPC6 currents in response to WNK1_1–491 under 0 or 70 nM intracellular calcium. OAG was applied as indicated. (G) TRPC6 currents were measured in cells with or without intracellular application of 3 nM WNK1_1–491. OAG was applied to the extracellular solution at indicated concentrations. Although this concentration of WNK1_1–491 by itself does not activate TRPC6, it potentiated M3R activation of the channel (see Figure 3B). (see also Figure S1).