Fig. 4.

WNK3 regulates the phosphorylation of NKCC1. Xenopus oocytes were injected with the indicated constructs and incubated at varying extracellular osmolarities. After incubation, oocytes were lysed, and Western blotting was performed by using the R5 (anti-Phospho-NKCC1) or T4 (anti-NKCC1) antibodies, as in Methods. Phosphorylation of NKCC1 normally increases from negligible levels in hypotonic conditions (180 mM) to complete phosphorylation in hypertonic conditions (220 mM). In contrast, coexpression of NKCC1 with kinase-active WNK3 results in robust phosphorylation of NKCC1 at all osmolarities. Conversely, expression of kinase-dead WNK3 results in marked reduction of NKCC1 phosphorylation.