Role of SPAK binding sites in SLC12 cotransporters basal activity and regulation by WNK3

With‐no‐lysine kinase 3 (WNK3) is a powerful activator of NKCC1/2 and NCC while is an inhibitor of KCC1 ‐ KCC4. WNKs lie upstream of the STE‐20 kinases SPAK/OSR1. It is known that interaction with SPAK occurs through the SPAK‐binding motif, RFx(V/I). WNK3 contains three such motifs, NCC and NKCC2 one; while KCC4 lacks this motif. In the present study we examined the relevance of SPAK binding sites for NCC, NKCC2, and KCC4 basal activity and regulation by WNK3. The sites of WNK3 (F242A, F745A, or F1337A; one at a time), NCC (F19A), and NKCC2 (F17A) were eliminated. Basal activity and effect of WNK3 was assessed by measuring the tracer 22 Na + or 86 Rb + uptake in Xenopus oocytes injected with wild type or mutant cRNAs. Basal activity of NCC‐F19A and NKCC2‐F17A was reduced by ~50%. However, the response to WNK3 and to intracellular chloride depletion was still present. The effect of WNK3 toward NKCC2, but not NCC or KCC4, was eliminated by WNK3‐F1337A. Interestingly, the WNK3 effect towards NCC or KCC4, but not NKCC2, was prevented in WNK3‐F242A. Biochemical analysis revealed that catalytic activity is preserved in both, WNK3‐F242A and WNK3‐F1337A. Thus, we conclude that basal activity of NCC and NKCC2, but not KCC4 requires the presence of an SPAK binding site on the cotransporter. Modulation of NCC, NKCC2, and KCC4 by WNK3 requires SPAK binding sites in the kinase, however, different sites are used for regulation of NKCC2 in contrast to NCC or KCC4.