WNK4 diverts the thiazide-sensitive NaCl cotransporter to the lysosome and stimulates AP-3 interaction.

With-no-lysine kinase 4 (WNK4) inhibits electroneutral sodium chloride reabsorption by attenuating the cell surface expression of the thiazide-sensitive NaCl cotransporter (NCC). The underlying mechanism for this effect remains poorly understood. Here, we explore how WNK4 affects the trafficking of NCC through its interactions with intracellular sorting machinery. An analysis of NCC cell surface lifetime showed that WNK4 did not alter the net rate of cotransporter internalization. In contrast, direct measurements of forward trafficking revealed that WNK4 attenuated the rate of NCC surface delivery, inhibiting the anterograde movement of cotransporters traveling to the plasma membrane from the trans-Golgi network. The response was paralleled by a dramatic reduction in NCC protein abundance, an effect that was sensitive to the lysosomal protease inhibitor leupeptin, insensitive to proteasome inhibition, and attenuated by endogenous WNK4 knockdown. Subcellular localization studies performed in the presence of leupeptin revealed that WNK4 enhanced the accumulation of NCC in lysosomes. Moreover, NCC immunoprecipitated with endogenous AP-3 complexes, and WNK4 increased the fraction of cotransporters that associate with this adaptor, which facilitates cargo transport to lysosomes. WNK4 expression also increased LAMP-2-positive lysosomal content, indicating that the kinase may act by a general AP-3-dependent mechanism to promote cargo delivery into the lysosomal pathway. Taken together, these findings indicate that WNK4 inhibits NCC activity by diverting the cotransporter to the lysosome for degradation by way of an AP-3 transport carrier.