WNK4 Kinase is a Negative Regulator of K + ‐Cl − Cotransporters

Abstract

WNK kinases (with no lysine kinase(K)) are regulators of several membrane transport proteins in which these kinases act as a molecular switches that coordinate the activity of several players. Members of the Solute Carrier Family number 12 are one of the main targets. WNK3 activates the Na+-driven NCC, NKCC1, and NKCC2 and inhibits the K+-driven KCC1 to KCC4. WNK4 inhibits the activity of NCC and NKCC1, while in the presence of the STE20-related proline-alanine rich kinase SPAK activates NKCC1. Nothing is known, however, regarding the effect of WNK4 upon the K+-Cl− cotransporters. Using the heterologous expression system of Xenopus laevis oocytes, here we show that WNK4 inhibits the activity of the K+-Cl− cotransporters KCC1, KCC3, and KCC4 by 50 to 60% under cell swelling, a condition in which these cotransporters are maximally active. The effect of WNK4 requires its catalytic activity because it was lost by the substitution of aspartate 318 for alanine (WNK4-D318A) that renders WNK4 catalytically inactive. WNK4 effect upon K+-Cl− cotransporters is not affected by co-injection with wild type SPAK cRNA or its catalytically inactive form (SPAK-K104R). In contrast, WNK4 missense mutations that cause pseudohypoaldosteronism type II do not affect the WNK4-induced inhibition of KCC4. Finally, we observed that catalytically inactive WNK4-D318A is able to bypass the tonicity requirements for KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K+-Cl− cotransporters.