WNK4 kinase is a negative regulator of K+-Cl- cotransporters.
WNK kinases [with no lysine (K) kinase] are emerging as regulators of several membrane transport proteins in which WNKs act as molecular switches that coordinate the activity of several players. Members of the cation-coupled chloride cotransporters family (solute carrier family number 12) are one of the main targets. WNK3 activates the Na(+)-driven cotransporters NCC, NKCC1, and NKCC2 and inhibits the K(+)-driven cotransporters 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 on 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 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. In contrast, three different 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 KCC2 and KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK, was prevented by the presence of protein phosphatase inhibitors, and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K(+)-Cl(-) cotransporters expressed in the kidney.
Garzón-Muvdi
Tomas
T
Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, México City, México.
Pacheco-Alvarez
Diana
D
Gagnon
Kenneth B E
KB
Vázquez
Norma
N
Ponce-Coria
José
J
Moreno
Erika
E
Delpire
Eric
E
Gamba
Gerardo
G
D000818:Animals; D015153:Blotting, Western; D016572:Cyclosporine / Q000494:pharmacology; D005260:Female; D008387:Marine Toxins; D020125:Mutation, Missense; D009865:Oocytes / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D011546:Pseudohypoaldosteronism / Q000235:genetics; D027981:Symporters / Q000037:antagonists & inhibitors / Q000502:physiology*; D029867:Xenopus Proteins / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis; D000096922:K Cl- Cotransporters
D016572:Cyclosporine; D008387:Marine Toxins; D010080:Oxazoles; D017346:Protein Serine-Threonine Kinases; D027981:Symporters; D029867:Xenopus Proteins; D000096922:K Cl- Cotransporters; C059041:calyculin A; C519694:STK39 protein, Xenopus; C519693:WNK4 protein, Xenopus