Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins.
Mutations in the WNK4 gene cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder of hyperkalemia and hypertension. The target molecules of this putative kinase and the molecular mechanisms by which the mutations cause the phenotypes are currently unknown. Although recent reports found that expression of WNK4 in Xenopus oocytes causes inhibition of the thiazide-sensitive NaCl cotransporter and the renal K channel ROMK, there may be additional targets of WNK4. For example, an increase in paracellular chloride permeability has been postulated to be a mediator of PHAII pathogenesis, a possibility supported by the localization of WNK4 at tight junctions in vivo. To determine the validity of this hypothesis, we measured transepithelial Na and Cl permeability in Madin-Darby canine kidney II cells stably expressing wild-type or a pathogenic mutant of WNK4. We found that transepithelial paracellular Cl permeability was increased in cells expressing a disease-causing mutant WNK4 (D564A) but that Na permeability was decreased slightly. Furthermore, WNK4 bound and phosphorylated claudins 1-4, major tight-junction membrane proteins known to be involved in the regulation of paracellular ion permeability. The increases in phosphorylation of claudins were greater in cells expressing the mutant WNK4 than in cells expressing wild-type protein. These results clearly indicate that the pathogenic WNK4 mutant possesses a gain-of-function activity and that the claudins may be important molecular targets of WNK4 kinase. The increased paracellular "chloride shunt" caused by the mutant WNK4 could be the pathogenic mechanism of PHAII.
Yamauchi
Kozue
K
Department of Nephrology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo, Tokyo 113-8519, Japan.
Rai
Tatemitsu
T
Kobayashi
Katsuki
K
Sohara
Eisei
E
Suzuki
Tatsunori
T
Itoh
Tomohiro
T
Suda
Shin
S
Hayama
Atsushi
A
Sasaki
Sei
S
Uchida
Shinichi
S
D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D003001:Cloning, Molecular; D004285:Dogs; D006801:Humans; D007668:Kidney; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000502:physiology; D019108:Tight Junctions / Q000502:physiology
D002712:Chlorides; D008565:Membrane Proteins; D012333:RNA, Messenger; D012964:Sodium; D017346:Protein Serine-Threonine Kinases; C433806:WNK4 protein, human