Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4.
In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl- cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl- cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2.
Gagnon
Kenneth B E
KB
Dept. of Anesthesiology, Vanderbilt Univ. Medical Center, T-4202 Medical Center North, 1161 21st Ave. South, Nashville, TN 37232, USA.
England
Roger
R
Delpire
Eric
E
D000818:Animals; D051379:Mice; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014982:Xenopus laevis; D000096922:K Cl- Cotransporters
D011188:Potassium; D017346:Protein Serine-Threonine Kinases; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; D000096922:K Cl- Cotransporters; C578878:Slc12a2 protein, mouse; C499024:Prkwnk4 protein, mouse; C486407:Stk39 protein, mouse