Background: WNK4 acts upstream of SPAK/OSR1 in the regulation of Na-K-2Cl cotransporters. Results: Here we show that WNK4 directly binds to NKCC1, and in the presence of Cab39 it stimulates NKCC1 activity. Conclusion: WNK4 regulates NKCC1 through SPAK/OSR1-dependent and SPAK/OSR1-independent pathways. Significance: We uncovered a novel mode of Na-K-2Cl cotransporter activation which involves a direct interaction of WNK4 with the cation-chloride cotransporter.

The Na-K-2Cl cotransporter-1 (NKCC1) is an electroneutral Na + -dependent transporter responsible for simultaneously translocating Na + , K + , and Cl − ions into cells. In human tissue, NKCC1 plays a critical role in regulating cytoplasmic volume, fluid intake, chloride homeostasis, and cell polarity. Here, we report four structures of human NKCC1 (hNKCC1), both in the absence and presence of loop diuretic (bumetanide or furosemide), using single-particle cryo–electron microscopy. These structures allow us to directly observe various novel conformations of the hNKCC1 dimer. They also reveal two drug-binding sites located at the transmembrane and cytosolic carboxyl-terminal domains, respectively. Together, our findings enable us to delineate an inhibition mechanism that involves a coupled movement between the cytosolic and transmembrane domains of hNKCC1.

Background: C-terminal SPAK fragments are found in kidney medulla. Results: We identified Dnpep as the protease responsible for SPAK cleavage, identified the sites of cleavage, and showed unusual preference for ␣ helices. Conclusion: C-terminal SPAK fragments originate from Dnpep-mediated proteolytic cleavage. Significance: SPAK and its cleavage are significant for the regulation of renal Na ϩ reabsorption and control of blood pressure.

Mammalian Ste20-related kinases modulate salt transport and ion homeostasis through physical interaction and phosphorylation of cation-chloride cotransporters. Identification of a sea urchin (Strongylocentrotus purpuratus) ortholog of the mouse Oxidative Stress Response 1 (OSR1) kinase prompted the cloning and testing of the functional effect of a non-mammalian kinase on a mammalian cotransporter. Heterologous expression of sea urchin OSR1 (suOSR1) cRNA with mouse WNK4 cRNA and mouse NKCC1 cRNA in Xenopus laevisoocytes activated the cotransporter indicating evolutionary conservation of the WNK4-OSR1-NKCC signaling pathway. However, expression of a suOSR1 kinase mutated to confer constitutive activity did not result in stimulation of the cotransporter. Using a chimeric strategy, we determined that both the mutated catalytic and regulatory domains of the suOSR1 kinase were functional, suggesting that the tertiary structure of full-length mutated suOSR1 must somehow adopt an inactive conformation. In order to identify the regions or residues which lock the suOSR1 kinase in an inactive conformation, we created and tested several additional chimeras by replacing specific portions of the suOSR1 gene with complimentary mouse OSR1 sequences. Co-expression of these chimeras identified several regions in both the catalytic and regulatory domain of suOSR1 which possibly prevented the kinase from acquiring an active conformation. Interestingly, non-functional suOSR1 chimeras were able to activate mouse NKCC1 when a mouse scaffolding protein, Cab39, was co-expressed in frog oocytes. Sea urchin/mouse OSR1 chimeras and kinase stabilization with mouse Cab39 has provided some novel insights into the activation mechanism of the Ste20-related kinases.

Calcium binding protein 39 (Cab39) is a scaffolding protein that stabilizes a heterotrimeric protein complex that modulates the activity of the adenosine mono‐phosphate kinase which regulates cellular energy homeostasis. Cab39 also modulates SPAK (Ste20 related Proline Alanine rich Kinase) and OSR1 (Oxidative stress responsive kinase 1), two kinases involved in activating the Na‐K‐2Cl cotransporter‐1 (NKCC1). In this study, we demonstrate that mouse Cab39 can substitute for WNK4 phosphorylation of a key serine residue (S383) in the SPAK‐T243E mutant, and even substitute for WNK4 phosphorylation of another key activating residue (threonine 243) when two wild‐type SPAK molecules are tethered together. Western blot analysis of frog oocyte lysates shows an intense signal for an endogenous Cab39. This observation raises the question of why the mouse isoform is necessary to observe the Cab39 effect on SPAK/OSR1 activation of NKCC1. To address this problem, we cloned the cDNA encoding the Xenopus Cab39 isoform and tested its effect on SPAK/OSR1 activation of NKCC1 in frog oocytes. Using site‐directed mutagenesis, we further examined residues that might be involved in the Cab39‐SPAK‐NKCC1 regulatory pathway.