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The sodium–potassium–chloride transporter NKCC1 of the SLC12 family performs Na+-dependent Cl−- and K+-ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+, K+, and 2 Cl−) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl− binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl−-ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl−-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure–function relationship of NKCC1 with broader implications for other SLC12 family members.

Background: A number of cAMP-elevating hormones stimulate phosphorylation (and hence activity) of the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Evidence suggests that protein phosphatase 1 (PP1) and other protein phosphatases modulate NCC phosphorylation, but little is known about PP1’s role and the mechanism regulating its function in the DCT. Methods: We used ex vivo mouse kidney preparations to test whether a DCT-enriched inhibitor of PP1, protein phosphatase 1 inhibitor–1 (I1), mediates cAMP’s effects on NCC, and conducted yeast two-hybrid and coimmunoprecipitation experiments in NCC-expressing MDCK cells to explore protein interactions. Results: Treating isolated DCTs with forskolin and IBMX increased NCC phosphorylation via a protein kinase A (PKA)–dependent pathway. Ex vivo incubation of mouse kidney slices with isoproterenol, norepinephrine, and parathyroid hormone similarly increased NCC phosphorylation. The cAMP-induced stimulation of NCC phosphorylation strongly correlated with the phosphorylation of I1 at its PKA consensus phosphorylation site (a threonine residue in position 35). We also found an interaction between NCC and the I1-target PP1. Moreover, PP1 dephosphorylated NCC in vitro, and the PP1 inhibitor calyculin A increased NCC phosphorylation. Studies in kidney slices and isolated perfused kidneys of control and I1-KO mice demonstrated that I1 participates in the cAMP-induced stimulation of NCC. Conclusions: Our data suggest a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA-dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway might be relevant for the physiologic regulation of renal sodium handling by cAMP-elevating hormones, and may contribute to salt-sensitive hypertension in patients with endocrine disorders or sympathetic hyperactivity.

The distal convoluted tubule (DCT) and the cortical collecting ducts (CCD) are portions of renal tubule that are partly responsible for maintaining the systemic concentrations of potassium, sodium, calcium and magnesium. Despite being structurally similar, DCT and CCD cells have different transport capabilities due to a variety of different membrane-associated transport proteins. However, DCT and CCD cells appear to be modulated via the same hormones. The objective of this study was assess the differential response of DCT and CCD cells to long-term exposure to the hormones vasopressin or angiotensin II, both of which modulate DCT and CCD cells differently. Mass spectrometry based quantitative proteomics was used to profile the differential proteome between DCT and CCD. Mouse kidney distal convoluted tubule cells (mpkDCT) were cultured in heavy SILAC medium (Lys+6, Arg+10) while cortical collecting duct cells (mpkCCD) were cultured in light SILAC medium (Lys+0, Arg+0). Four passages of labelled cells were used to generate four biological replicates for statistical analysis. In addition to control conditions, cells were also stimulated for 4 days with the vasopressin type II receptor agonist 1-desamino-8-D-arginine vasopressin (dDAVP, 1nM) or angiotensin II (ANGII, 1nM). Cells were harvested, equally pooled and subjected to offline high-pH fractionation based two dimensional LC-MS/MS analysis (Q-Exactive). Identification and quantification of proteins was performed by MaxQuant. Proteins that had at least three ratios out of four biological replicates were defined as quantifiable proteins. Proteins that had at least three iBAQ values out of four biological replicates in light or heavy channel and zero iBAQ values in the other channel were defined as unique proteins in a particular cell type. Out of the some four thousand quantifiable proteins under basal, dDAVP and AngII conditions, 1101, 1566 and 1294 proteins passed the quantitative Benjamini-Hochberg (BH) FDR 1% thr