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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., (©2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

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., (Copyright © 2019 by the American Society of Nephrology.)

We and others have recently shown that angiotensin II can activate the sodium chloride cotransporter (NCC) through a WNK4-SPAK-dependent pathway. Because WNK4 was previously shown to be a negative regulator of NCC, it has been postulated that angiotensin II converts WNK4 to a positive regulator. Here, we ask whether aldosterone requires angiotensin II to activate NCC and if their effects are additive. To do so, we infused vehicle or aldosterone in adrenalectomized rats that also received the angiotensin receptor blocker losartan. In the presence of losartan, aldosterone was still capable of increasing total and phosphorylated NCC twofold to threefold. The kinases WNK4 and SPAK also increased with aldosterone and losartan. A dose-dependent relationship between aldosterone and NCC, SPAK, and WNK4 was identified, suggesting that these are aldosterone-sensitive proteins. As more functional evidence of increased NCC activity, we showed that rats receiving aldosterone and losartan had a significantly greater natriuretic response to hydrochlorothiazide than rats receiving losartan only. To study whether angiotensin II could have an additive effect, rats receiving aldosterone with losartan were compared with rats receiving aldosterone only. Rats receiving aldosterone only retained more sodium and had twofold to fourfold increase in phosphorylated NCC. Together, our results demonstrate that aldosterone does not require angiotensin II to activate NCC and that WNK4 appears to act as a positive regulator in this pathway. The additive effect of angiotensin II may favor electroneutral sodium reabsorption during hypovolemia and may contribute to hypertension in diseases with an activated renin-angiotensin-aldosterone system.