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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. Synopsis: The Na+‐K+‐2Cl− cotransporter NKCC1 (SLC12A2) performs Na+‐dependent uptake of Cl− and K+ ions and is an important factor in ion homeostasis and osmotic control. Its mechanisms of ion recognition and release are addressed here by cryo‐EM microscopy, ion uptake studies, and molecular dynamics simulations. A 2.6 Å resolution cryo‐EM map reveals lipid molecules at a dimeric interface and three cholesterol molecules per protomer that regulate its transport activity.Analysis of the revealed intracellular water networks and bound ions indicates that the Cl– ion at a Cl2 site is released reversibly prior to Na+ release.Intracellular Na+ ion release is modeled and indicates guidance along transmembrane helix 5 by two negatively charged glutamate residues.The Cl2 site, which overlaps with a conserved glutamate residue in the SLC6 transporter, may sense chloride to regulate transport. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]