biostudies-literatureZhao YNIDDK NIH HHSNIGMS NIH HHS2747https://www.ebi.ac.uk/biostudies/studies/S-EPMC9117670biostudies-literatureUnknown13(1)Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na⁺, 1 K⁺, and 2 Cl^- across cell membranes. NKCC1 mediates trans-epithelial Cl^- secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.Nature communicationsStructural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide.PMC9117670U24 GM129547U24 GM129539R01 DK128592Roy KVidossich PCancedda LDe Vivo MForbush BCao EZhao YfalseStructural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide.Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na⁺, 1 K⁺, and 2 Cl^- across cell membranes. NKCC1 mediates trans-epithelial Cl^- secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.2022-01-01T00:00:00Z2022 May2024-11-10T08:44:24.157Z2024-11-10T08:44:24.157ZS-EPMC91176703558505310.1038/s41467-022-30407-3