Abstract
The sodium–chloride cotransporter (NCC) is critical for kidney physiology1. The NCC has a major role in salt reabsorption in the distal convoluted tubule of the nephron2,3, and mutations in the NCC cause the salt-wasting disease Gitelman syndrome4. As a key player in salt handling, the NCC regulates blood pressure and is the target of thiazide diuretics, which have been widely prescribed as first-line medications to treat hypertension for more than 60 years5,6,7. Here we determined the structures of human NCC alone and in complex with a commonly used thiazide diuretic using cryo-electron microscopy. These structures, together with functional studies, reveal major conformational states of the NCC and an intriguing regulatory mechanism. They also illuminate how thiazide diuretics specifically interact with the NCC and inhibit its transport function. Our results provide critical insights for understanding the Na–Cl cotransport mechanism of the NCC, and they establish a framework for future drug design and for interpreting disease-related mutations.
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Data availability
The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank. The accession numbers are: EMD-29103, EMD-29097, EMD-29096, EMD-29098, EMD-29099 and EMD-29100. The coordinates have been deposited in the Protein Data Bank. The accession numbers are: 8FHT, 8FHO, 8FHN, 8FHP, 8FHQ and 8FHR.
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Acknowledgements
We thank L. Montabana and M. Zaoralová at Stanford-SLAC Cryo-EM facilities for help with EM data collection. This work was made possible by support from NIH R01 GM138590, Stanford University, and the Harold and Leila Y. Mathers Charitable Foundation to L.F. Some of this work was performed at the Stanford-SLAC Cryo-EM Center (S2C2), which is supported by the US National Institutes of Health Common Fund Transformative High-Resolution Cryo-Electron Microscopy program (U24 GM129541). The content in this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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M.F., Jianxiu Zhang, C.-L.L. and Jinru Zhang carried out biochemical, functional and cryo-EM studies. L.F. directed the project. M.F. and L.F. wrote the manuscript with input from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Biochemical and functional characterizations of human NCC.
a, Representative size-exclusion chromatography profiles of the purified NCC. b, Uptake activities of NCC constructs (mean ± s.d., n = 4 independent experiments). The NCC-mediated I− uptake activity is sensitive to the thiazide diuretic metolazone. The P-value (Control vs. NCCcryo2) was derived from ordinary one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. The P-value (with vs. without metolazone) was derived from two-sided unpaired t test with multiple testing correction using the Bonferroni method. c, Uptake activities of NCC in the presence and absence of extracellular Na+ (normalized to the activity of NCCwt in a sodium-containing condition; mean ± s.d., n = 3 independent experiments). d, I− uptake by NCC at various concentrations of extracellular I− (normalized to the activity with 20 mM extracellular I−; mean ± s.d., n = 4 independent experiments).
Extended Data Fig. 2 Cryo-EM data processing of NCCcryo1.
a, Workflow for NCCcryo1 data processing. Representative cryo-EM image, 2D averages, and workflow for classification and refinement. b, Local resolution of the NCCcryo1 map. c, Gold-standard FSC curves of the NCCcryo1 map. d, Angular distribution of particles for the final reconstruction. e, Map vs. model FSC. f, Cryo-EM density maps of NCCcryo1. g, Density maps of the Na+-binding site and the Cl−-binding site.
Extended Data Fig. 3 Cryo-EM data processing of NCCcryo2.
Workflow for NCCcryo2 data processing. Representative cryo-EM image, 2D class averages, and workflow for classification and refinement.
Extended Data Fig. 4 Quality of the cryo-EM density maps of NCCcryo2.
Local resolutions of the NCC maps of class 1, 2, 3-1, 3-2, and 3-3 (left), corresponding gold-standard FSC curves (middle), and angular distributions of particles for the final reconstructions (right).
Extended Data Fig. 5 Quality of the cryo-EM density maps of NCCcryo2.
a, Cryo-EM density maps of NCCcryo2. b, Map vs. model FSC for NCCcryo2. c, Density maps of the nucleotide and polythiazide in NCCcryo2. d, Density map of the Na+-binding site in NCCcryo2.
Extended Data Fig. 6 NCC architecture and ion binding sites.
a, Organization of the NCC dimer. b, Conformational differences of the TMDs in three classes of NCCcryo2 structures (class 3-2 as a representative for class 3). Within class 3, three sub-classes (3-1, 3-2, and 3-3) superimpose well onto each other, with only slight relative rotation between two protomers. c, Superposition of the NCC, NKCC1 (PDB: 7S1X), and KCC1 (PDB: 7TTI) TMD in an outward-facing conformation. d, Proposed Na+-binding site of NCCcryo2 (green) superimposed onto the Na+-binding site of NKCC1 (cyan) and Na2 site of the Na+-coupled sialic acid symporter SiaT40 (yellow). Dashed lines denote possible Na+ coordination. Na+ is shown as a purple sphere. e, Proposed Cl−-binding site of NCCcryo1 (green) superimposed onto the SCl2 site of NKCC1 (cyan) and KCC1 (wheat). Dashed lines denote possible Cl− coordination. Cl− is shown as an orange sphere. f, Uptake activities of NCC variants with mutations in the substrate-binding pocket (normalized to WT; mean ± s.d., n = 4 independent experiments for variants, n = 8 independent experiments for WT and control).
Extended Data Fig. 7 Chemical structures of thiazides and the binding site of polythiazide.
a, Chemical structures of representative thiazide diuretics. The 6-postion chloro or –CF3 group and the 7-position sulfamoyl group are coloured green and red, respectively. b, Polythiazide overlaps with the Cl−-binding site of NCC. Space that a Cl− ion could otherwise occupy is shown as orange dots. c, Binding site of polythiazide. The 2-position methyl group of polythiazide points to a hydrophilic cavity. The surface is coloured by electrostatic potential (red, −5 kT e−1; blue, +5 kT e−1).
Extended Data Fig. 8 NCC gates and functional characterizations.
a, Intracellular gate. b, Extracellular gate. In a and b, the salt bridge and hydrogen bond interactions are shown as dashed lines. c, Uptake activities of NCC variants with mutations at the extracellular or intracellular gate (normalized to WT; mean ± s.d., n = 4 independent experiments except for WT and control, n = 12 independent experiments). d, Uptake activities of NCC variants with mutations at the nucleotide-binding site (normalized to WT; mean ± s.d., n = 4 independent experiments). e, Surface expression levels of NCC WT and variants (normalized to WT; mean ± s.d., n = 3 independent experiments for variants. For WT and control, in the upper panels, n = 4 independent experiments; in the lower middle and right panels, n = 5 independent experiments. In the lower left panel, n = 6 independent experiments for WT, n = 5 independent experiments for control).
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Fan, M., Zhang, J., Lee, CL. et al. Structure and thiazide inhibition mechanism of the human Na–Cl cotransporter. Nature 614, 788–793 (2023). https://doi.org/10.1038/s41586-023-05718-0
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DOI: https://doi.org/10.1038/s41586-023-05718-0
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