Cation–chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we determined the cryo-electron microscopy structure of the Na–K–Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiological functions of CCCs and interpreting disease-related mutations.
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The three-dimensional cryo-EM density maps have been deposited into the Electron Microscopy Data Bank under accession numbers EMD-0470, EMD-0471, EMD-0472, EMD-0473, EMD-0474 and EMD-0475. The coordinates are deposited into the Protein Data Bank with accession numbers 6NPH, 6NPJ, 6NPK and 6NPL. All simulation starting models and trajectories are available upon request.
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We thank D. Bushnell and L. Montabana at Stanford-SLAC Cryo-EM facilities, Z. Li at HMS, C. Xu and K. Song at the UMass cryo-EM facility, and C. López and C. Yoshioka at the OHSU Multiscale Microscopy Core for help with electron microscopy data collection. We thank the staff at the Beckman Center Cell Sciences and Imaging Facility for their help with fluorescence imaging. This work was made possible by support from Stanford University and the Harold and Leila Y. Mathers Charitable Foundation to L.F., an NIH Kirschstein-NRSA fellowship and Medical Scientist Training Program funding to T.A.C., a Dean’s fellowship to J.Z., National Science Foundation Graduate Research Fellowships to N.R.L. and A.W., and a Stanford Graduate Fellowship to A.W. We thank A. Brunger and M. Maduke for helpful comments.
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Biochemical characterization of NKCC1 and cryo-EM data processing workflow for N-terminal truncated NKCC1.
a, NKCC1 in size-exclusion chromatography. Experiments were repeated six times independently with similar results. b, Representative negative-stain EM micrograph of purified NKCC1 in digitonin. Experiments were repeated six times independently with similar results. c, Two-dimensional class averages from negative-stained particles reveal the dimeric structure of NKCC1, with two soluble domains visible beneath the TMD and detergent micelle. Experiments were repeated six times independently with similar results. d, Representative cryo-EM micrograph of N-terminal truncated NKCC1 particles purified in digitonin. e, Two-dimensional class averages of N-terminal truncated NKCC1. f, Classification and refinement workflow used to obtain final cryo-EM maps of N-terminal truncated NKCC1. Two processing strategies were followed to obtain a high-resolution map of the TMD (left), or a composite map of the TMD and soluble domain following Relion3 multibody refinement (right).
a, Representative cryo-EM micrograph of full-length NKCC1 particles purified in digitonin. b, Two-dimensional class averages of full-length NKCC1. Several averages show high resolution features in the TMD; however, the soluble domain is fuzzy and disordered, indicative of conformational flexibility between these two domains. c, Classification and refinement workflow used to obtain final cryo-EM maps of full-length NKCC1. Two processing strategies were followed to obtain a high-resolution map of the TMD (left), or a composite map of the TMD and soluble domain following Relion3 multibody refinement (right).
a, Angle distributions for particles contributing to the high-resolution map of the TMD for full-length NKCC1. b, FSC curves: gold-standard FSC curve between the two half maps with indicated resolution at FSC = 0.143 (red); FSC curve between the atomic model of the TMD and the final map with indicated resolution at FSC = 0.5 (blue); FSC curve between half map 1 (orange) or half map 2 (green) and the atomic model refined against half map 1. c, Local resolution of the high-resolution TMD map as determined with ResMap. d, Slice view of the angle distributions for particles contributing to the map of the soluble domain for full-length NKCC1 obtained from Relion3 multibody refinement. e, FSC curves: gold-standard FSC curve between the two half maps with indicated resolution at FSC = 0.143 (red); FSC curve between the atomic model of the soluble domain and the final map with indicated resolution at FSC = 0.5 (blue); FSC curve between half map 1 (orange) or half map 2 (green) and the atomic model refined against half map 1. f, Local resolution of the soluble domain map as determined with ResMap. g, The cryo-EM density maps are of high quality throughout the TMD and show clear side chain features in CTD. Densities for all transmembrane helices, as well as helices and strands in the CTD are shown as wire mesh (6σ). The transporter is shown as sticks.
a, Contribution of individual eigenvectors to the total variance in rotation and translation between the TMD and soluble domain. Eigenvectors 1–3 contribute more than 50% of the total variance in the rotations and translations between domains. b–d, Histograms of amplitude along eigenvector 1 (b), eigenvector 2 (c) and eigenvector 3 (c). All eigenvector amplitude histograms are monomodal, suggesting that the rotations and/or translations are continuous in nature. e–g, Representation of the extremes of rotation and translation between TMD and soluble domain along eigenvector 1 (e), eigenvector 2 (f) and eigenvector 3 (g). For simplicity of visualization, the maps at either extreme of an individual eigenvector were aligned on the TMDs. Blue arrows indicate the direction of movement of the soluble domain relative to the TMD.
a, A single subunit of the NKCC soluble domain in ribbon representation. The N- and C-terminal halves of the CTD that show a similar structure are coloured in green and orange, respectively. b, The dimer of the soluble domain. One subunit is shown as a surface and the other is shown as a ribbon. c, Close-up view of the TMD–soluble domain interface. Two subunits are coloured in dark green and gold, respectively. The TMD is shown as a surface representation and the soluble domain as a ribbon representation. The NTD and the C-terminal end of the protein are located near the interface. d, The intracellular surface of the TMD. The surface is coloured according to electrostatic potential (red, −10 kT e−1; blue, +10 kT e−1).
Extended Data Fig. 6 Uptake activities of interface mutants and characterizations of all NKCC1 mutants in this study.
a, Uptake activities of NKCC1 mutants at the TMD–cytosolic domain interface. 86Rb+ uptake of NKCC1 mutants was normalized to that of the wild type (mean ± s.e.m., n = 4 independent experiments except for wild type, n = 7 independent experiments and for wild type with bumetanide, n = 3 independent experiments). b, Wild-type NKCC1 and mutants (also including those in Figs. 1e, 3e, 4f) in size-exclusion chromatography. The GFP fusion protein is monitored by fluorescence. Experiments were repeated three times independently with similar results. c, The expression level of wild-type NKCC1 and mutants as shown by western blot. Experiments were repeated three times independently with similar results. For gel source data, see Supplementary Fig. 2. d, The membrane localization of wild-type NKCC1 and mutants. The fluorescence images are shown for HEK293 cells expressing NKCC1–GFP. Experiments were repeated three times independently with similar results.
a, Ion probability densities of ions within translocation pathway (top) and individual traces (bottom) for simulations performed with K+, Na+, and Cl− initially present in the translocation pathway (model 1, see Methods). b, Ion probability densities of ions within translocation pathway (top) and individual traces (bottom) for simulations performed with only K+ and Na+ initially present in the translocation pathway (model 1, see Methods). In the latter set of simulations, Cl– spontaneously explores regions that form stable chloride-binding sites. Probability density maps were calculated as described in Methods. The top four simulation traces represent the minimum distance of an ion of a particular type from the pre-determined centre of each binding site (see Methods). The bottom simulation trace shows the number of Cl− ions bound within any of the three chloride-binding sites at once.
a–d, Comparison of relative stabilities of ions across multiple simulation conditions. Here, stability refers to the amount of time an ion resides within 3 Å of the pre-determined centre of each binding site (see Methods). Each dot corresponds to a result from a single simulation. Closed circles indicate simulations with each cation placed in its predicted site; open circles indicate simulations with either cation swapped into the other cation’s predicted binding site. Blue circles correspond to measurements for Na+ and pink circles for K+. For a and b, we examined the relative stabilities of cations to determine whether the predicted Na+ and K+ binding sites exhibit a preference for their respective cations. a, For simulations started with all ions bound, Na+ resided longer in the Na+ site compared to K+ when K+ was initially placed within the Na+ site (‘Na+/K+/2Cl−’ vs ‘Na+/K+ swapped, 2 Cl−’). The same trend appeared for simulations with just cations initially bound within the cavity (‘Na+/K+’ vs ‘Na+/K+ swapped’). b, For simulations started with all ions bound, K+ resided for longer in the K+ site compared to Na+ when Na+ was initially placed within the K+ site (‘Na+/K+/2Cl−’ vs ‘Na+/K+ swapped, 2 Cl−’). For simulations with just cations initially bound, K+ and Na+ appeared to leave after similar amounts of time—perhaps because, particularly in the absence of Cl−, the K+ site is quite accessible to the intracellular solvent, and ions of either type could dissociate rapidly from this area. c, K+ remained within its initial position much longer compared to Na+ in the presence of two Cl− ions. In simulations with Na+ and 2 Cl− ions bound, Na+ often immediately dissociated from its site, perhaps indicating that the Na+ site on its own has a weaker affinity for binding cations in this state compared to the K+ site, the increased proximity of which to Cl− site 1 probably helps to stabilize K+ in the site. d, Chloride ions spontaneously visited and remained within 3 Å of the primary intracellular chloride site (site 2) for longer in the presence of both cations compared to in the presence of either cation alone. e, In one simulation, in which Na+ and K+ were initially placed in a swapped configuration with no Cl− ions bound, we observed escape of K+ (pink) followed by destabilization of the Na+ ion (blue) within the K+ site (123 ns) accompanied by Cl− binding. Na+ then proceeded to move down towards the intracellular side (220 ns) before rebinding for a 25-ns period within the proposed Na+-binding site.
This file contains: Supplementary Figure 1 - sequence alignment of CCC transporters; Supplementary Figure 2 - source image for gel electrophoresis in Extended Data Figure 6c; Supplementary Table 1 -structural mapping of NCC mutations in Gitelman Syndrome and Supplementary References.
Visualization of movement between NKCC1 TM and CTD along eigenvector 1, revealing a swiveling type motion between individual domains.
Visualization of movement between NKCC1 TM and CTD along eigenvector 2, revealing a rocking type motion between individual domains.
Visualization of movement between NKCC1 TM and CTD along eigenvector 3, revealing a rocking type motion between individual domains.
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Chew, T.A., Orlando, B.J., Zhang, J. et al. Structure and mechanism of the cation–chloride cotransporter NKCC1. Nature 572, 488–492 (2019). https://doi.org/10.1038/s41586-019-1438-2
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