Structure and mechanism of the cation-chloride cotransporter NKCC1

Abstract

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.

Extended Data Figure 1 |. Biochemical characterization of NKCC1 and cryo-EM data processing workflow for N-terminal truncation NKCC1.

a, NKCC1 in size exclusion chromatography. Experiments were repeated 6 times independently with similar results. b, Representative negative-stain EM micrograph of purified NKCC1 in digitonin. Experiments were repeated 6 times independently with similar results. c, 2D class averages from negative-stained particles reveal the dimeric structure of NKCC1, with two soluble domains visible beneath the TM domain and detergent micelle. Experiments were repeated 6 times independently with similar results. d, Representative cryo-EM micrograph of N-terminal truncated NKCC1 particles purified in digitonin. e, 2D class averages of N-terminal truncated NKCC1. f, Classification and refinement workflow utilized to obtain final cryo-EM maps of N-terminal truncated NKCC1. Two processing strategies were followed in order to obtain a high-resolution map of the TM domain (left), or a composite map of the TM and soluble domains following Relion3 multibody refinement (right).

Extended Data Figure 2 |. Cryo-EM data processing workflow for full-length NKCC1.

a, Representative cryo-EM micrograph of full-length NKCC1 particles purified in digitonin. b, 2D class averages of full-length NKCC1. Several averages show high resolution features in the TM domain, however the soluble domain is fuzzy and disordered, indicative of conformational flexibility between these two domains. c, Classification and refinement workflow utilized to obtain final cryo-EM maps of full-length NKCC1. Two processing strategies were followed in order to obtain a high-resolution map of the TM domain (left), or a composite map of the TM and soluble domains following Relion3 multibody refinement (right).

Extended Data Figure 3 |. Cryo-EM 3D reconstruction analysis, statistics and representative density.

a, Angle distributions for particles contributing to the high-resolution map of the TM domain for full-length NKCC1. b, Fourier shell correlation (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 TM 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. c, Local resolution of the high-resolution TM domain 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, Fourier shell correlation (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 TM domain and show clear side chain features in CTD. Densities for all TM helices, as well as helices and strands in CTD are shown as wire mesh (6 σ). The transporter is shown as sticks.

Extended Data Figure 4 |. Principal Component Analysis of domain movements from multibody refinement.

a, Contribution of individual eigenvectors to the total variance in rotation and translation between the TM 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/translations are continuous in nature. e-g, Representation of the extremes of rotation/translation between TM and soluble domains 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 TM domains. Blue arrows indicate the direction of movement of the soluble domain relative to the TM domain.

Extended Data Figure 5 |. Structure of soluble domain.

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 TM and soluble domain interface. Two subunits are coloured in dark green and gold, respectively. The TM domain 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 at the interface. d, The intracellular surface of the TM domain. The surface is coloured according to electrostatic potential (red, −10 kT e⁻¹; blue, +10 kT e⁻¹).

Extended Data Figure 6 |. Uptake activities of interface mutants and characterizations of all NKCC1 mutants in this study.

a, Uptake activities of NKCC1 mutants at the TM and the cytosolic domain interface. ⁸⁶Rb⁺ uptake of NKCC1 mutant was normalized to that of WT (mean± s.e.m., n=4 independent experiments except for WT, n=7 independent experiments and for WT with bumetanide, n=3 independent experiments). b, NKCC1 WT and mutants (also including those in Fig. 1e, 3e, 4f) in size exclusion chromatography. The GFP-fusion protein is monitored by fluorescence. Experiments were repeated 3 times independently with similar results. c, The expression level of NKCC1 WT and mutants as shown by Western blot. Experiments were repeated 3 times independently with similar results. For gel source data, see Supplementary Figure 2. d, The membrane localization of NKCC1 WT and mutants. The fluorescence images are shown for HEK293 cell expressing NKCC1-GFP fusion. Experiments were repeated 3 times independently with similar results.

Extended Data Figure 7 |. Molecular dynamics simulations.

a, Ion probability densities of ions within translocation pathway (top) and individual traces (bottom) for all simulations performed with K⁺, Na⁺, and Cl^– initially present in the translocation pathway. b, Ion probability densities of ions within translocation pathway (top) and individual traces (bottom) for all simulations performed with only K⁺ and Na⁺ initially present in the translocation pathway. 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.

Extended Data Figure 8 |. Ion stability in MD simulations.

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, whose increased proximity to Cl⁻ site 1 likely 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 toward the intracellular side (220 ns) before rebinding for a 25-ns period within the proposed Na⁺-binding site.

Figure 1 |. Overall structure and functional characterization of NKCC1.

a, Full-length NKCC1 density map coloured by protomer. b, Overall structure of NKCC1 dimer. Ribbon representations viewed at two angles. c, Topology diagram. d, Ribbon representation of NKCC1 protomer, coloured by structural element. e, Uptake activities of zNKCC1. The activity was normalized to WT (mean±s.e.m., n=4 independent experiments except for WT and control, n=10 independent experiments).

Figure 2 |. TM domain and dimer interface.

a, TM domain structure in one subunit, viewed from membrane (left) or intracellular side (right). b, Slab view of one TM domain showing partially inward-open conformation. Surface coloured by electrostatic potential (red, −5 kT e⁻¹; blue, +5 kT e⁻¹). c, Lipids and dimer of TMDs, viewed from intracellular side. d, Ordered lipid molecules at dimer interface. Densities attributed to lipids shown as blue meshes.

Figure 3 |. Potassium- and sodium-binding sites.

a, Potassium-binding site density map (10.0 σ, blue mesh). Strong extra density is attributed to K⁺ (pink sphere). b, Potassium-binding site. Dashed lines denote possible coordination. c, Sodium-binding site. NKCC1’s proposed Na⁺-binding site (yellow) is superimposed onto SiaT’s Na2 site (green). Dashed lines denote the sodium coordination in SiaT. d, The sequence alignment around key K⁺- or Na⁺-coordinating residues. Human and zebrafish proteins are denoted by “h” and “z”, respectively. Highlighted positions show Y305 (potassium-coordinating), S538 and S539 (sodium-coordinating). e, Uptake activities of the substrate-binding pocket mutants, normalized to WT (mean±s.e.m., n=4 independent experiments; WT and control are the same as in Fig. 1e).

Figure 4 |. Chloride-binding sites.

a, Probability density for potassium (pink), sodium (blue), or chloride (green) across two simulations initiated with bound cations. Spheres indicate the initial position of cations. b, Three regions of chloride binding identified in simulation. c, Ion probability densities (top), as shown in a, for a simulation initiated with all four ions bound. The initial position of K⁺, Na⁺, and Cl⁻ are indicated as pink, blue, and green (dashed boundaries) spheres, respectively. The positions of Cl⁻ based on the cryo-EM are indicated by green spheres with solid boundaries. First five traces (bottom) show distance from ion-binding site to nearest ion of that type (see Methods). In the presence of stably bound chlorides, cations reside longer within the translocation pore. Light grey lines at 5 Å offer visual guide. Bottom trace shows number of chlorides occupying the chloride-binding sites. d, Upper chloride-binding site (site 1) and potassium-binding site density map (11.0 σ, blue mesh). e, Lower chloride-binding site (site 2) density map (6.0 σ, blue mesh). f, Mutant uptake activities, normalized to WT (mean±s.e.m., n=4 independent experiments; WT and control are the same as in Fig. 1e).

Figure 5 |. Disease-related mutations and interconnected ion-binding sites.

a, Mutations on NCC linked to Gitelman syndrome mapped onto the structure through sequence alignment. Mutations are shown as spheres and grouped into six categories: ion translocation pathway (purple), cap domain (blue), TM domain dimer interface (green), TM-soluble interface (red), CTD dimer interface (cyan), and structural/folding (yellow) mutations. Other mutations in grey. b, Schematic of ion-binding sites: coordination, dashed lines; core domain helices, light blue; and scaffold domain helices, yellow.