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Structure of a CLC chloride ion channel by cryo-electron microscopy

Nature volume 541, pages 500–505 (2017)Cite this article

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Abstract

CLC proteins transport chloride (Cl−) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC proteins are channels that conduct Cl− ions passively, whereas others are secondary active transporters that exchange two Cl− ions for one H+. The structural basis underlying these distinctive transport mechanisms is puzzling because CLC channels and transporters are expected to share the same architecture on the basis of sequence homology. Here we determined the structure of a bovine CLC channel (CLC-K) using cryo-electron microscopy. A conserved loop in the Cl− transport pathway shows a structure markedly different from that of CLC transporters. Consequently, the cytosolic constriction for Cl− passage is widened in CLC-K such that the kinetic barrier previously postulated for Cl−/H+ transporter function would be reduced. Thus, reduction of a kinetic barrier in CLC channels enables fast flow of Cl− down its electrochemical gradient.

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Figure 1: Functional and cryo-EM analysis of a bovine CLC-K channel.
Figure 2: Flexibility of the CLC-K dimer arrangement.
Figure 3: Architecture of the CLC-K channel.
Figure 4: Cl− ion transport pathway.
Figure 5: Cl− ion accessibility in the pore region.
Figure 6: Models for the mechanisms of ion transport by CLC.

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Acknowledgements

We thank M. Ebrahim at the Rockefeller University Cryo-EM Resource Center for help with microscope operation, staff at the Memorial Sloan Kettering Cancer Center Antibody & Bioresource Core Facility for hybridoma generation, Y. C. Hsiung for help with large-scale cell culture, members of the MacKinnon laboratory for helpful discussions, and J. Chen for critical reading of the manuscript. E.P. is supported by the Jane Coffin Childs Memorial Fund fellowship (#61-1513). R.M. is a Howard Hughes Medical Institute investigator.

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Authors and Affiliations

  1. Laboratory of Molecular Neurobiology and Biophysics and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, 10065, New York, USA

    Eunyong Park, Ernest B. Campbell & Roderick MacKinnon

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  1. Eunyong Park
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  3. Roderick MacKinnon
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Contributions

E.P. performed experiments. E.B.C. assisted in development of monoclonal antibodies. E.P. and R.M. designed experiments, analysed and interpreted results, and wrote the manuscript.

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Correspondence to Roderick MacKinnon.

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Reviewer Information Nature thanks C. Miller, M. Pusch, S. Scheres and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Functional characterization of the bovine CLC-K channel.

a, Representative two-electrode voltage clamp (TEVC) recordings of bovine CLC-K channel in X. laevis oocytes. Clamping voltages were from −60 to +60 mV (10-mV steps). b–e, I–V curves of recordings in a. Shown are means and standard deviations (s.d.; error bars) of 5, 9, 10 and 11 independent oocyte recordings, respectively. f, I–V curves of whole-cell patch recordings on Chinese hamster ovary (CHO) cells expressing CLC-K and barttin. The pipette and bath solutions contain 144 and 52 mM Cl−, respectively. Shown are means and s.d. (error bars) of 3 or 5 independent recordings using different cells. g, Immunofluorescence staining of the bovine CLC-K channel expressed on the plasma membrane. CHO cells were transiently transfected with a green fluorescent protein (GFP)-tagged channel construct alone or together with barttin, and then cell-surface-targeted channels were probed by non-permeabilized immunofluorescence (IF) staining using monoclonal anti-CLC-K antibodies (clone 16E3), which specifically recognize an extracellular epitope of the CLC-K channel. Hoechst 33342 was used to stain nuclei. The same exposure parameters were used for the left and right panels. Shown are representative images of reproducible results. Scale bar, 10 μm.

Extended Data Figure 2 Cryo-EM image processing procedure.

a, A representative micrograph of the CLC-K–Fab complex (scale bar, 50 nm). Boxed regions (white squares) are magnified on the right to show representative particle images. Note that in the top/bottom views, Fab signals are evident while the channel part is barely visible because of low contrast (also see b). b, Images of selected two-dimensional classes from reference-free two-dimensional classification by RELION. Note that the distal half of the Fab fragment shows much flexibility, resulting in blurred features. Scale bar, 10 nm. c, Summary of the image processing procedure (see Methods).

Extended Data Figure 3 Three-dimensional reconstruction of CLC-K.

a, Angular distribution histogram of class 1 particle projections. b, Fourier shell correlation (FSC) of class 1 half maps before (black) and after (blue and red) masking. Two soft masks were used (see also c): one (red) including only the channel portion and the other (blue) including the channel and the variable domain (VH/VL) of the Fab fragments but excluding the constant domain (CH1/CL) of Fab. When a mask was used, the FSC curve was corrected for masking effects during the RELION postprocessing procedure (phase randomization above 7.8 Å). c, Masks used in FSC calculations (red and blue) in b are shown with the unsharpened, unfiltered map (grey) superimposed. A contour level of 0.7 was used for the surface representation of the masks. d, Local resolution map of the class 1 reconstruction estimated by the blocres program. Shown is the combined map, which is not sharpened or filtered. e, FSC between model and map of the class 1 particles. The black curve shows FSC between the final refined atomic model and the combined map that the model was refined against. The blue and red curves show FSC between the atomic model and the half map it was refined against (half 1) and FSC between the atomic model and the other half map it was not refined against (half 2), respectively (see also Methods). f–j, As in a–e, but with the class 2 structure. For the FSC calculations with the masks in g, the FSC curves were corrected for masking effects (phase randomization above 8.7 Å).

Extended Data Figure 4 EM density of the CLC-K channel.

EM density segments (mesh) of the class-1 CLC-K reconstruction are superimposed with the model in a ribbon representation. Numbers indicate amino acid positions of segments. The density map was sharpened with a B-factor of −100 Å2 and low-pass filtered at 3.7 Å. CTD, cytosolic domain. VL, variable domain of the IgG light chain. VH, variable domain of the IgG heavy chain. Note that a model for the distal half of the Fab fragment was not built because of poor density features in this region caused by its high flexibility (see also Extended Data Fig. 2b).

Extended Data Figure 5 Comparison between the class 1 and 2 structures of CLC-K.

a, The dimer interfaces (dashed boxes) of the class 1 and 2 structures were compared. Views from the extracellular side were aligned based on the upper (red) subunit. In addition, the right panel shows superposition of a class 1 monomer (grey) onto a class 2 monomer (red). b, N-terminal amphipathic helix of class 2 structure. A side view of the class 2 structure is shown in a ribbon representation superimposed with density (mesh) of the N-terminal helix. The two subunits are shown in pale green and blue. The TM helix αB, which is visible in both class 1 and 2 structures is shown in dark green. A 12-amino-acid amphipathic helix preceding αB, visible only in class 2, is shown in yellow. The density map was sharpened with a B-factor of −120 Å2 and low-pass filtered at 4.1 Å. The approximate membrane boundaries are indicated by grey lines.

Extended Data Figure 6 Structural features of the CLC-K channel.

a, Comparison between the CLC-K (class 1; grey and magenta) and EcCLC (yellow) structures. The TM domain of an EcCLC monomer was superposed onto that of CLC-K. α-Helices are represented as cylinders. b, The density of the extracellular loops αI–J of the CLC-K channel (class 1) is shown in blue and orange. The density of the remaining parts is shown in pale blue and yellow. In the top right panel, the model of one subunit is shown in the ribbon representation. αN and αF are highlighted, and Val166 and Tyr520 are shown in a ball-and-stick representation (green). The two ends of the αI–J link are indicated as cyan spheres with amino acid positions. The bottom panels show side views of one subunit with the αI–J link in blue density. The density map (5σ cut-off) was sharpened with a B-factor of −100 Å2 and low-pass filtered at 4.0 Å. c, As in the top left panel of b, but additionally showing a model of the αI–J link of EcCLC in a ball-and-stick representation (yellow). d, The same view as in the bottom right panel of b, but with superposition of an EcCLC model (pink; PDB accession 1OTS) onto the CLC-K model. A model for the αI–J linker of EcCLC is shown in yellow balls and sticks. e, Sequence alignment of the extracellular segments between TM helices αK and αM from various CLC proteins. The segments forming α-helices in the extracellular domain of bovine CLC-K are indicated by red coils. Red and blue dots indicate positions of mutations causing Bartter syndrome (CLC-Kb) and myotonia congenita (CLC-1), respectively. The A349D (ref. 5) mutation in CLC-Kb causes Bartter syndrome. The P408A (ref. 72), Q412P (ref. 73), F413C (ref. 4), A415V (ref. 74), E417G (ref. 75) or W433R (ref. 76) mutations in CLC-1 (corresponding to positions 342, 346, 347, 349, 351 and 367 of CLC-K, respectively) cause myotonia congenita. In addition, mutations at R438 (R438C in CLC-Kb or R496S in CLC-1), of which the side chain directly interacts with W367 (W433 in CLC-1) cause Bartter syndrome5 or myotonia congenita77 (see Fig. 4c).

Extended Data Figure 7 Structure of the ion transport pathway.

a, Stereo images of the atomic model of the CLC-K channel’s Cl−-selective filter (class 1; light magenta) and comparisons with that of CmCLC (cyan) and EcCLC (yellow) are shown in a stick representation. In the cases of CmCLC and EcCLC, bound Cl− ions are shown as spheres. Predicted polar interactions are indicated by grey dashed lines. b, EM density and model of the αC–D loop are shown for the class-2 CLC-K structure. Also, density and model were shown for the Tyr520 side chain. The density map (grey mesh) was sharpened with a B-factor of −120 Å2 and low-pass filtered at 4.1 Å.

Extended Data Figure 8 Density in the Scen Cl−-binding site.

A view into Scen of CLC-K (from Sext towards the cytosol) is shown in a stereo diagram. Predicted polar interactions between bound Cl− ions and the protein, including anion–quadrupole interactions, are indicated by green dashed lines. The shown EM density map is of the class 1 structure, sharpened with a B-factor of −100 Å2 and low-pass filtered at 3.7 Å. Density for protein (grey mesh) and possible ions (magenta mesh) are shown at contour levels of 5.8σ and 3.2σ, respectively.

Extended Data Figure 9 Surface electrostatic potential.

Surface electrostatics of the CLC-K channel (class 1) compared with the EcCLC and CmCLC transporters. Left panels show side views with one subunit in a surface representation and the other in a ribbon representation. Electrostatic potential is indicated by the colour scale (bottom left). Views from the extracellular side (blue arrow) and intracellular side (red arrow) are shown in the middle and right panels, respectively. The entrances to the Cl− ion pathway are indicated by dashed circles. Mobile monovalent cation and anion species at 0.15 M each were included in the electrostatic potential calculations.

Extended Data Table 1 Model refinement statistics
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Park, E., Campbell, E. & MacKinnon, R. Structure of a CLC chloride ion channel by cryo-electron microscopy. Nature 541, 500–505 (2017). https://doi.org/10.1038/nature20812

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  • DOI: https://doi.org/10.1038/nature20812

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