Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM

Abstract

The calcium-activated chloride channel TMEM16A is a ligand-gated anion channel that opens in response to an increase in intracellular Ca2+ concentration1,2,3. The protein is broadly expressed4 and contributes to diverse physiological processes, including transepithelial chloride transport and the control of electrical signalling in smooth muscles and certain neurons5,6,7. As a member of the TMEM16 (or anoctamin) family of membrane proteins, TMEM16A is closely related to paralogues that function as scramblases, which facilitate the bidirectional movement of lipids across membranes8,9,10,11. The unusual functional diversity of the TMEM16 family and the relationship between two seemingly incompatible transport mechanisms has been the focus of recent investigations. Previous breakthroughs were obtained from the X-ray structure of the lipid scramblase of the fungus Nectria haematococca (nhTMEM16)12,13, and from the cryo-electron microscopy structure of mouse TMEM16A at 6.6 Å (ref. 14). Although the latter structure disclosed the architectural differences that distinguish ion channels from lipid scramblases, its low resolution did not permit a detailed molecular description of the protein or provide any insight into its activation by Ca2+. Here we describe the structures of mouse TMEM16A at high resolution in the presence and absence of Ca2+. These structures reveal the differences between ligand-bound and ligand-free states of a calcium-activated chloride channel, and when combined with functional experiments suggest a mechanism for gating. During activation, the binding of Ca2+ to a site located within the transmembrane domain, in the vicinity of the pore, alters the electrostatic properties of the ion conduction path and triggers a conformational rearrangement of an α-helix that comes into physical contact with the bound ligand, and thereby directly couples ligand binding and pore opening. Our study describes a process that is unique among channel proteins, but one that is presumably general for both functional branches of the TMEM16 family.

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Figure 1: TMEM16A structures.
Figure 2: Pore and Ca2+-binding site in the Ca2+-bound structure.
Figure 3: Ca2+-free structure and conformational changes upon Ca2+ release.
Figure 4: Functional characterization of conformational changes.
Figure 5: Activation mechanism.

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Acknowledgements

We thank O. Medalia and M. Eibauer, the Center for Microscopy and Image Analysis (ZMB) of the University of Zurich, and the Mäxi foundation for support and access to electron microscopes. J. D. Walter is acknowledged for comments on the manuscript and all members of the Dutzler laboratory for help at various stages of the project. This research was supported by a grant from the European Research Council (number 339116, AnoBest). C.P. was supported by a postdoctoral fellowship (Forschungskredit) of the University of Zurich.

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Contributions

V.K. and Y.N. expressed and purified the protein for cryo-EM and functional reconstitution and performed the flux assay. C.P. prepared the sample for cryo-EM, collected electron microscopy data and proceeded with structure determination. A.K.M.L. generated mutants, performed electrophysiological recordings and fitted data. C.P., V.K., A.K.M.L., Y.N. and R.D. jointly planned experiments, analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Raimund Dutzler.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks C. Hartzell and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Functional characterization and cryo-EM reconstruction of mTMEM16A in a Ca2+-bound form.

a, Left, Ca2+ concentration–response relationships measured from inside-out patches excised from a stable cell line expressing mTMEM16A. Data were recorded at −80 and 80 mV and show averages of normalized currents of five biological replicates, errors are s.e.m. Solid lines, fit to a Hill equation. Right, a representative current trace. Numbers above the current traces indicate the Ca2+ concentration (μM); blue and violet dots, reference pulses used for rundown correction at 80 and −80 mV, respectively. b, Fluorescence-based Cl flux assay into proteoliposomes containing mTMEM16A in the presence (green) and absence (red) of Ca2+. Transport into protein-free liposomes is shown for comparison (blue). H+ transport into proteoliposomes accompanying concentration-gradient driven influx of Cl is monitored by the decrease in the fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA). Time zero corresponds to the addition of the H+ ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Experiments show average of four technical replicates, errors are s.e.m. c, d, Representative cryo-EM image (c) and 2D-class averages (d) of vitrified mTMEM16A in a Ca2+-bound state. e, Angular distribution plot of particles included in the final C2-symmetric 3D reconstruction. The number of particles with the respective orientations is represented by length and colour of the cylinders. f, 3D classification of the final map performed to identify structural variances. Predominant classes are shown as maps with differences highlighted in red. The percentage of total particles in each class is indicated and the final resolution after refinement and masking is shown where applicable. 3D classification revealed three predominant classes. Structural variations between these marginally different populations concern α3, which is generally less well defined than the other transmembrane segments, and the connecting α2–α3 and α3–α4 loops. g, Final reconstruction map coloured by local resolution as calculated by BlocRes from the Bsoft software package37. h, FSC plot of the final refined unmasked (red) and masked (green) mTMEM16A map in the presence of calcium ions. The resolution at which the curve drops below the 0.143 threshold is indicated. A thumbnail of the mask used for FSC calculation overlaid on the atomic model is shown in the upper right corner.

Extended Data Figure 2 Cryo-EM reconstruction of mTMEM16A in a Ca2+-free form.

a, b, Representative cryo-EM image (a) and 2D-class averages (b) of vitrified mTMEM16A in a Ca2+-free state. c, Angular distribution plot of particles included in the final C2-symmetric 3D reconstruction. The number of particles with the respective orientations is represented by length and colour of the cylinders. d, 3D classification of the final map performed to identify structural variances. Predominant classes are shown as maps with differences highlighted in red. The percentage of total particles in each class is indicated and the final resolution after refinement and masking is shown where applicable. As for the Ca2+-bound structure, 3D classification revealed three predominant classes with pronounced variation in the less well-defined α3 and the connecting α2–α3 and α3–α4 loops. e, Superposition of cryo-EM density at the extracellular part of α3 of two major classes. f, Final reconstruction map coloured by local resolution as calculated by BlocRes from the Bsoft software package37. g, FSC plot of the final refined unmasked (red) and masked (violet) mTMEM16A in the absence of calcium ions. The resolution at which the curve drops below the 0.143 threshold is indicated. A thumbnail of the mask used for FSC calculation overlaid on the atomic model is shown in the upper right corner. h, Projections of Ca2+-free and Ca2+-bound conformations of mTMEM16A orthogonal to the membrane. The movement of α6 has perturbed the detergent micelle in the Ca2+-free structure, which has become partially discontinuous near the intracellular vestibule.

Extended Data Figure 3 Cryo-EM density.

a, Sections of the cryo-EM density of the Ca2+-bound (grey, 5.5σ) and Ca2+-free (blue, 4.5σ) densities superimposed on part of the respective refined structures. Structures are shown as sticks in unique colours, structural elements and datasets (Ca2+, no Ca2+) are labelled. b, Electron density (grey, 4.5σ) of the Ca2+-bound protein around disulfide bonds in the extracellular domain superimposed on the refined model.

Extended Data Figure 4 Cryo-EM density of selected regions.

a, Stereo view of cryo-EM density (3.5σ) around the α2–α3 and the α4–α5 loop of the Ca2+-bound structure shown superimposed on the refined model. b, Stereo view of the occupied Ca2+-binding site with cryo-EM density (5.5σ) shown superimposed on the refined model of the Ca2+-bound state. c, Stereo view of the vacant Ca2+ binding region in the Ca2+-free structure of mTMEM16A with cryo-EM density (4.5σ) shown superimposed on the refined model. Selected α-helices are labelled.

Extended Data Figure 5 mTMEM16A sequence and structural features.

a, Sequence of the mTMEM16A(ac) isoform (UniProt Q8BHY3.2) used in this study. Secondary structure elements: green, transmembrane domain; violet, N-terminal domain; grey, C-terminal region; blue, α1–α2 loop; black, α2–α3 loop; beige, α5–α6 loop; red, α9–α10 loop. Yellow inverted triangles, position of cysteines involved in disulfide bonds; green circles, glycines in α6 that serve as hinges in conformational transitions; red triangles, residues of the Ca2+-binding site. The sequence inserted in c isoforms is highlighted in yellow and the preceding stretch of glutamates in red. b, Ribbon representation of the mTMEM16A subunit. Colouring as in a, and disulfide bridges are indicated in yellow. c, Sequence alignment of a section of α6 in different family members; human orthologues, mTMEM16 and fungal family members are shown. Selected residues are highlighted. Asterisk, insertion in TMEM16A and TMEM16B. d, Dimer interface. A Cα-representation of the symmetry-related extracellular part of α10 is shown. Interacting residues are labelled and their side-chains displayed as sticks. The interface is predominantly hydrophobic but contains a pair of H-bonds between Q872 and N873 at the inner end of the contact region located at the centre of the membrane, equivalent to interactions between conserved glutamate and histidine residues formed in TMEM16 scramblases13. e, Ribbon representation of the extracellular domain. Green, extracellular parts of transmembrane helices; blue, α1–α2 loop; beige, α5–α6 loop; red, α9–α10 loop. The extracellular domain is stabilized by at least three disulfide bridges (yellow), two within the α1–α2 loop and another connecting the α1–α2 with the α9–α10 loop, which probably explains why mutation of any of these six cysteine residues causes a loss of function of the channel18. A fourth disulfide bridge is potentially formed between two adjacent cysteines in the α5–α6 loop. In d, e, the view is rotated by 90° around the dimer axis compared to Fig. 1. f, N-terminal domain. Top, ribbon representation of the N-terminal domain of mTMEM16A; bottom, a Cα-representation of a superposition of the equivalent domains of mTMEM16A (green) and nhTMEM16 (red). g, Ribbon representation of the interacting α2–α3 (black) and α4–α5 (green) loops. Yellow, sequence corresponding to the c segment (Glu-Ala-Val-Lys) of mTMEM16 splice variants; red, preceding stretch of glutamates (Glu-Glu-Glu-Glu). The short α4–α5 loop has been previously proposed to carry residues that distinguish TMEM16 channels from scramblases52; the proximity of the α2–α3 loop to the α4–α5 loop suggests a potential coupling to the transmembrane domain. Although the α2–α3 loop is not directly involved in high-affinity Ca2+ binding leading to channel activation, a stretch of glutamates adjacent to the c region might still interact with Ca2+ and thus be responsible for the observed increase in the open probability at high ligand concentration23. Selected secondary structure elements are labelled in eg.

Extended Data Figure 6 Superposition of TMEM16 structures.

a, Stereo view of a superposition of the Ca2+-bound (green) and Ca2+-free (violet) structures of the mTMEM16A ion channel. The proteins are displayed as Cα representations. b, Root mean square deviations (RMSDs) of Cα atoms in the superposition shown in a. The resemblance between the ligand-bound and ligand-free structures is reflected in the low RMSD of 0.5 Å for 692 Cα positions that encompass the entire subunit, except for the inner half of α6. In contrast to the bulk of the protein, the RMSD of 6.5 Å for 17 Cα atoms of the inner part of α6 indicates a large conformational change upon binding of Ca2+. Apart from the specified movements, no obvious distortions in the structure of the Ca2+-bound conformation are observed compared to the protein that was never exposed to Ca2+ during purification. This is despite the fact that in patch-clamp experiments, mTMEM16A becomes inactive on prolonged exposure to the ligand28. c, Stereo view of a superposition of the Ca2+-bound structures of the mTMEM16A ion channel (green) and the nhTMEM16 lipid scramblase (brown). Proteins are shown as ribbons. d, RMSDs of Cα atoms from a superposition of conserved secondary structure elements of mTMEM16A on nhTMEM16 shown in c. The general resemblance of the mTMEM16A structure to the structure of the lipid scramblase nhTMEM16 is illustrated by the RMSD of 4.0 Å between 447 Cα positions. a, c, Blue spheres, bound Ca2+ ions. b, d, Individual positions are displayed (b, green x; d, orange x). Transmembrane helices are indicated below for mTMEM16A (b) and nhTMEM16 (d).

Extended Data Figure 7 Pore geometry.

a, Ion conduction pore of mTMEM16A in the Ca2+-bound conformation. Grey mesh, accessible surface of the pore (generated with a solvent probe of diameter 2.2 Å); blue spheres, bound Ca2+. b, Pore diameter along the pore axis of mTMEM16A in the Ca2+-bound conformation, as calculated with the program HOLE53. c, View of interactions between the extracellular parts of α4 and α6 in the Ca2+-bound structure. d, Ion conduction pore of mTMEM16A in the Ca2+-free conformation. The accessible surface was generated as in a. e, Pore diameter along the pore axis of mTMEM16A in the Ca2+-free conformation (violet) compared to the diameter of the Ca2+-bound pore (green dashed line). f, Difference in the pore diameter between Ca2+-free and Ca2+-bound conformations. Negative values reflect the tighter geometry of the pore in the Ca2+-free conformation. g, Stereo view of a superposition of the pore regions in the Ca2+-bound (green) and Ca2+-free (violet) conformations of mTMEM16A. a, c, e, g, α-Helices constituting the ion conduction pore are shown in Cα-representation, side-chains of pore-lining residues as sticks. In a, b and df, circles, triangles and inverted triangles indicate equivalent locations in the pore.

Extended Data Figure 8 Pore electrostatic properties and Ca2+-binding site mutants.

a, Left, structure of mTMEM16A in the Ca2+-bound state used for calculation of the electrostatic potential. Orange spheres, positions along which the pore potential was plotted. Right, close-up of the Ca2+-binding site. Blue spheres, bound Ca2+ ions. b, Electrostatic potential along the pore of mTMEM16A in the Ca2+-bound conformation, as determined by a numerical solution of the Poisson–Boltzmann equation. c, Left, structure of the Ca2+-free state used for calculation of the electrostatic potential in a similar representation to that in a. Right, close-up of the vacant ligand binding-site. d, Electrostatic potential along the pore of mTMEM16A in the Ca2+-free conformation determined as in b (violet), compared to the potential of the Ca2+-bound state (green dashed line). a, c, The protein is shown as ribbon, the view is from within the membrane on one of the two pores in the dimeric protein. The membrane boundary is indicated by black lines, with the hydrophobic core in the centre and the two headgroup regions above and below. Violet spheres in the close-ups correspond to the position at which the electrostatic potential in the Ca2+-free conformation is at its minimum. Asterisk, triangle and inverted triangle indicate equivalent locations in the pore. eg, Ca2+ concentration–response relationships (left) and representative current traces (right) of mTMEM16A, wild type (e), mTMEM16A(N651A) (f) and mTMEM16A(N730A) (g). In eg, data in the left panels were measured from inside-out patches at −80 and 80 mV and show averages of normalized currents of five (wild type, mTMEM16A(N651A)) and seven (mTMEM16A(N730A)) biological replicates, errors are s.e.m. Solid lines, fit to a Hill equation. Dashed lines in f and g indicate the relations of wild type at the same voltages. Numbers above the current traces indicate the Ca2+ concentration (μM); blue and violet dots, reference pulses used for rundown correction at 80 and −80 mV, respectively.

Extended Data Figure 9 Electrophysiology.

a, b, Ca2+ concentration–response relationships (left) and representative current traces (right) of mTMEM16A(G644A) (a) and mTMEM16A(G644P) (b). c, Rectification of the basal current of mTMEM16A(G644P). Left, representative current recorded at 80 (I80) and −80 mV (I−80) at 0 (blue) and 2 μM Ca2+ (orange). Right, rectification index (defined as I80/−I−80) at 0 and 2 μM Ca2+. Values and averages of seven biological replicates are displayed, errors are s.e.m. dg, Ca2+ concentration–response relationships (left) and representative current traces (right) of mTMEM16A(G656A) (d), mTMEM16A(G656P) (e), mTMEM16A(P658A) (f) and mTMEM16A(P658G) (g). h, Left, time dependence of modification of a mutant of mTMEM16A that only contained 6 essential cysteines18 (mTMEM16A(6C)) with 5 mM MTSEA in the presence of 1 mM Ca2+, monitored by the change of the rectification index (−I−100/I120). Right, time dependence of the modification of mTMEM16A(6C-K588C) by 2.5 mM MTSEA in the presence of 1 mM Ca2+. Experiments with 5 mM of the positively charged MTSEA (Fig. 4f) are shown as dashed line for comparison. Data show the average of five (mTMEM16A(6C) and Ca2+) or seven (mTMEM16A(6C-K588C) and Ca2+, 2.5 mM MTSEA) biological replicates, errors are s.e.m. i, Normalized I–V relationships obtained using a ramp protocol before (beige) and after (blue) application of MTSEA to different constructs in the presence of 1 mM Ca2+ (unless stated otherwise). From left to right; mTMEM16A(6C); mTMEM16A(6C-K588C); mTMEM16A(6C-K588C), no Ca2+; and mTMEM16A(6C-K588Q/S592C). j, Normalized I–V relationships obtained using a ramp protocol before (beige) and after (blue) application of the negatively charged MTSES in the presence of 1 mM Ca2+ for wild type (left) and mTMEM16A(S592C) (right). In i and j, averages of four (mTMEM16A(6C-S592C) and Ca2+, wild type and Ca2+), five (mTMEM16A(6C) and Ca2+, mTMEM16A(S592C) and Ca2+), seven (mTMEM16A(6C-K588C) and Ca2+) and eight (mTMEM16A(6C-K588C) and no Ca2+) biological replicates are shown. k, Time-dependent changes of the rectification index (RI) after incubation with 10 mM MTSES. Data show the average of six (wild type and Ca2+) or five (mTMEM16A(S592C) and Ca2+) biological replicates, errors are s.e.m. l, Time-dependent changes of the current amplitude at 80 mV for wild type (left) and mTMEM16A(S592C) (right) after incubation with 10 mM MTSES. Dashed line, decay of the current owing to rundown. Data show the average of nine (wild type and Ca2+) or seven (mTMEM16A(S592C) and Ca2+) biological replicates, errors are s.e.m. m, Ca2+ concentration–response relationships (left) and representative current traces (right) of mTMEM16A(I550A). In a, b, dg and m, data in the left panels were measured from inside-out patches at −80 and 80 mV and show averages of normalized currents of six (mTMEM16A(G644A), mTMEM16A(P658A)), seven (mTMEM16A(G644P), mTMEM16A(G656P), mTMEM16A(I550A)) or eight (mTMEM16A(G656A), mTMEM16A(P658G)) biological replicates, errors are s.e.m. Solid lines, fit to a Hill equation. Data recorded at 80 and −80 mV are shown in blue and violet, respectively. Dashed lines, relations of wild type at the same voltages (Extended Data Fig. 8e). Numbers above the current traces indicate the Ca2+ concentration (μM); blue and violet dots, reference pulses used for rundown correction at 80 and −80 mV, respectively. Source data

Extended Data Figure 10 Statistics and validation.

a, Statistics of cryo-EM data collection, 3D reconstruction and model refinement. b, c, FSC curves of refined models versus maps of mTMEM16A in the presence (b) and absence (c) of calcium ions for cross-validation. The green (b) and violet (c) curves show the FSC curves for the refined model compared to the full masked dataset (FSCsum). Blue, FSC curve for the refined model compared to the masked half-map 1 (FSCwork, used during validation refinement); grey, refined model compared to the masked half-map 2 (FSCfree, not used during validation refinement). Dashed lines, FSC threshold used for FSCsum of 0.5 and for FSCfree/work of 0.143.

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Ligand-bound mTMEM16A structure.

cryo-EM density map of the mTMEM16A ion channel obtained in presence of calcium ions with the modelled structure superimposed. Only one subunit is shown and the two bound Ca2+ are coloured in blue. (AVI 29113 kb)

Ligand-free mTMEM16A structure.

cryo-EM density map of the mTMEM16A ion channel obtained in absence of calcium ions with the modelled structure superimposed. Only one subunit is shown. (AVI 24123 kb)

Comparison of the Ca2+-bound and Ca2+-free state.

Superimposition of the mTMEM16A ion channel structure in the Ca2+-bound (green) and the Ca2+-free (violet) state. (MP4 25410 kb)

Ca2+-induced conformational changes.

Morph between the Ca2+-bound and the Ca2+-free state (violet) superimposed on the structure in presence of calcium (green, Ca2+ shown as blue spheres). The structure is viewed from within the membrane rotated by approximately 70 degrees about the y axis compared to Figure 1. (AVI 19351 kb)

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Paulino, C., Kalienkova, V., Lam, A. et al. Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM. Nature 552, 421–425 (2017). https://doi.org/10.1038/nature24652

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