Abstract
The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer’s disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.
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Cryo-EM maps and structural coordinates generated during this study have been deposited in the Electron Microscopy Data Bank and Protein Data Bank with accession codes: EMD-21143 and PDB 6VAM (chCALHM1), EMD-21141 and PDB 6VAK (hCALHM2), EMD-21140 and PDB 6VAI (hCALHM2 gap junction) and EMD-21142 and PDB 6VAL (chCALHM1–hCALHM2 chimera). Source data for Figs. 1b and 3e are available with the paper online.
Change history
20 February 2020
The article was incorrectly linked to a Nature article (10.1038/s41586-019-1781-3). The link has now been removed.
17 February 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41594-020-0396-6
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Acknowledgements
We thank D. Thomas and M. Wang for managing the cryo-EM facility and the computing facility at Cold Spring Harbor Laboratory, respectively. A. Hoffmann and T. Malinauskas were involved in the early phase of the research related to hCALHM2. This work was supported by NIH (NS113632 and MH085926), Robertson funds at Cold Spring Harbor Laboratory, the Doug Fox Alzheimer’s fund, Austin’s Purpose, and Heartfelt Wing Alzheimer’s fund (to H.F.), the Howard Hughes Medical Institute (to N.G.) and the Biotechnology and Biological Sciences Research Council (to S.J.T.). J.L.S. is supported by the Charles H. Revson Senior Fellowship in Biomedical Science.
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J.L.S., T.-H.C., T.G., N.S., N.G. and H.F. designed and conducted experiments involving cryo-EM. K.M. conducted electrophysiology experiments. S.R. and S.J.T. conducted computational simulations. All authors wrote the manuscript.
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Extended data
Extended Data Fig. 1 Reconstitution of chCALHM1 and hCALHM2 into lipid nanodiscs.
a, Representative Superose-6 SEC chromatograph of chCALHM1 in MSP2N2 nanodiscs with soy polar extract. b, SDS-PAGE of the fractions collected from SEC. The band for chCALHM1 has a tendency to spread out in SDS-PAGE. Fractions that eluted between 13.5-15.5 ml were pooled, concentrated and subjected to cryo-EM. c, Representative Superose 6 SEC chromatograph of hCALHM2 in MSP2N2 nanodiscs with soy polar extract. d, SDS-PAGE of the fractions collected from SEC. Fractions that eluted between 14.5-16.5 ml were pooled, concentrated and subjected to cryo-EM.
Extended Data Fig. 2 Single particle analysis of chCALHM1.
a, A representative micrograph (scale bar = 38.8 nm), representative 2D class averages, and the 3D classification workflow are shown. b, The FSC plots of the two half maps (top) and the map vs model (bottom) are shown. c, The angular distribution plot for class 3. d, Local resolutions of class 3 were calculated using ResMap.
Extended Data Fig. 3 Representative cryo-EM density of chCALHM1.
a, Cryo-EM density of the overall octameric assembly (left) and the cross-sectional view of the central cavity (right). b–c, Representative density for a monomer (b), and individual TMDs (c), and a CTH and a TMD4-CTH linker.
Extended Data Fig. 4 Presence of extra cryo-EM density in the chCALHM1 pore.
a, Extra cryo-EM density is observed in the middle of the pore-like structure of chCALHM1. Here, the pore-density and the density for only subunit H are shown for clarity. TMD1 and the pore-density are continuous (arrow). b, The density observed from the top of the extracellular region. The diameter of the pore is 19.5 Å.
Extended Data Fig. 5 Single particle analysis of hCALHM2.
a, A representative micrograph (scale bar = 38.8 nm), representative 2D class averages, and the 3D classification workflow are shown. b, The FSC plots of the two half maps (top) and the map vs. model (bottom) are shown for class 8. c, The angular distribution plot for class 8. d, Local resolutions of class 8 were calculated using ResMap.
Extended Data Fig. 6 Representative cryo-EM density of hCALHM2.
a, Cryo-EM density of the overall 11-mer assembly (left) and the cross-sectional view of the central cavity (right). b–c, Representative density for a monomer (b), and individual TMDs (c) and a CTH and a TMD4-CTH linker.
Extended Data Fig. 7 Interaction of hCALHM2 subunits.
a–b, The hCALHM2 structure viewed from the top of extracellular region (a) and the side of the membrane (b). Shown in spheres are the Arg124 residues at the equivalent position to chCALHM1 Asp120 or hCALHM1 Asp121. c, Arg124 (sphere) and surrounding residues (sticks) form polar and hydrophobic interactions to mediate inter-subunit interactions. d–e, The inter-subunit interactions between TMD2 and TMD4 (d) and CTHs (e). f, The schematic presentation of the interactions between two CTHs (magenta and slate blue) in hCALHM2 (top) and chCALHM1 (bottom). Polar and van der Waals interactions mediated by hydrophobic residues (ovals) are shown as dashed and solid lines, respectively. The lines in magenta are the conserved interactions between chCALHM1 and hCALHM2. The residues in italic are the equivalent ones in hCALHM1.
Extended Data Fig. 8 Single particle analysis of CALHM1-2.
a, A representative micrograph (scale bar = 40.5 nm), representative 2D class averages, and the 3D classification workflow are shown. b, The FSC plots of the two half maps (top) and the map vs. model (bottom) are shown for class 8. c, The angular distribution plot for class 8. d, Local resolutions of class 8 were calculated using ResMap.
Extended Data Fig. 9 Single particle analysis of 22-meric hCALHM2.
a, A representative micrograph (scale bar = 38.8 nm), representative 2D class averages, and the 3D classification workflow are shown. b, The FSC plots of the two half maps (top) and the map vs. model (bottom) are shown for class 8. c, The angular distribution plot for class 8. d, Local resolutions of class 8 were calculated using ResMap.
Extended Data Fig. 10 Structure of hCALHM2 gap junction.
a, Cryo-EM density of the 22-meric hCALHM2 viewed from the side of the membrane and from the cytoplasm. b, The structural models in the same orientation as the cryo-EM density in (a), showing locations of the TMD2-4 and the CTH. There is little or no structural change between the 22-mer and 11-mer structures except for the extracellular region (due to the inter-11-mer interaction). The interaction between the two hemichannels is mediated by His147, His152, and Glu145 in the extracellular loop between TMD3 and TMD4. Density between His147 and His152 is continuous implying the potential presence of a divalent cation. Residues from the upper and lower hemichannels are annotated with black and gray fonts. Ovals are placed at the interaction sites.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2.
Source data
Source Data Fig. 1
Statistical source data for Fig. 1b.
Source Data Fig. 3
Uncropped western blots for Fig. 3e.
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Syrjanen, J.L., Michalski, K., Chou, TH. et al. Structure and assembly of calcium homeostasis modulator proteins. Nat Struct Mol Biol 27, 150–159 (2020). https://doi.org/10.1038/s41594-019-0369-9
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DOI: https://doi.org/10.1038/s41594-019-0369-9
