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Structure and assembly of calcium homeostasis modulator proteins

A Publisher Correction to this article was published on 17 February 2020

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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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Fig. 1: Structure and function of chCALHM1.
Fig. 2: Intersubunit interface of chCALHM1.
Fig. 3: Structure and function of hCALHM2.
Fig. 4: Structure of the CALHM1–CALHM2 chimera.
Fig. 5: Comparison of pore properties between chCALHM1 and hCALHM2.

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Data availability

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

References

  1. Jun, M. et al. Calhm2 governs astrocytic ATP releasing in the development of depression-like behaviors. Mol. Psychiatry 23, 883–891 (2018).

    Google Scholar 

  2. Vingtdeux, V. et al. CALHM1 deficiency impairs cerebral neuron activity and memory flexibility in mice. Sci. Rep. 6, 24250 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Dreses-Werringloer, U. et al. A polymorphism in CALHM1 influences Ca2+ homeostasis, Aβ levels, and Alzheimer’s disease risk. Cell 133, 1149–1161 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Taruno, A. et al. CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes. Nature 495, 223–226 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Ma, Z. et al. Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability. Proc. Natl Acad. Sci. USA 109, E1963–E1971 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Vingtdeux, V. et al. Effect of the CALHM1 G330D and R154H human variants on the control of cytosolic Ca2+ and Aβ levels. PLoS One 9, e112484 (2014).

    PubMed  PubMed Central  Google Scholar 

  7. Workman, A. D. et al. CALHM1-mediated ATP release and ciliary beat frequency modulation in nasal epithelial cells. Sci. Rep. 7, 6687 (2017).

    PubMed  PubMed Central  Google Scholar 

  8. Ma, Z. et al. CALHM3 is essential for rapid ion channel-mediated purinergic neurotransmission of GPCR-mediated tastes. Neuron 98, 547–561.e10 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Tanis, J. E. et al. CLHM-1 is a functionally conserved and conditionally toxic Ca2+-permeable ion channel in Caenorhabditis elegans. J. Neurosci. 33, 12275–12286 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

    CAS  PubMed  Google Scholar 

  11. Regan, M. C. et al. Structural mechanism of functional modulation by gene splicing in NMDA receptors. Neuron 98, 521–529.e3 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Harauz, G. & van Heel, M. Exact filters for general geometry three dimensional reconstruction. Optik 73, 146–156 (1986).

    Google Scholar 

  13. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    CAS  PubMed  Google Scholar 

  14. Slabinski, L. et al. XtalPred: a web server for prediction of protein crystallizability. Bioinformatics 23, 3403–3405 (2007).

    CAS  PubMed  Google Scholar 

  15. Siebert, A. P. et al. Structural and functional similarities of calcium homeostasis modulator 1 (CALHM1) ion channel with connexins, pannexins, and innexins. J. Biol. Chem. 288, 6140–6153 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Tanis, J. E., Ma, Z. M. & Foskett, J. K. The NH2 terminus regulates voltage-dependent gating of CALHM ion channels. Am. J. Physiol. Cell Physiol. 313, C173–C186 (2017).

    PubMed  PubMed Central  Google Scholar 

  17. Oshima, A. et al. Atomic structure of the innexin-6 gap junction channel determined by cryo-EM. Nat. Commun. 7, 13681 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Karakas, E. & Furukawa, H. Crystal structure of a heterotetrameric NMDA receptor ion channel. Science 344, 992–997 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Lee, C. H. et al. NMDA receptor structures reveal subunit arrangement and pore architecture. Nature 511, 191–197 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Maeda, S. et al. Structure of the connexin 26 gap junction channel at 3.5 Å resolution. Nature 458, 597–602 (2009).

    CAS  PubMed  Google Scholar 

  21. Deneka, D., Sawicka, M., Lam, A. K. M., Paulino, C. & Dutzler, R. Structure of a volume-regulated anion channel of the LRRC8 family. Nature 558, 254–259 (2018).

    CAS  PubMed  Google Scholar 

  22. Kasuya, G. et al. Cryo-EM structures of the human volume-regulated anion channel LRRC8. Nat. Struct. Mol. Biol. 25, 797–804 (2018).

    CAS  PubMed  Google Scholar 

  23. Vingtdeux, V. et al. CALHM1 ion channel elicits amyloid-beta clearance by insulin-degrading enzyme in cell lines and in vivo in the mouse brain. J. Cell Sci. 128, 2330–2338 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. de Jong, D. H. et al. Improved parameters for the Martini coarse-grained protein force field. J. Chem. Theory Comput. 9, 687–697 (2013).

    PubMed  Google Scholar 

  25. Stansfeld, P. J. et al. MemProtMD: automated insertion of membrane protein structures into explicit lipid membranes. Structure 23, 1350–1361 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Bhattacharyya, M. et al. Molecular mechanism of activation-triggered subunit exchange in Ca2+/calmodulin-dependent protein kinase II. Elife 5, e13405 (2016).

    PubMed  PubMed Central  Google Scholar 

  27. Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M. & Henderson, R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259, 393–421 (1996).

    CAS  PubMed  Google Scholar 

  28. Eisenberg, D., Schwarz, E., Komaromy, M. & Wall, R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J. Mol. Biol. 179, 125–142 (1984).

    CAS  PubMed  Google Scholar 

  29. Klesse, G., Rao, S., Sansom, M. S. P. & Tucker, S. J. CHAP: a versatile tool for the structural and functional annotation of ion channel pores. J. Mol. Biol. 431, 3353–3365 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Ritchie, T. K. et al. Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Schorb, M., Haberbosch, I., Hagen, W. J. H., Schwab, Y. & Mastronarde, D. N. Software tools for automated transmission electron microscopy. Nat. Methods 16, 471–477 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Tegunov, D. & Cramer, P. Real-time cryo-electron microscopy data preprocessing with Warp. Nat. Methods 16, 1146–1152 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Grant, T., Rohou, A. & Grigorieff, N. cisTEM, user-friendly software for single-particle image processing. Elife 7, e35383 (2018).

    PubMed  PubMed Central  Google Scholar 

  34. Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. Elife 4, e06980 (2015).

    PubMed  PubMed Central  Google Scholar 

  35. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    PubMed  PubMed Central  Google Scholar 

  36. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    CAS  PubMed  Google Scholar 

  39. Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

    Google Scholar 

  40. Jorgensen, W. L., Maxwell, D. S. & Tirado-Rives, J. Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J. Am. Chem. Soc. 118, 11225–11236 (1996).

    CAS  Google Scholar 

  41. Abascal, J. L. F. & Vega, C. A general purpose model for the condensed phases of water: TIP4P/2005. J. Chem. Phys. 123, 234505 (2005).

    CAS  PubMed  Google Scholar 

  42. Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101 (2007).

    PubMed  Google Scholar 

  43. Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: a new molecular dynamics method. J. Appl. Phys. 52, 7182–7190 (1981).

    CAS  Google Scholar 

  44. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: a linear constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472 (1997).

    CAS  Google Scholar 

  45. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

    CAS  Google Scholar 

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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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Contributions

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.

Corresponding author

Correspondence to Hiro Furukawa.

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The authors declare no competing interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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). bc, 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). bc, 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.

ab, 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. de, 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.

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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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