Structural basis for Ca2+ selectivity of a voltage-gated calcium channel


Voltage-gated calcium (CaV) channels catalyse rapid, highly selective influx of Ca2+ into cells despite a 70-fold higher extracellular concentration of Na+. How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial NaV channel NaVAb. Our results reveal interactions of hydrated Ca2+ with two high-affinity Ca2+-binding sites followed by a third lower-affinity site that would coordinate Ca2+ as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining Ca2+ selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations Cd2+ and Mn2+, with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca2+ ions bound in the selectivity filter and supports a ‘knock-off’ mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our CaVAb model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate CaV channels.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Structure and function of the CaVAb channel.
Figure 2: Ca2+-binding sites in and near the selectivity filter of NaVAb, CaVAb and their derivatives.
Figure 3: Ion binding and block of CaVAb and its derivatives.
Figure 4: Catalytic cycle for Ca2+ conductance by CaVAb.

Accession codes


Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes: 4MS2 (TLDDWSN, 15 mM Ca2+), 4MTF (TLDDWSN, 0.5 mM Ca2+), 4MTG (TLDDWSN, 2.5 mM Ca2+), 4MTO (TLDDWSN, 5 mM Ca2+), 4MVM (TLDDWSN, 10 mM Ca2+), 4MVO (TLDDWSN, 15 mM Ca2+), 4MVQ (TLDDWSD, 15 mM Ca2+), 4MVR (TLDDWSD, 100 mM Mn2+), 4MVS (TLDDWSD, 100 mM Cd2+), 4MVZ (TLEDWSD, 15 mM Ca2+), 4MW3 (TLDDWSM, 15 mM Ca2+), 4MVU (TLEDWSM, 15 mM Ca2+), 4MW8 (NavAb, 15 mM Ca2+).


  1. 1

    Sather, W. A. & McCleskey, E. W. Permeation and selectivity in calcium channels. Annu. Rev. Physiol. 65, 133–159 (2003)

    CAS  Article  Google Scholar 

  2. 2

    Catterall, W. A. Voltage-gated calcium channels. Cold Spring Harb. Perspect. Biol. 3, a003947 (2011)

    Article  Google Scholar 

  3. 3

    Almers, W. & McCleskey, E. W. Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore. J. Physiol. (Lond.) 353, 585–608 (1984)

    CAS  Article  Google Scholar 

  4. 4

    Almers, W., McCleskey, E. W. & Palade, P. T. A non-selective cation conductance in frog muscle membrane blocked by micromolar external calcium ions. J. Physiol. (Lond.) 353, 565–583 (1984)

    CAS  Article  Google Scholar 

  5. 5

    Hess, P. & Tsien, R. W. Mechanism of ion permeation through calcium channels. Nature 309, 453–456 (1984)

    CAS  ADS  Article  Google Scholar 

  6. 6

    Armstrong, C. M. & Neyton, J. Ion permeation through calcium channels. A one-site model. Ann. NY Acad. Sci. 635, 18–25 (1991)

    CAS  ADS  Article  Google Scholar 

  7. 7

    Dang, T. X. & McCleskey, E. W. Ion channel selectivity through stepwise changes in binding affinity. J. Gen. Physiol. 111, 185–193 (1998)

    CAS  Article  Google Scholar 

  8. 8

    Lopin, K. V., Obejero-Paz, C. A. & Jones, S. W. Evaluation of a two-site, three-barrier model for permeation in CaV3.1 (α1G) T-type calcium channels: Ca2+, Ba2+, Mg2+, and Na+ . J. Membr. Biol. 235, 131–143 (2010)

    CAS  Article  Google Scholar 

  9. 9

    Heinemann, S. H., Terlau, H., Stuhmer, W., Imoto, K. & Numa, S. Calcium channel characteristics conferred on the sodium channel by single mutations. Nature 356, 441–443 (1992)

    CAS  ADS  Article  Google Scholar 

  10. 10

    Ellinor, P. T., Yang, J., Sather, W. A., Zhang, J. F. & Tsien, R. W. Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions. Neuron 15, 1121–1132 (1995)

    CAS  Article  Google Scholar 

  11. 11

    Yang, J., Ellinor, P. T., Sather, W. A., Zhang, J. F. & Tsien, R. W. Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. Nature 366, 158–161 (1993)

    CAS  ADS  Article  Google Scholar 

  12. 12

    Kim, M. S., Morii, T., Sun, L. X., Imoto, K. & Mori, Y. Structural determinants of ion selectivity in brain calcium channel. FEBS Lett. 318, 145–148 (1993)

    CAS  Article  Google Scholar 

  13. 13

    Cibulsky, S. M. & Sather, W. A. The EEEE locus is the sole high-affinity Ca2+ binding structure in the pore of a voltage-gated Ca2+ channel: block by Ca2+ entering from the intracellular pore entrance. J. Gen. Physiol. 116, 349–362 (2000)

    CAS  Article  Google Scholar 

  14. 14

    Cloues, R. K., Cibulsky, S. M. & Sather, W. A. Ion interactions in the high-affinity binding locus of a voltage-gated Ca2+ channel. J. Gen. Physiol. 116, 569–586 (2000)

    CAS  Article  Google Scholar 

  15. 15

    Payandeh, J., Scheuer, T., Zheng, N. & Catterall, W. A. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011)

    CAS  Article  Google Scholar 

  16. 16

    Payandeh, J., Gamal El-Din, T. M., Scheuer, T., Zheng, N. & Catterall, W. A. Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature 486, 135–139 (2012)

    CAS  ADS  Article  Google Scholar 

  17. 17

    Zhang, X. et al. Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. Nature 486, 130–134 (2012)

    CAS  ADS  Article  Google Scholar 

  18. 18

    Ren, D. et al. A prokaryotic voltage-gated sodium channel. Science 294, 2372–2375 (2001)

    CAS  ADS  Article  Google Scholar 

  19. 19

    Yu, F. H. & Catterall, W. A. The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis. Sci. STKE 2004, re15 (2004)

    PubMed  Google Scholar 

  20. 20

    Koishi, R. et al. A superfamily of voltage-gated sodium channels in bacteria. J. Biol. Chem. 279, 9532–9538 (2004)

    CAS  Article  Google Scholar 

  21. 21

    Yue, L., Navarro, B., Ren, D., Ramos, A. & Clapham, D. E. The cation selectivity filter of the bacterial sodium channel, NaChBac. J. Gen. Physiol. 120, 845–853 (2002)

    CAS  Article  Google Scholar 

  22. 22

    Morais-Cabral, J. H., Zhou, Y. & MacKinnon, R. Energetic optimization of ion conduction rate by the K+ selectivity filter. Nature 414, 37–42 (2001)

    CAS  ADS  Article  Google Scholar 

  23. 23

    Alam, A. & Jiang, Y. Structural analysis of ion selectivity in the NaK channel. Nature Struct. Mol. Biol. 16, 35–41 (2009)

    CAS  Article  Google Scholar 

  24. 24

    Alam, A., Shi, N. & Jiang, Y. Structural insight into Ca2+ specificity in tetrameric cation channels. Proc. Natl Acad. Sci. USA 104, 15334–15339 (2007)

    CAS  ADS  Article  Google Scholar 

  25. 25

    Derebe, M. G. et al. Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites. Proc. Natl Acad. Sci. USA 108, 598–602 (2011)

    CAS  ADS  Article  Google Scholar 

  26. 26

    McCleskey, E. W. & Almers, W. The Ca channel in skeletal muscle is a large pore. Proc. Natl Acad. Sci. USA 82, 7149–7153 (1985)

    CAS  ADS  Article  Google Scholar 

  27. 27

    Chen, X. H. & Tsien, R. W. Aspartate substitutions establish the concerted action of P-region glutamates in repeats I and III in forming the protonation site of L-type Ca2+ channels. J. Biol. Chem. 272, 30002–30008 (1997)

    CAS  Article  Google Scholar 

  28. 28

    Cibulsky, S. M. & Sather, W. A. Control of ion conduction in L-type Ca2+ channels by the concerted action of S5–6 regions. Biophys. J. 84, 1709–1719 (2003)

    CAS  ADS  Article  Google Scholar 

  29. 29

    Williamson, A. V. & Sather, W. A. Nonglutamate pore residues in ion selection and conduction in voltage-gated Ca2+ channels. Biophys. J. 77, 2575–2589 (1999)

    CAS  Article  Google Scholar 

  30. 30

    McCusker, E. C. et al. Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing. Nature Commun. 3, 1102 (2012)

    ADS  Article  Google Scholar 

  31. 31

    Shaya, D. et al. Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels. J. Mol. Biol (published online 10 October 2013)

  32. 32

    Faham, S. & Bowie, J. U. Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J. Mol. Biol. 316, 1–6 (2002)

    CAS  Article  Google Scholar 

  33. 33

    Faham, S. et al. Crystallization of bacteriorhodopsin from bicelle formulations at room temperature. Protein Sci. 14, 836–840 (2005)

    CAS  Article  Google Scholar 

  34. 34

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  35. 35

    Collaborative Computational Project, 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  36. 36

    Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  37. 37

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    CAS  Article  Google Scholar 

  38. 38

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

    CAS  Article  Google Scholar 

  39. 39

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  40. 40

    Laskowski, R. A., Moss, D. S. & Thornton, J. M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993)

    CAS  Article  Google Scholar 

  41. 41

    DeLano, W. L. PyMOL molecular viewer (V.1. 2r3pre) ( (2002)

  42. 42

    Gamal El-Din, T. M., Martinez, G. Q., Payandeh, J., Scheuer, T. & Catterall, W. A. A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb. J. Gen. Physiol. 142, 181–190 (2013)

    Article  Google Scholar 

Download references


We are grateful to the beamline staff at the Advanced Light Source (BL8.2.1 and BL8.2.2) for their assistance during data collection. Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) under award number R01NS015751 (W.A.C.), the National Heart, Lung, and Blood Institute (NHLBI) of the NIH under award number R01HL112808 (W.A.C. and N.Z.) and a National Research Service Award from training grant T32GM008268 (T.M.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work was also supported by the Howard Hughes Medical Institute (N.Z.).

Author information




L.T., T.M.G.E.-D., J.P., T.S., N.Z. and W.A.C. designed the experiments. J.P. initiated the experimental work. L.T. conducted the protein purification, crystallization and diffraction experiments. L.T., J.P. and N.Z. determined and analysed the structures of the apo and cation-bound forms of CaVAb and the intermediate CaVAb constructs. T.M.G.E.-D. and T.S. performed physiological studies of CaVAb and related constructs. G.Q.M. and T.M.H. made the constructs and performed the preliminary data collection. All authors interpreted the structures in light of the physiological data. L.T., N.Z. and W.A.C. wrote the manuscript with input from all co-authors. W.A.C. and N.Z. are co-senior authors.

Corresponding authors

Correspondence to Ning Zheng or William A. Catterall.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 and Supplementary Table 1. (PDF 13277 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tang, L., Gamal El-Din, T., Payandeh, J. et al. Structural basis for Ca2+ selectivity of a voltage-gated calcium channel. Nature 505, 56–61 (2014).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing