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Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain–Ca2+/calmodulin complex

Nature Structural & Molecular Biology volume 12, pages 1108–1115 (2005)Cite this article

Abstract

Changes in activity-dependent calcium flux through voltage-gated calcium channels (CaVs) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca2+/calmodulin (Ca2+/CaM) and a CaV channel consensus isoleucine-glutamine (IQ) motif: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the high-resolution structure of the Ca2+/CaM–CaV1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca2+/CaM lobes through sets of conserved 'aromatic anchors.' Ca2+/N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca2+/N lobe–IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca2+/N lobe aromatic anchors uncovers their role in CaV1.2 CDF. Together, our data suggest that CaV subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe–specific regulation of CaVs.

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Figure 1: Structure of the Ca2+/CaM–CaV1.2 IQ domain complex.
Figure 2: Lobe-specific Ca2+/CaM–CaV1.2 IQ domain interactions.
Figure 3: ITC characterization of Ca2+/CaM–CaV1.2 IQ domain interactions.
Figure 4: Lobe-specific interactions affect CDI and CDF.
Figure 5: Schematic of Ca2+/CaM lobe multiple binding modes on the CaV1.2 IQ domain.

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References

  1. Hille, B. Ion Channels of Excitable Membranes (Sinauer Associates, Inc., Sunderland, Massachusetts, USA, 2001).

    Google Scholar 

  2. Catterall, W.A. Structure and regulation of voltage-gated Ca2+ channels. Annu. Rev. Cell Dev. Biol. 16, 521–555 (2000).

    Article  CAS  Google Scholar 

  3. Saimi, Y. & Kung, C. Calmodulin as an ion channel subunit. Annu. Rev. Physiol. 64, 289–311 (2002).

    Article  CAS  Google Scholar 

  4. Kang, M.G. & Campbell, K.P. Gamma subunit of voltage-activated calcium channels. J. Biol. Chem. 278, 21315–21318 (2003).

    Article  CAS  Google Scholar 

  5. Sheng, Z.H., Westenbroek, R.E. & Catterall, W.A. Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery. J. Bioenerg. Biomembr. 30, 335–345 (1998).

    Article  CAS  Google Scholar 

  6. Walker, D. & De Waard, M. Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function. Trends Neurosci. 21, 148–154 (1998).

    Article  CAS  Google Scholar 

  7. Kandel, E.R., Schwartz, J.H. & Jessell, T.M. Principles of Neural Science (McGraw-Hill, New York, USA, 2000).

  8. Budde, T., Meuth, S. & Pape, H.C. Calcium-dependent inactivation of neuronal calcium channels. Nat. Rev. Neurosci. 3, 873–883 (2002).

    Article  CAS  Google Scholar 

  9. DeMaria, C.D., Soong, T.W., Alseikhan, B.A., Alvania, R.S. & Yue, D.T. Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels. Nature 411, 484–489 (2001).

    Article  CAS  Google Scholar 

  10. Lee, A. et al. Ca2+/calmodulin binds to and modulates P/Q-type calcium channels. Nature 399, 155–159 (1999).

    Article  CAS  Google Scholar 

  11. Lee, A., Zhou, H., Scheuer, T. & Catterall, W.A. Molecular determinants of Ca2+/calmodulin-dependent regulation of Cav2.1 channels. Proc. Natl. Acad. Sci. USA 100, 16059–16064 (2003).

    Article  CAS  Google Scholar 

  12. Peterson, B.Z., DeMaria, C.D., Adelman, J.P. & Yue, D.T. Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type calcium channels. Neuron 22, 549–558 (1999).

    Article  CAS  Google Scholar 

  13. Qin, N., Olcese, R., Bransby, M., Lin, T. & Birnbaumer, L. Ca2+-induced inhibition of the cardiac Ca2+ channel depends on calmodulin. Proc. Natl. Acad. Sci. USA 96, 2435–2438 (1999).

    Article  CAS  Google Scholar 

  14. Zühlke, R.D., Pitt, G.S., Deisseroth, K., Tsien, R.W. & Reuter, H. Calmodulin supports both inactivation and facilitation of L-type calcium channels. Nature 399, 159–162 (1999).

    Article  Google Scholar 

  15. Zühlke, R.D., Pitt, G.S., Tsien, R.W. & Reuter, H. Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the (alpha)1C subunit. J. Biol. Chem. 275, 21121–21129 (2000).

    Article  Google Scholar 

  16. Alseikhan, B.A., DeMaria, C.D., Colecraft, H.M. & Yue, D.T. Engineered calmodulins reveal the unexpected eminence of Ca2+ channel inactivation in controlling heart excitation. Proc. Natl. Acad. Sci. USA 99, 17185–17190 (2002).

    Article  CAS  Google Scholar 

  17. Pitt, G.S. et al. Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels. J. Biol. Chem. 276, 30794–30802 (2001).

    Article  CAS  Google Scholar 

  18. Erickson, M.G., Liang, H., Mori, M.X. & Yue, D.T. FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation. Neuron 39, 97–107 (2003).

    Article  CAS  Google Scholar 

  19. Romanin, C. et al. Ca(2+) sensors of L-type Ca(2+) channel. FEBS Lett. 487, 301–306 (2000).

    Article  CAS  Google Scholar 

  20. Tang, W. et al. Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel. Biophys. J. 85, 1538–1547 (2003).

    Article  CAS  Google Scholar 

  21. Pate, P. et al. Determinants for calmodulin binding on voltage-dependent Ca2+ channels. J. Biol. Chem. 275, 39786–39792 (2000).

    Article  CAS  Google Scholar 

  22. Black, D.J. et al. Calmodulin interactions with IQ peptides from voltage-dependent calcium channels. Am. J. Physiol. Cell Physiol. 288, C669–C676 (2005).

    Article  CAS  Google Scholar 

  23. Liang, H. et al. Unified mechanisms of Ca(2+) regulation across the Ca(2+) channel family. Neuron 39, 951–960 (2003).

    Article  CAS  Google Scholar 

  24. Osawa, M. et al. A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin-dependent kinase kinase. Nat. Struct. Biol. 6, 819–824 (1999).

    Article  CAS  Google Scholar 

  25. Kurokawa, H. et al. Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide. J. Mol. Biol. 312, 59–68 (2001).

    Article  CAS  Google Scholar 

  26. Bahler, M. & Rhoads, A. Calmodulin signaling via the IQ motif. FEBS Lett. 513, 107–113 (2002).

    Article  CAS  Google Scholar 

  27. Jurado, L.A., Chockalingam, P.S. & Jarrett, H.W. Apocalmodulin. Physiol. Rev. 79, 661–682 (1999).

    Article  CAS  Google Scholar 

  28. Dunitz, J.D. Win some, lose some: enthalpy-entropy compensation in weak intermolecular interactions. Chem. Biol. 2, 709–712 (1995).

    Article  CAS  Google Scholar 

  29. Gao, T., Bunemann, M., Gerhardstein, B.L., Ma, H. & Hosey, M.M. Role of the C terminus of the alpha 1C (CaV1.2) subunit in membrane targeting of cardiac L-type calcium channels. J. Biol. Chem. 275, 25436–25444 (2000).

    Article  CAS  Google Scholar 

  30. Lee, A., Scheuer, T. & Catterall, W.A. Ca2+/calmodulin-dependent facilitation and inactivation of P/Q-type Ca2+ channels. J. Neurosci. 20, 6830–6838 (2000).

    Article  CAS  Google Scholar 

  31. Zühlke, R.D. & Reuter, H. Ca2+-sensitive inactivation of L-type Ca2+ channels depends on multiple cytoplasmic amino acid sequences of the alpha1C subunit. Proc. Natl. Acad. Sci. USA 95, 3287–3294 (1998).

    Article  Google Scholar 

  32. Kim, J., Ghosh, S., Nunziato, D.A. & Pitt, G.S. Identification of the components controlling inactivation of voltage-gated Ca2+ channels. Neuron 41, 745–754 (2004).

    Article  CAS  Google Scholar 

  33. Deschenes, I. et al. Isoform-specific modulation of voltage-gated Na(+) channels by calmodulin. Circ. Res. 90, E49–E57 (2002).

    Article  Google Scholar 

  34. Kim, J. et al. Calmodulin mediates Ca2+ sensitivity of sodium channels. J. Biol. Chem. 279, 45004–45012 (2004).

    Article  CAS  Google Scholar 

  35. Mori, M. et al. Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity? Biochemistry 39, 1316–1323 (2000).

    Article  CAS  Google Scholar 

  36. Clackson, T. & Wells, J.A. A hot spot of binding energy in a hormone-receptor interface. Science 267, 383–386 (1995).

    Article  CAS  Google Scholar 

  37. Leavitt, S. & Freire, E. Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Biol. 11, 560–566 (2001).

    Article  CAS  Google Scholar 

  38. Jencks, W.P. On the attribution of additivity of binding energies. Proc. Natl. Acad. Sci. USA 78, 4046–4050 (1981).

    Article  CAS  Google Scholar 

  39. Mouton, J., Feltz, A. & Maulet, Y. Interactions of calmodulin with two peptides derived from the c-terminal cytoplasmic domain of the Ca(v)1.2 Ca2+ channel provide evidence for a molecular switch involved in Ca2+-induced inactivation. J. Biol. Chem. 276, 22359–22367 (2001).

    Article  CAS  Google Scholar 

  40. Xiong, L., Kleerekoper, Q.K., He, R., Putkey, J.A. & Hamilton, S.L. Sites on calmodulin that interact with the C-terminal tail of Cav1.2 channel. J. Biol. Chem. 280, 7070–7079 (2005).

    Article  CAS  Google Scholar 

  41. Hoeflich, K.P. & Ikura, M. Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108, 739–742 (2002).

    Article  CAS  Google Scholar 

  42. Van Petegem, F., Clark, K.A., Chatelain, F.C. & Minor, D.L., Jr . Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain. Nature 429, 671–675 (2004).

    Article  CAS  Google Scholar 

  43. Kholod, N. & Mustelin, T. Novel vectors for co-expression of two proteins in E. coli. Biotechniques 31, 322–323 326–328 (2001).

    Article  CAS  Google Scholar 

  44. Kapust, R.B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14, 993–1000 (2001).

    Article  CAS  Google Scholar 

  45. Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993).

    Article  CAS  Google Scholar 

  46. McPherson, A. Crystallization of Biological Macromolecules (Cold Spring Harbor Press, Cold Spring Harbor, New York, USA, 1999).

    Google Scholar 

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

    Article  CAS  Google Scholar 

  48. Schneider, T.R. & Sheldrick, G.M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002).

    Article  Google Scholar 

  49. d Fortelle, E.l. & Bricogne, G. Maximum-likelihood heavy atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).

    Article  Google Scholar 

  50. Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000).

    Article  CAS  Google Scholar 

  51. Perrakis, A., Morris, R. & Lamzin, V.S. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999).

    Article  CAS  Google Scholar 

  52. McRee, D.E. XtalView/Xfit–A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    Article  CAS  Google Scholar 

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

  54. Edelhoch, H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948–1954 (1967).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank K. Brejc and D. Fass for comments on the manuscript; J. Holton at Beamline 8.3.1 at the Advanced Light Source for help with data collection; C.B. Klee (US National Institutes of Health, Bethesda, Maryland, USA) for the calmodulin clone; R.W. Tsien (Stanford University School of Medicine, Stanford, California, USA) and D.T. Yue (Johns Hopkins University School of Medicine, Baltimore, USA) for calcium channel clones; and members of the Minor laboratory for support at all stages of this work. This work was supported by awards to D.L.M. from the McKnight Foundation for Neuroscience, the Rita Allen Foundation, the Alfred P. Sloan Foundation and the US National Institutes of Health and to F.V.P. from the American Heart Association Western States Affiliate. D.L.M. is a McKnight Scholar in Neurosciences, an Alfred P. Sloan Research Fellow and a Rita Allen Foundation Scholar.

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  1. Department of Biochemistry and Biophysics and Department of Cellular and Molecular Pharmacology, Cardiovascular Research Institute, California Institute for Quantitative Biomedical Research, University of California, San Francisco, 1700 4th St., Box 2532, San Francisco, 94143-2532, California, USA

    Filip Van Petegem, Franck C Chatelain & Daniel L Minor, Jr

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Supplementary information

Supplementary Fig. 1

Experimentally phased and density modified electron density map for the IQ domain in complex A. (PDF 669 kb)

Supplementary Fig. 2

Details of interactions of Cav1.2 IQ domain residues with Ca2+/CaM. (PDF 663 kb)

Supplementary Fig. 3

Overview of main chain and side chain interactions in complexes A and C. (PDF 281 kb)

Supplementary Fig. 4

Sequence alignment of the IQ domains of Cav1, Cav2 and Nav1 channels. (PDF 222 kb)

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Van Petegem, F., Chatelain, F. & Minor,, D. Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain–Ca2+/calmodulin complex. Nat Struct Mol Biol 12, 1108–1115 (2005). https://doi.org/10.1038/nsmb1027

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