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> Ca 2+ ATPase
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+ Phospholamban, Sarcolipin
+ Brody Disease
+ Cardiomyopathy
+ Ca2+ Release Channel
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+ Ca2+ Binding Proteins

o OMIM SERCA1
o OMIM SERCA2
o OMIM SERCA3

 

Structure/Function Relationships in the Calcium ATPase

Sarco(endo)plasmic reticulum calcium-ATPases pump calcium from the cytoplasm of mammalian cells into organellar structures such as the sarcoplasmic reticulum in muscle or the endoplasmic reticulum in non-muscle cells. Their threshold of activation by calcium is of the order of 100-200 nM, so that they set the resting level of cytoplasmic calcium. In fast-twitch muscle, the SERCA1 isoform is expressed at high levels and is highly concentrated in the sarcoplasmic reticulum. Full or partial reactions of calcium transport can be carried out with vesicles isolated from this source, making it a popular system for analysis of the mechanism of calcium transport.

[Picture: Bill Rice analyzing 3D model of SERCA2 transmembrane segments] Our research in this field has focused on SERCA molecules. We purified SERCA1 and cloned cDNA encoding SERCA1, SERCA2 and SERCA3. From the deduced amino acid sequences, we proposed a model of SERCA structure. The key features include a transmembrane domain made up of 10 transmembrane alpha helices, a stalk sector made up of helical extensions of transmembrane helices, and cytopasmic beta-strand, phosphorylation and nucleotide binding domains attached to the stalk domain at a distance of 60 Å from the transmembrane domain. This model is verified and refined as new structural data emerge.

[J Biol Chem Picture: Cystine scanning of SERCA2 transmembrane helices]We expressed all three proteins in COS-1 or HEK-293 cells under conditions where we could isolate microsomal fractions and carry out analysis of full or partial reactions of calcium transport. Then we initiated a program of determination of the functional consequences of site-directed mutagenesis. This program has allowed us to localize the two calcium binding sites to three (or possibly four) transmembrane helices, to localize ATP binding sites to the cytoplasmic nucleotide binding domain and to identify highly conserved residues throughout the molecule that are involved in conformational changes.

From these analyses, we have proposed a model of calcium transport which begins when calcium binds to two high affinity sites in the transmembrane domain that are formed by the precise juxtaposition of three helices. These sites are accessible from the cytoplasm, but not from the lumen. Phosphorylation of the enzyme by ATP initiates a series of conformational changes that, through long range interactions, change the conformation of the transmembrane helices so that access of calcium to the cytoplasm is lost, access to the lumen is gained and calcium binding sites are destroyed, releasing calcium to the lumen.

Our efforts to refine this concept of calcium transport include both functional and structural analysis. We are using site-directed disulfide mapping of transmembrane helices to determine their orientation relative to each other at a few Å resolution, we are analysing the significance of a calcium binding motif that we defined recently and we are attempting to purify cytoplasmic domains for eventual crystallization and structural determination.

 


> Ca 2+ ATPase
^ Top of Page
+ Phospholamban, Sarcolipin
+ Brody Disease
+ Cardiomyopathy
+ Ca2+ Release Channel
+ MH/CCD
+ Ca2+ Binding Proteins

o OMIM SERCA1
o OMIM SERCA2
o OMIM SERCA3



SERCA Publications

  1. Rice, W.J., Green, N.M. and MacLennan, D.H. (1997) Site-directed disulfide mapping of helices M4 and M6 in the Ca2+ binding domain of SERCA1a, the Ca2+ ATPase of fast twitch skeletal muscle sarcoplasmic reticulum. J Biol Chem 272(50):31412-9
  2. Liu, L.H., Paul, R.J., Sutliff, R.L., Miller, M.L., Lorenz, J.N., Pun, R.Y., Duffy, J.J., Doetschman, T., Kimura, Y., MacLennan, D.H., Hoying, J.B. and Shull, G.E. (1997) Defective endothelium-dependent relaxation of vascular smooth muscle and endothelial cell Ca2+ signaling in mice lacking sarco(endo)plasmic reticulum Ca2+-ATPase isoform 3. J Biol Chem 272(48):30538-45
  3. MacLennan, D.H., Rice, W.J. and Odermatt, A. (1997) Structure/function analysis of the Ca2+ binding and translocation domain of SERCA1 and the role in Brody disease of the ATP2A1 gene encoding SERCA1. Ann N Y Acad Sci 834175-85
  4. MacLennan, D.H., Rice, W.J. and Green, N.M. (1997) The mechanism of Ca2+ transport by sarco(endo)plasmic reticulum Ca2+- ATPases. J Biol Chem 272(46):28815-8
  5. MacLennan, D.H. and Becker, S.J. (1996) Sarcoplasmic reticulum Ca2+ ATPases, in Molecular Physiology and Pharmacology of Cardiac Ion Channels and Transporters (Morad, M., Ebashi, S., Trautwein, W. and Kurachi, Y.) Kluwer Academic Publishers Dordrecht, Holland
  6. Rice, W.J. and MacLennan, D.H. (1996) Scanning mutagenesis reveals a similar pattern of mutation sensitivity in transmembrane sequences M4, M5 and M6, but not in M8 of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a). Journal of Biological Chemistry 271(49):31412-31419
  7. Odermatt, A., Kurzydlowski, K. and MacLennan, D.H. (1996) The vmax of the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) is not altered by Ca2+/calmodulin-dependent phosphorylation or by interaction with phospholamban. Journal of Biological Chemistry 271(24):14206-13
  8. Toyofuku, T., Curotto Kurzydlowski, K., Narayanan, N. and MacLennan, D.H. (1994) Identification of Ser38 as the site in cardiac sarcoplasmic reticulum Ca(2+)-ATPase that is phosphorylated by Ca2+/calmodulin-dependent protein kinase. Journal of Biological Chemistry 269(42):26492-6
  9. Skerjanc, I.S., Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1993) Deletion of NH2- and COOH-terminal sequences destroys function of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. FEBS Letters 336(1):168-70
  10. Clarke, D.M., Loo, T.W., Rice, W.J., Andersen, J.P., Vilsen, B. and MacLennan, D.H. (1993) Functional consequences of alterations to hydrophobic amino acids located in the M4 transmembrane sector of the Ca(2+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 268(24):18359-64
  11. Skerjanc, I.S., Toyofuku, T., Richardson, C. and MacLennan, D.H. (1993) Mutation of glutamate 309 to glutamine alters one Ca(2+)-binding site in the Ca(2+)-ATPase of sarcoplasmic reticulum expressed in Sf9 cells. Journal of Biological Chemistry 268(21):15944-50
  12. MacLennan, D.H., Toyofuku, T. and Lytton, J. (1992) Structure-function relationships in sarcoplasmic or endoplasmic reticulum type Ca2+ pumps. Annals of the New York Academy of Sciences 6711-10
  13. Toyofuku, T., Kurzydlowski, K., Lytton, J. and MacLennan, D.H. (1992) The nucleotide binding/hinge domain plays a crucial role in determining isoform-specific Ca2+ dependence of organellar Ca(2+)-ATPases. Journal of Biological Chemistry 267(20):14490-6
  14. MacLennan, D.H., Clarke, D.M., Loo, T.W. and Skerjanc, I.S. (1992) Site-directed mutagenesis of the Ca2+ ATPase of sarcoplasmic reticulum. Acta Physiologica Scandinavica. Supplementum 607141-50
  15. Lytton, J., Westlin, M., Burk, S.E., Shull, G.E. and MacLennan, D.H. (1992) Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. Journal of Biological Chemistry 267(20):14483-9
  16. Clarke, D.M., Maruyama, K., Loo, T.W. and MacLennan, D.H. (1992) Molecular biology of sarcoplasmic reticulum proteins involved in EC coupling, in Molecular Biology of the Myocardium (Tada, M.) Japan Scientific Societies Press Tokyo
  17. Andersen, J.P., Vilsen, B. and MacLennan, D.H. (1992) Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 267(4):2767-74
  18. Vilsen, B., Andersen, J.P. and MacLennan, D.H. (1991) Functional consequences of alterations to hydrophobic amino acids located at the M4S4 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 266(28):18839-45
  19. Vilsen, B., Andersen, J.P. and MacLennan, D.H. (1991) Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 266(24):16157-64
  20. Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2(+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 265(36):22223-7
  21. MacLennan, D.H. (1990) Molecular tools to elucidate problems in excitation-contraction coupling. Biophysical Journal 58(6):1355-65
  22. Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) The epitope for monoclonal antibody A20 (amino acids 870-890) is located on the luminal surface of the Ca2(+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 265(29):17405-8
  23. Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2(+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 265(24):14088-92
  24. Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) Functional consequences of alterations to polar amino acids located in the transmembrane domain of the Ca2(+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 265(11):6262-7
  25. Vilsen, B., Andersen, J.P., Clarke, D.M. and MacLennan, D.H. (1989) Functional consequences of proline mutations in the cytoplasmic and transmembrane sectors of the Ca2(+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 264(35):21024-30
  26. Andersen, J.P., Vilsen, B., Leberer, E. and MacLennan, D.H. (1989) Functional consequences of mutations in the beta-strand sector of the Ca2(+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 264(35):21018-23
  27. Burk, S.E., Lytton, J., MacLennan, D.H. and Shull, G.E. (1989) cDna cloning, functional expression, and mRna tissue distribution of a third organellar Ca2+ pump. Journal of Biological Chemistry 264(31):18561-8
  28. Maruyama, K., Clarke, D.M., Fujii, J., Loo, T.W. and MacLennan, D.H. (1989) Expression and mutation of Ca2+ ATPases of the sarcoplasmic reticulum. Cell Motility & the Cytoskeleton 14(1):26-34
  29. Maruyama, K., Clarke, D.M., Fujii, J., Inesi, G., Loo, T.W. and MacLennan, D.H. (1989) Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 264(22):13038-42
  30. Clarke, D.M., Maruyama, K., Loo, T.W., Leberer, E., Inesi, G. and MacLennan, D.H. (1989) Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 264(19):11246-51
  31. Clarke, D.M., Loo, T.W., Inesi, G. and MacLennan, D.H. (1989) Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature 339(6224):476-8
  32. Lytton, J., Zarain-Herzberg, A., Periasamy, M. and MacLennan, D.H. (1989) Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. Journal of Biological Chemistry 264(12):7059-65
  33. Green, N.M. and MacLennan, D.H. (1989) ATP driven ion pumps: an evolutionary mosaic. Biochemical Society Transactions 17(5):819-22
  34. Maruyama, K. and MacLennan, D.H. (1988) Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells. Proceedings of the National Academy of Sciences of the United States of America 85(10):3314-8
  35. Green, N.M., Taylor, W.R. and MacLennan, D.H. (1988) A consensus structure for cation pumps, in Ion Pumps, Structure, Function and Regulation (Stein, W.D. and Karlish, S.J.) Alan R. Liss New York
  36. Lytton, J. and MacLennan, D.H. (1988) Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca2+-ATPase gene. Journal of Biological Chemistry 263(29):15024-31
  37. Brandl, C.J., deLeon, S., Martin, D.R. and MacLennan, D.H. (1987) Adult forms of the Ca2+ATPase of sarcoplasmic reticulum. Expression in developing skeletal muscle. Journal of Biological Chemistry 262(8):3768-74
  38. MacLennan, D.H., Brandl, C.J., Korczak, B. and Green, N.M. (1986) Calcium ATPases: Contribution of molecular genetics to our understanding of structure and function, in Proteins of Excitable Membranes (Hille, B. and Fambrough, D.) Wiley New York
  39. Brandl, C.J., Green, N.M., Korczak, B. and MacLennan, D.H. (1986) Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell 44(4):597-607
  40. MacLennan, D.H., Brandl, C.J., Korczak, B. and Green, N.M. (1985) Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature 316(6030):696-700
  41. MacLennan, D. and Reithmeier, R. (1985) Structural analysis of the Ca2++Mg2+-ATPase of sarcoplasmic reticulum, in Structure and Function of Sarcoplasmic Reticulum (Fleischer, S. and Tonomura, Y.) Academic Press New York
  42. MacLennan, D.H. and Reithmeier, R.A.F. (1982) The structure of the Ca2+/Mg2+-ATPase of sarcoplasmic reticulum, in Membranes and Transport (Martonosi, A.N.) Plenum Press New York
  43. Reithmeier, R.A. and MacLennan, D.H. (1981) The NH2 terminus of the (Ca2+ + Mg2+)-adenosine triphosphatase is located on the cytoplasmic surface of the sarcoplasmic reticulum membrane. Journal of Biological Chemistry 256(12):5957-60
  44. Reithmeier, R.A., de Leon, S. and MacLennan, D.H. (1980) Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin. Journal of Biological Chemistry 255(24):11839-46
  45. Klip, A., Reithmeier, R.A. and MacLennan, D.H. (1980) Alignment of the major tryptic fragments of the adenosine triphosphatase from sarcoplasmic reticulum. Journal of Biological Chemistry 255(14):6562-8
  46. Stewart, P.S. and MacLennan, D.H. (1976) Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum. Journal of Biological Chemistry 251(3):712-9
  47. Stewart, P.S. and MacLennan, D.H. (1974) Surface particles of sarcoplasmic reticulum membranes. Structural features of the adenosine triphosphatase. Journal of Biological Chemistry 249(3):985-93
  48. MacLennan, D.H., Seeman, P., Iles, G.H. and Yip, C.C. (1971) Membrane formation by the adenosine triphosphatase of sarcoplasmic reticulum. Journal of Biological Chemistry 246(8):2702-10
  49. MacLennan, D.H. (1970) Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum. Journal of Biological Chemistry 245(17):4508-18

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Revised
1998Apr09