title: Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.
abstract: We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca(2+) activation of the transport process. In this study, yeast membranes, expressing wild type and mutant Ca(2+)-ATPases, were reacted with Cr small middle dotATP and tested for their ability to occlude (45)Ca(2+) by HPLC analysis, after cation resin and C(12)E(8) treatment. We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca(2+) to the same extent as wild type ATPase. Using NMR and mass spectrometry we have analyzed the conformational properties of the synthetic L6-7 loop and demonstrated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum. All three aspartate Asp(813)/Asp(815)/Asp(818) were required to coordinate the trivalent lanthanide ion. Overall these observations suggest a dual function of the loop: in addition to mediating contact between the intramembranous Ca(2+)-binding sites and the cytosolic phosphorylation site (Zhang, Z., Lewis, D., Sumbilla, C., Inesi G., and Toyoshima, C. (2001) J. Biol. Chem. 276, 15232-15239), the L6-7 loop, in a preceding step, participates in the formation of an entrance port, before subsequent high affinity binding of Ca(2+) inside the membrane.
authors.affiliation: Section de Biophysique des Fonctions Membranaires, DBJC, CEA et CNRS URA 2096 and LRA17V Université de Paris XI, CE Saclay, 91191 Gif sur Yvette Cedex, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Menguy
authors.forename: Fabienne
authors.initials: F
authors.lastname: Corre
authors.forename: Birte
authors.initials: B
authors.lastname: Juul
authors.forename: Laurence
authors.initials: L
authors.lastname: Bouneau
authors.forename: Daniel
authors.initials: D
authors.lastname: Lafitte
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Derrick
authors.forename: Parveen S
authors.initials: PS
authors.lastname: Sharma
authors.forename: Pierre
authors.initials: P
authors.lastname: Falson
authors.forename: Barry A
authors.initials: BA
authors.lastname: Levine
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Møller
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D017136:Ion Transport; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium