Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca²⁺ through the sarcoplasmic reticulum Ca²⁺ ATPase

T Menguy, F Corre, B Juul, L Bouneau, D Lafitte, PJ Derrick, Parveen Sharma, P Falson, Baruch Levine, JV Moller, M le Maire

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21 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)13016-13028
Number of pages13
JournalJournal of Biological Chemistry
Volume277
Issue number15
Early online date18 Jan 2002
DOIs
Publication statusPublished - 5 Apr 2002

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