Involvement of the Cytoplasmic Loop L6-7 in the Entry Mechanism for Transport of Ca super(2+) through the Sarcoplasmic Reticulum Ca super(2+)-ATPase

• Published in: The Journal of biological chemistry Vol. 277; no. 15; pp. 13016 - 13028
• Main Authors: Menguy, T, Corre, F, Juul, B, Bouneau, L, Lafitte, D, Derrick, P J, Sharma, P S, Falson, P, Levine, BA, Moeller, J V, le Maire, M
• Format: Journal Article
• Language: English
• Published: 12-04-2002
• ISSN: 0021-9258

We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca super(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca super(2+) activation of the transport process. In this study, yeast membranes, expressing wild type and mutant Ca super(2+)-ATPases, were reacted with Cr super(.)ATP and tested for their ability to occlude super(45)Ca super(2+) by HPLC analysis, after cation resin and C sub(12)E sub(8) treatment. We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca super(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 super(813)/Asp super(815)/Asp super(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 super(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 super(2+) inside the membrane.