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

We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca2+-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca2+ activation of the transport process. In this study, yeast membranes, expressing wild type and mutant Ca2+-ATPases, were reacted with Cr(.)ATP and tested for their ability to occlude 45 Ca2+ by HPLC analysis, after cation resin and Cl,E, treatment. We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca2+ 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 Ca2+-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 Ca2+ inside the membrane.