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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 Cr⅐ATP 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