CRATP-INDUCED CA2+ OCCLUSION IN MUTANTS OF THE CA2+-ATPASE OF SARCOPLASMIC-RETICULUM

Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca2+-occluded form of the sarcoplasmic reticulum Ca2+-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca2+-ATPase mutants Gly-233 --> Glu, Gly-233 --> Val, Glu-309 --> Gln, Gly-310 --> Pro, Pro-312 --> Ala, Ile-315 --> Arg, Leu-319 --> Arg, Asp-703 --> Ala, Gly-770 --> Ala, Glu-771 --> Gln, Asp-800 --> Asn, and Gly-801 --> Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca2+-ATPase. The "phosphorylation-negative" mutant Asp-703 --> Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770 --> Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca2+-site mutants") and mutant Gly-801 --> Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310 --> Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J. P., Vilsen, B., and MacLennan, D. H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca2+-activated phosphorylation from MgATP took place in this mutant.