Mutation of glutamate 309 to glutamine alters one Ca²⁺-binding site in the Ca²⁺-ATPase of sarcoplasmic reticulum expressed in Sf9 cells

Sf9 cells infected with a baculovirus vector containing SERCA1 cDNA expressed immunoreactive rabbit fast-twitch muscle Ca²⁺-ATPase at levels up to 3 mg/liter. The microsomal fraction isolated from infected Sf9 cells catalyzed Ca²⁺ transport at rates 6-fold above control values. To obtain direct evidence for the postulate (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H., et al. (1989) Nature 339, 476-478) that Glu³⁰⁹ contributes to a Ca²⁺-binding site in the transmembrane sector of the Ca²⁺-ATPase, Ca²⁺ binding to wild type and mutant (Glu³⁰⁹ to Gln) Ca²⁺-ATPases was measured. The wild type Ca²⁺-ATPase, expressed in Sf9 cells and purified using a monoclonal antibody bound to Sepharose beads, bound ~1.6-1.7 mol Ca²⁺/mol of enzyme at 2 μM Ca²⁺ in a buffer favoring the E₁ conformation of the enzyme and at 10 μM Ca²⁺ in a buffer favoring the E₂ conformation. Under identical conditions, the mutant Ca²⁺-ATPase bound less than 0.1 mol of Ca²⁺/mol of enzyme in E₁ buffer, but 0.8 mol Ca²⁺/mol in the E₂ buffer. In spite of the ability of the Glu³⁰⁹ to Gln mutant enzyme to bind about 1 mol of Ca²⁺/mol of enzyme, E₂P formation was not inhibited by up to 100 μM Ca²⁺, and E₁P formation from ATP and Ca²⁺ was not observed with up to 100 μM Ca²⁺ in intact microsomal vesicles from Sf9 cells. Nevertheless, with detergent-solubilized and purified mutant Ca²⁺-ATPases, E₂P formation was inhibited with a K_0.5 of 2 μM Ca²⁺. These observations are consistent with the view that a single intact Ca²⁺-binding site is present in the mutant Ca²⁺- ATPase, which is accessible to Ca²⁺ only from the lumenal side and only in the E₂ conformation. Transition from E₂ to E₁-Ca²⁺ may occur during or following Ca²⁺ binding, accounting for the relatively high Ca²⁺ affinity and inhibition by Ca²⁺ of phosphorylation from P(i).