DIRECT DEMONSTRATION OF CA2-ENDOPLASMIC RETICULUM CA2+ ATPASE MUTANTSOVEREXPRESSED IN COS-1 CELLS TRANSFECTED WITH ADENOVIRUS VECTORS( BINDING DEFECTS IN SARCO)

Single mutations of specific amino acids within the membrane-bound reg ion of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by P-i (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficie ncy by means of recombinant adenovirus vectors, yielding sufficient ex pression of wild type and mutant SERCA-1 ATPase for measurements of Ca 2+ binding to the microsomal fraction of the transfected cells. We fin d that in the presence of 20 mu M Ca2+ and in the absence of ATP, the Glu(771) --> Gln, Thr(799), Ala, Asp(800) --> Asn, and Glu(908) --> Al a mutants exhibit negligible binding, indicating that the oxygen funct ions of Glu(771), Thr(799), Asp(800) and Glu(908) are involved in inte ractions whose single disruption causes major changes in the highly co operative ''duplex'' binding. Total loss of Ca2+ binding is accompanie d by loss of Ca2+ inhibition of the P-i reaction. We also find that, a t pK 7.0, the Glu(309) --> Gln and the Asn(796) --> APa mutants bind a pproximately half as much Ca2+ as the wild type ATPase and do not inte rfere with Ca2+ inhibition of the P-i reaction. At pH 6.2, the Glu(309 ), Gln mutant does not bind any Ca2+, and its phosphorylation by P-i i s not inhibited by Ca2+, On the contrary, the Asn(796) --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Gl u(309) --> Gln mutant, ionization of acidic functions in other amino a cids (e.g. Glu(771) and Asp(800)) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn(796) --> Ala mutant, on t he other hand, the Glu(309) carboxylic function allows binding of inhi bitory Ca2+ even at pH 6.2, In all eases mutational interference with the inhibition of the P-i reaction by Ca2+ can be overcome by raising the Ca2+ concentration to the mM range, consistent with a general effe ct of mutations on the affinity of the ATPase for Ca2+.