Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors

Single mutations of specific amino acids within the membrane-bound region 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 Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find 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) --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu(771), Thr(799), Asp(800) and Glu(908) are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the P-i reaction. We also find that, at pK 7.0, the Glu(309) --> Gln and the Asn(796) --> APa mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere 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 is 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 Glu(309) --> Gln mutant, ionization of acidic functions in other amino acids (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 the other hand, the Glu(309) carboxylic function allows binding of inhibitory 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 effect of mutations on the affinity of the ATPase for Ca2+.