Functional consequences of alterations to polar amino acids located in the transmembrane domain of the Ca2(+)-ATPase of sarcoplasmic reticulum

Abstract

Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908 (ligands 1 to 6, respectively) appear to form the high affinity Ca2(+)-binding sites of the Ca2(+)-ATPase. The plasticity of the Ca2(+)-binding sites was tested by separate replacement of each of the ligands with a structurally similar oxygen-containing residue using site-specific mutagenesis. Mutant cDNAs were transfected into COS-1 cells, and ATP-dependent Ca2+ transport or partial reactions were studied in microsomes containing the expressed Ca2(+)-ATPases. In most cases where amino acid substitutions were carried out, the expressed enzymes lacked Ca2+ transport function and Ca2(+)-dependent phosphorylation by ATP. Furthermore, the mutant enzymes were phosphorylated by inorganic phosphate, even in the presence of Ca2+, which inhibits phosphorylation of the wild-type enzyme possessing intact Ca2(+)-binding sites. On mutant, however, containing an isosteric replacement of Glu by Gln at ligand 6, exhibited wild-type levels of Ca2+ transport activity and Ca2+ affinity. Two mutants exhibited properties consistent with a reduction in Ca2+ affinity. In the mutant in which Thr was replaced by Ser at ligand 4, Ca2+ transport activity was 70% of wild-type, while half-maximal activation by Ca2+ occurred at 0.8 microM as compared to 0.3 microM for the wild-type enzyme. In the mutant Glu309----Asp at ligand 1, Ca2+ transport activity was lost, but Ca2(+)-activated phosphorylation by ATP was retained. The concentration of Ca2+ required to activate phosphorylation was increased about 10-fold, however, compared to wild type. These results support our hypothesis that ligands 1 to 6, believed to reside within the transmembrane domain, interact with Ca2+ ions during the transport process. The roles of 12 other oxygen-containing residues and of His278 located in the transmembrane domain were also examined by mutation. Although the oxygen-containing side chains of these residues are potential Ca2+ ligands, their replacement with nonpolar amino acids did not abolish Ca2+ transport function, leading to the conclusion that they are not essential ligands for high affinity Ca2+ binding by the Ca2+ pump.