2 RESIDUES THAT MAY LIGATE CA2+ IN TRANSMEMBRANE DOMAIN 6 OF THE PLASMA-MEMBRANE CA2+-ATPASE

In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn(879), Met(882), Asp(883), and Ser(887) were singly altered, Asn(879), Met(882), and Asp(883) were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D, M,, Loo, T, W,, and MacLennan, D, H, (1990) J, Biol, Chem, 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding, The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [gamma-P-32]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands, However, all the mutants at the position of Met(882) showed some activity, Indeed, the Met(882) --> Ile mutant was fully active at a saturating Ca2+ concentration and only the K-1/2, for Ca2+ activation was shifted slightly upward, Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps, The mutant Ser(887) --> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met(882).