title: Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase.
abstract: Cation pumps bind and translocate ions with the intermediate formation of a phosphoenzyme. In spite of extensive knowledge of the primary and even secondary structures of several of these cation transport enzymes, however, no high affinity cation binding sites have yet been determined. Here we report the use of oligonucleotide-directed, site-specific mutagenesis to identify the amino acids involved in Ca2+ binding in one of these transport enzymes, the Ca2+-ATPase of sarcoplasmic reticulum. Alteration of Glu 309, Glu 771, Asn 796, Thr 799, Asp 800 or Glu 908, each of which is predicted to lie near the centre of the transmembrane domain in putative transmembrane sequences M4, M5, M6 and M8 resulted in complete loss of Ca2+ transport function and of Ca2+-dependent phosphorylation of the enzyme by ATP. Phosphorylation of each of the mutant enzymes with inorganic phosphate was observed, however, even in the presence of Ca2+, which inhibits phosphorylation in the wild-type enzyme possessing an intact high affinity Ca2+-binding site. These results suggest that at least six polar, oxygen-containing residues lying near the centre of the transmembrane domain provide ligands for one or both of the two high affinity Ca2+ binding sites in the Ca2+-ATPase.
authors.affiliation: Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, Ontario, Canada.
authors.forename: D M
authors.initials: DM
authors.lastname: Clarke
authors.forename: T W
authors.initials: TW
authors.lastname: Loo
authors.forename: G
authors.initials: G
authors.lastname: Inesi
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D004247:DNA / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D014162:Transfection
chemical_list: D004247:DNA; D000252:Calcium-Transporting ATPases; D002118:Calcium