title: The nucleotide binding/hinge domain plays a crucial role in determining isoform-specific Ca2+ dependence of organellar Ca(2+)-ATPases.
abstract: Several isoforms of organellar Ca(2+)-ATPases have been identified, each of which is expressed in a tissue-specific manner. In order to examine the functional properties of fast-twitch (SERCA 1a), cardiac/slow-twitch (SERCA 2a), and non-muscle (SERCA 3) isoforms of the Ca(2+)-ATPase, cDNAs of each type were expressed transiently in COS-1 cells. A study of the Ca2+ dependence of Ca2+ uptake showed that SERCA 1 and SERCA 2 have identical Ca2+ dependences (K0.5 = pCa 6.87 +/- 0.03 and pCa 6.87 +/- 0.02, respectively), but SERCA 3 has a lower Ca2+ dependence (K0.5 = pCa 6.32 +/- 0.03). A study of the ATP dependence of Ca2+ uptake showed that SERCA 1, 2, and 3 have almost identical ATP dependences. Average Hill coefficients derived from Ca2+ uptake curves ranged from 1.7 to 1.8 for the three isoforms. In order to identify which regions of the linear sequence determine this difference in Ca2+ dependence, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed. Chimeric Ca(2+)-ATPases containing the nucleotide binding/hinge domain of SERCA 2 had SERCA 2 type Ca2+ dependence, but both nucleotide binding/hinge and COOH-terminal transmembrane domains of SERCA 3 were required for SERCA 3 type Ca2+ dependence. Accordingly, structural interactions between the nucleotide binding/hinge and COOH-terminal transmembrane domains appear to determine isoform-specific Ca2+ dependences.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, C.H. Best Institute, Ontario, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Toyofuku
authors.forename: K
authors.initials: K
authors.lastname: Kurzydlowski
authors.forename: J
authors.initials: J
authors.lastname: Lytton
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002678:Chimera; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology; D009838:Oligodeoxyribonucleotides; D015388:Organelles / Q000201:enzymology*; D011487:Protein Conformation; D015183:Restriction Mapping; D014162:Transfection
chemical_list: D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D004247:DNA; D000252:Calcium-Transporting ATPases; D002118:Calcium