title: Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3.
abstract: The sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca(2+) concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca(2+); however, Ca(2+) affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b. We used a fluorescence-based assay for monitoring continuous Ca(2+) uptake into microsomes to examine the properties of endogenous human SERCA3 and SERCA2b. The kinetic parameters were derived using a cooperative two-component uptake model for Ca(2+) activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody PL/IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca(2+) affinity for SERCA3 compared with SERCA2b (1.10 +/- 0.04 vs. 0.26 +/- 0.01 microM), and using mixtures of recombinant protein isoforms, we validated the two-component uptake model. Then we determined apparent Ca(2+) affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca(2+) affinity in these two preparations was 1.04 +/- 0.07 and 1.1 +/- 0.2 muM for SERCA3 and 0.27 +/- 0.02 and 0.26 +/- 0.01 microM for SERCA2b, respectively. Our data demonstrate, for the first time, that affinity for Ca(2+) is inherently lower for SERCA3 expressed in situ than for other SERCA isoforms.
authors.affiliation: University of Calgary Health Science Centre, Calgary, AB, Canada T2N 4N1.
authors.forename: P Charukeshi
authors.initials: PC
authors.lastname: Chandrasekera
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Kargacin
authors.forename: Julie P
authors.initials: JP
authors.lastname: Deans
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lytton
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000493:pharmacokinetics*; D020013:Calcium Signaling / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007668:Kidney / Q000166:cytology; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D014066:Palatine Tonsil / Q000166:cytology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; C506949:ATP2A3 protein, human; D002118:Calcium