title: Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function.
abstract: A molecularly tagged form of calreticulin (CR), a low affinity-high capacity Ca2+ binding protein that resides in the ER lumen, was transiently transfected into HeLa cells to specifically modify the Ca2+ buffering capacity of the intracellular Ca2+ stores. Fluorescence and confocal microscope immunocytochemistry revealed the tagged protein to be expressed by over 40% of the cells and to overlap in its distribution the endogenous CR yielding a delicate cytoplasmic network, i.e., the typical pattern of ER. In contrast, no signal was observed associated with the plasmalemma (marked by ConA) and within the nucleus. One- and two-dimensional Western blots revealed the transfected to exceed the endogenous CR of approximately 3.5-fold and to maintain its Ca2+ binding ability, whereas the expression of other ER proteins was unchanged. Ca2+ homeostasis in the transfected cells was investigated by three parallel approaches: (a) 45Ca equilibrium loading of cell populations; (b) [Ca2+]c measurement with fura-2 followed by quantitative immunocytochemistry of single cells and iii) [Ca2+]c measurement of cell population upon cotransfection with the Ca(2+)-sensitive photoprotein, aequorin. The three approaches revealed different aspects of Ca2+ homeostasis, yielding results which were largely complementary. In particular, the following conclusions were established: (a) both endogenous and transfected CR participate in Ca2+ buffering within the IP3-sensitive, rapidly exchanging, Ca2+ stores; the other pools of the cells were in contrast unaffected by CR transfection; (b) the Ca2+ capacity of the stores is not the main limiting factor of individual IP3-mediated Ca2+ release responses triggered by receptor agonists; (c) in control cells, the contribution of CR to Ca2+ buffering within the IP3-sensitive stores accounts for approximately 45% of the total, the rest being probably contributed by the other lumenal (and also membrane) Ca2+ binding proteins; (d) the free [Ca2+] within the lumen of the IP3-sensitive stores, revealed by the degree of Ca2+ binding to the transfected CR protein, amounts to values in (or approaching) the millimolar range; and (e) Ca2+ influx across the plasmalemma activated by depletion of the stores is directly dependent on the lumenal [Ca2+].
authors.affiliation: Department of Biomedical Sciences, University of Padova, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Bastianutto
authors.forename: E
authors.initials: E
authors.lastname: Clementi
authors.forename: F
authors.initials: F
authors.lastname: Codazzi
authors.forename: P
authors.initials: P
authors.lastname: Podini
authors.forename: F
authors.initials: F
authors.lastname: De Giorgi
authors.forename: R
authors.initials: R
authors.lastname: Rizzuto
authors.forename: J
authors.initials: J
authors.lastname: Meldolesi
authors.forename: T
authors.initials: T
authors.lastname: Pozzan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D015415:Biomarkers; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D037282:Calreticulin; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
chemical_list: D015415:Biomarkers; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D002118:Calcium