Rat basophiliv leukemia cells as model system for inositol 1,4,5-trisphosphate receptor IV, a receptor of the type II family: functional comparison and immunological detection

1 is a second messenger used by many cell types to release Ca 2ϩ from internal stores (1). Cytosolic Ca 2ϩ has a bell-shaped effect on the IP 3 receptor (IP 3 R), with low concentrations stimulating the release and high concentrations inhibiting it (2-5). Calmodulin, a Ca 2ϩ -binding protein abundantly present in many cell types (6), binds to the modulatory region of the IP 3 R in a Ca 2ϩ -dependent way (7,8). Calmodulin also interacts with the IP 3 R in a Ca 2ϩ -independent way (9 -11), with one of the interaction sites located within the IP 3 -binding domain (11

Section: Resultsmentioning

confidence: 99%

The Bell-shaped Ca2+ Dependence of the Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Is Modulated by Ca2+/Calmodulin

Missiaen

Parys

Weidema

et al. 1999

“…Microsomal Preparations-Total microsomal fractions from non-transfected control COS-1 cells and from Pmr1-and SERCA-transfected COS-1 cells as well as from CHO cells were essentially prepared as previously described (22). CHO cells were chosen as positive control for IP 3 receptor expression, because we previously reported the presence of the three IP 3 receptor isoforms in those cells (23).…”

Section: Methodsmentioning

confidence: 99%

Baseline Cytosolic Ca2+ Oscillations Derived from a Non-endoplasmic Reticulum Ca2+Store

Missiaen¹,

Acker²,

Parys³

et al. 2001

) is an intracellular second messenger used by many cells to release Ca 2ϩ from their internal stores (6). IP 3 acts on the IP 3 receptor, which is an important Ca 2ϩ release channel in the endoplasmic reticulum (7). There is, however, increasing evidence that IP 3 receptors are also localized in the Golgi apparatus (8 -12). The role of this latter organelle in the generation of cytosolic Ca 2ϩ signals is not well known, partly because the mechanism by which the Golgi complex takes up Ca 2ϩ is less well understood compared with the Ca 2ϩ transport into the endoplasmic reticulum mediated by the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA). The Golgi apparatus expresses a specific Ca 2ϩ pump belonging to the Pmr1 family of Ca 2ϩ -transport ATPases (13). Pmr1 is insensitive to the inhibitor thapsigargin (14), in contrast to the SERCA Ca 2ϩ pumps in the sarco-and endoplasmic reticulum (15).We expressed the Pmr1 Ca 2ϩ pump of Caenorhabditis elegans (12) in COS-1 cells and pretreated the cells with thapsigargin to prevent SERCA-mediated Ca 2ϩ uptake in the endoplasmic reticulum. We demonstrate that a Ca 2ϩ store filled by the Pmr1 Ca 2ϩ pump and probably belonging to the Golgi compartment can set up baseline Ca 2ϩ spiking. EXPERIMENTAL PROCEDURESCell Culture and Transfection-COS-1 cells and HeLa cells were cultured as described previously (12, 16). The culture medium was replaced every 2 to 3 days. The cells were plated at a density of 2500 cells/cm 2 in Coverglass Chambers (Nunc Inc., Naperville, IL) or at a density of 10,000 cells/cm 2 in 12-well clusters (Costar, MA). Four days after plating, COS-1 cells were transiently transfected with Pmr1 from C. elegans or with the rabbit SERCA1a in pMT2 vector and investigated 3 days later (12). The full-length rabbit SERCA1a clone was kindly provided by Drs. J. P. Andersen and B. Vilsen (Department of Physiology, University of Aarhus, Denmark). Some cells were transfected with pig SERCA2a or SERCA2b in pSV57 (17).Immunofluorescence-Immunofluorescence microscopy was done as described previously (12). The primary antiserum Celpmrloop against Pmr1 was raised against a recombinant protein corresponding to the putative large cytoplasmic loop between transmembrane segments 4 and 5 of Pmr1 from C. elegans (12). The primary antiserum 809-27 against rabbit SERCA1 was kindly provided by Dr. J. Møller (Department of Biophysics, University of Aarhus, Denmark) (18). The antiserum against SERCA2a was described previously (19). The antibody against the 58K protein of the Golgi apparatus (clone 58K-9) was purchased from Abcams (Cambridge, UK).45 Ca 2ϩ Uptake-Fluxes were performed on saponin-permeabilized cells at 25°C as described previously (20). The cells were permeabilized by treating them for 10 min with 20 g/ml saponin at 25°C in a medium containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 2 mM MgCl 2 , 1 mM ATP and 1 mM EGTA. The non-mitochondrial Ca 2ϩ stores were loaded for 45 min in loading medium containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM MgCl 2 , 5 mM ATP, ...

“…Full-length IP 3 R-1 was detected with the polyclonal antibody Rbt03. This antibody is directed against the same epitope as the previously described Rbt04 (23)(24)(25)(26) and has specificity and affinity identical to those of Rbt04. A second antibody was raised against the Ca 2ϩ -binding domain cyt3b present between amino acids 378 and 450 of the mouse IP 3 R-1 (21).…”

Section: Ip 3 Binding-[mentioning

confidence: 99%

Modulation of Inositol 1,4,5-Trisphosphate Binding to the Recombinant Ligand-binding Site of the Type-1 Inositol 1,4,5-Trisphosphate Receptor by Ca2+ and Calmodulin

Sipma¹,

Smet²,

Sienaert³

et al. 1999

(14,15).The N-terminal part of the IP 3 R contains all the structural determinants responsible for specific and selective binding of its physiological agonist, IP 3 (17-20). We have therefore expressed the N-terminal ligand-binding site (first 581 amino acids) of the mouse IP 3 R-1 in Escherichia coli, using a strategy of growth and expression at low temperatures, as described previously by Yoshikawa et al. (20). This protein contains a previously identified Ca 2ϩ -binding region located between amino acids 304 -450 (21). We now demonstrate that Ca 2ϩ and calmodulin can both inhibit IP 3 binding to this recombinant protein and that these inhibitors act independently and additively. Our data indicate that the N-terminal ligand-binding domain of IP 3 R-1 contains regulatory regions directly interacting with Ca 2ϩ and calmodulin. EXPERIMENTAL PROCEDURES Expression of IP 3 R-1 in Sf9Insect Cells-The full-length neuronal mouse IP 3 R-1 cDNA clone containing the S1 splice domain in p400C1 plasmid vector (22) was kindly provided by Drs. K. Mikoshiba and A. Miyawaki (University of Tokyo, Tokyo, Japan). The 5Ј-untranslated region of the original p400C1 clone was removed by polymerase chain reaction (PCR) by amplification of the 5Ј-terminal part up to the Cel-II restriction site (nucleotide 555), before subcloning the IP 3 R-1 cDNA in the baculovirus (Autographa californica) transfer vector pVL 1393 (Invitrogen). Recombinant virus was produced in Spodoptera frugiperda (Sf9) cells by cotransfection of the pVL 1393 IP 3 R-1 construct and the linearized A. californica nuclear polyhydrosis virus DNA (BaculoGold, Pharmingen). The recombinant viruses were purified by isolating individual plaques of transfected cells. These clonal viral populations were amplified by infecting Sf9 cells. The recombinant protein was harvested