©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of a Stable Chinese Hamster Ovary Cell Line Overexpressing the Plasma Membrane Ca-ATPase (*)

Danilo Guerini , Stefan Schröder , Davide Foletti , Ernesto Carafoli (§)

From the (1)Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stable Chinese hamster ovary (CHO) cell lines overexpressing the human plasma membrane Ca-ATPase (PMCA) were generated, and three independent cell clones were characterized in details. They overexpressed high amounts of active PMCA pump (15-20 times over the amount of endogenous PMCA) as indicated by experiments in which the formation of the phosphoenzyme intermediate and the uptake of Ca by microsomes were measured. Immunocytochemistry experiments coupled to the biotinylation of the pump in the intact cells indicated the correct delivery of the expressed pump to the plasma membrane. The expressed pump was purified by affinity chromatography on calmodulin sepharose. The PMCA of transfected CHO cells promoted an increase of Ca into the medium, after induction of Ca release from the internal stores by activation of a purinergic receptor. An evident decrease of the activity of the endogenous sarcoplasmic reticulum Ca-ATPase pump was observed, probably related to the down-regulation of its expression. The cells overexpressing the PMCA pump had delayed recovery after trypsinization and plating. Their doubling time was, however, the same as CHO cells.


INTRODUCTION

The plasma membrane Ca-ATPase (PMCA)()(Carafoli, 1991, 1992) and the Na/Ca-exchanger are responsible for the ejection of Ca from eucaryotic cells (Carafoli, 1991). At variance with the Na/Ca-exchanger, which is predominantly present in excitable tissues (Blaustein et al., 1991), the plasma membrane Ca pump is present in all tissues studied (Carafoli and Guerini, 1993). An important difference between the two systems is the affinity for Ca. That of the pump is much higher, i.e. the pump is likely to be responsible for the fine regulation of intracellular Ca (Carafoli, 1991). An exact quantification of the respective role of the two systems is difficult, mostly because specific inhibitors are not available, as they are, for example for the sarcoplasmic reticulum Ca-ATPase (SERCA); thapsigargin has been important in the definition of the role of the system in the contraction of myocytes (Wrzosek et al., 1992) and of other systems. Most of the studies on the physiological role of the PMCA pump were performed on erythrocytes (Schatzmann, 1982), i.e. on a system lacking internal organelles and in which the PMCA is the only enzyme that pump Ca across the plasma membrane. The pump was first purified from erythrocytes, characterized biochemically (Carafoli, 1992), and eventually cloned (Shull and Greeb, 1988; Verma et al., 1988). It is encoded by four human genes (Strehler, 1991; Carafoli and Guerini, 1993), the number of possible isoforms being increased by a complex pattern of alternative splicing (Brandt et al., 1992; Keeton et al., 1993; Stauffer et al., 1993). The different isoforms are expressed in a striking tissue-specific way (Stahl et al., 1992; Stauffer et al., 1993).

To study the physiological function of the isoforms, it was necessary to develop systems which would allow their expression and eventually purification. Successful overexpression of the PMCA4CI (for the isoforms nomenclature, see Carafoli(1994)) has been achieved in different systems (Adamo et al., 1992a; Heim et al., 1992; Enyedi et al., 1993). They have permitted the study of the properties of this isoform and provided the tool for creating mutated variants (Enyedi et al., 1993, 1994). They were all based on the transient overexpression of the pump in COS cells or in viral systems (Baculovirus, Heim et al., 1992), i.e. they were unsuitable for the study of the long term physiological effects of pump overexpression.

To overcome the problem, stable CHO cells overexpressing the PMCA4CI isoform were generated after transfection with a glutamine synthetase-based vector (Cockett et al., 1990). Of the different clones which were isolated, three expressed high amounts of active PMCA pump and correctly delivered it to the plasma membrane. The cells were able to survive with a 15-20-fold excess of PMCA activity as compared with the parental cells. Interestingly, when grown to confluence they displayed a decreased level (4-5-fold) of the endogenous SERCA pump as compared with nontransfected cells. Although the duplication time of the transfected cells was similar to that of the nontransfected counterparts, they had a 12-16 h longer lag phase.


EXPERIMENTAL PROCEDURES

Materials

Glasgow minimal Eagle's medium, dialyzed FCS (fetal calf serum), antibiotics, and other cell culture media supplements were purchased from Life Technologies, Inc. (Basel, Switzerland). Other reagents were of the highest quality commercially available.

Methods

DNA Manipulations

The PMCA4CI was cloned in pTZ18/U between the BamHI and the KpnI sites. The resulting vector was cut with KpnI, blunt-ended with Klenow polymerase, and cut with XbaI (this site is present in the polylinker of pTZ18/U). The 3600-bp PMCA4CI cDNA fragment was ligated into the XbaI-SmaI cut pEE14 vector (Celltech Ltd., 4EN Berkshire, United Kingdom). The pEE14 vector contains the glutamine synthetase gene, which can be used for the selection of stable transfected cells (Cockett et al., 1990). The plasmid DNA was purified by CsCl gradient centrifugation prior to transfection (Sambrook et al., 1989).

Genomic DNA was prepared from cells according to standard procedures (Sambrook et al., 1989). It was digested twice overnight with the desired restriction enzyme, precipitated, and its concentration determined spectrophotometrically. Southern blotting was performed according to Sambrook et al.(1989) and hybridization was performed with a randomly primed labeled EcoRI-EcoRI 1200-bp fragment (corresponding to the nucleotides 2177-3403 of PMCA4CI, Strehler et al., 1990) under stringent conditions.

Isolation of Stable Transfected Cells

CHO.K1 cells (ATCC CCL 61) were maintained in Glasgow minimal Eagle's medium, 10% dialyzed FCS, 100 µg/ml gentamicin supplemented with amino acids, nucleotides, and pyruvate as described earlier (Cockett et al., 1990). Transfections were performed by the Ca(PO) method (Cockett et al., 1990) on semiconfluent CHO cells. One day after transfection, 25 µM MSX (methionine sulfoximide, Sigma) was added to the medium. Clones were collected and analyzed for the integration of the PMCA4CI gene. Positive clones were subjected to additional selection-amplification rounds at 150, 300, and 500 µM MSX.

Preparation of Membranes

Crude membranes were prepared as follows: cells were resuspended at 5-10 10 cells/ml in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml pepstatin after two washings with TBS (25 mM Tris-HCl, pH 7.4, and 150 mM NaCl), and subjected to three cycles of freeze and thaw. The particulate fraction was sedimented at 15,000 g for 15 min. In some case the supernatant was centrifuged for 1 h at 110,000 g. The resulting protein pellet was resuspended in 4 mM Tris-HCl, pH 8.0, 10% sucrose and frozen at -70 °C.

Microsomes were prepared as follows: after homogenization of the cells in 10 mM Tris-HCl, pH 7.4 (40 strokes of a Dounce homogenizer on ice), sucrose and NaCl were added to a final concentration of 10% and 150 mM, respectively, and centrifuged 5 min at 750 g. The postnuclear pellet was sedimented by 20-min centrifugation at 10,000 g. The microsomes were collected from the postnuclear supernatant by centrifuging it twice for 45 min at 100,000 g. The relative content of plasma membrane was determined by measuring the Na/K-ATPase activity in the preparation essentially as described by Jewell and Lingrel(1991). Following assay buffer was used: 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2 mM EGTA, 2 mM [-P]ATP (10-30 Ci/mmol) in the presence or in the absence of 5 mM ouabain.

Western Blotting

Proteins were separated by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) and transferred to nitrocellulose membranes (Towbin et al., 1979). The membranes were incubated with the polyclonal antibody 94.2 (diluted 1/500) or with the monoclonal antibody 5F10. After incubation with alkaline phosphatase-coupled secondary antibodies (Bio-Rad), the immunocomplexes were visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Bio-Rad).

Formation of the Phosphoenzyme Intermediate from ATP

Membrane proteins were resuspended in 20 mM MOPS-KOH, pH 6.8, 100 KCl in the presence of 100 µM Ca, 100 µM Ca plus 100 µM La, or 1 mM EGTA as described in the legend for the figures. The reaction was started by adding 0.2-2 µM [P]ATP (50-500 Ci/mmol) on ice and stopped 30 s later by the addition of 7% trichloroacetic acid. The proteins in the washed pellet were separated on acidic gels (Sarkadi et al., 1986), stained with Coomassie Brilliant Blue, dried, and exposed for 1-3 days at -70 °C.

Immunoprecipitation

200-250 µg of membranes were phosphorylated as described above in the presence of 0.2 µM ATP, 200 µM CaCl, and 200 µM LaCl. After precipitation with trichloroacetic acid, the pellet was washed with 200 µl of cold HO and resuspended in 200 µl of MEN buffer (50 mM MES-NaOH, pH 6.4, 150 mM NaCl, 1 mM EDTA, 0.2% gelatin, 0.1% NaN, 0.1% SDS, 0.1% Nonidet P-40). The resuspended proteins were centrifuged at 10,000 g for 30 min at 4 °C and divided in three aliquots: 20 µl were kept aside as control for the phosphoenzyme intermediate stability, two aliquots of 90 µl were diluted with 1 ml of MEN buffer and mixed with 2 µl of the monoclonal antibody 5F-10 (specific for the PMCA, Borke et al., 1989) or 4 µl of polyclonal serum of rabbits immunized against the SERCA2b pump (Wuytack et al., 1989). The samples were incubated under gentle rocking for 1 h at 4 °C, before addition of 15 µl of packed protein A-Sepharose (protein A-Sepharose CL-4B, Pharmacia Fine Chemicals, Uppsala, Sweden). After another 2-h incubation at 4 °C, the immunocomplexes bound to the protein A-Sepharose were recovered by centrifugation. The pellet was washed four times with MEN buffer, twice with 20 mM MOPS-KOH, pH 6.8, 0.1% Nonidet P-40, and finally twice with 20 mM MOPS-KOH, pH 6.8. The pellet was resuspended at room temperature for 30 min in 30-40 µl of 70 mM Tris-PO, pH 6.4, 5% SDS, 5% dithiothreitol and 8 M urea, separated on acidic gels (Sarkadi et al., 1986) and further processed as described above.

Ca Uptake

Microsomes were prepared as described above (see ``Preparation of Membranes''). The uptake of Ca was measured by the Millipore fast filtration method (Enyedi et al., 1993): 30-36 µg of microsomal proteins were resuspended in the uptake buffer (25 mM TES-KOH, pH 7.2, 100 mM KCl, 7 mM MgCl, 40 mM KHPO, 5 mM NaN, 4 µg/ml oligomycin, 0.5 mM ouabain, 200 nM thapsigargin and 200 nM calmodulin). The free Ca concentration was adjusted using the program of Fabiato and Fabiato (1979). Immediately after the addition of Ca (2-3 10 cpm/ml), the uptake was initiated by the addition of 6 mM ATP. At given times, 100 µl of the mixture were rapidly filtered through 0.45-µm nitrocellulose filters (Millipore SA, Molsheim, France), which were washed four times with cold 150 mM KCl and 1 mM CaCl. The filters were then counted in a Beckman scintillation counter.

Indirect Immunofluorescence Microscopy

The cells were plated on coverslips and analyzed by indirect immunofluorescence microscopy 48-60 h after plating. The cells were washed twice with PBS (phosphate-buffered saline, 150 mM NaCl, 20 mM NaHPO, pH 7.4, 0.1 mM CaCl, 0.1 mM MgCl), fixed for 20 min in 3% paraformaldehyde, washed four times with PBS, and then incubated for 30 min in 0.1 M glycine. After four washes with PBS, the cells were permeabilized in 0.1% Triton X-100 for 3 min, washed four times with PBS, and incubated in blocking buffer (5% FCS, 0.1% bovine serum albumin, 5% glycerol, 0.04% NaN in PBS) for 1 h.

The coverslips were overlaid with the primary antibody (antiserum p94.2) diluted 1:50 in blocking buffer. After five washes with the blocking buffer, the cells were treated with the secondary antibody for 1 h (swine anti-rabbit fluorescein-conjugated antibodies diluted 1:30 to 1:50 in blocking buffer, Dako A/S, Glostrup, Denmark). The coverslips were washed five times with blocking buffer prior to mounting in a medium containing 80% glycerol, 2.5% DABCO (2,4-diazabicyclo-[2,2,2]-octan) in PBS, pH 8.0. The cells were observed in an Axiovert 10 microscope (Carl Zeiss, Oberkochen, Germany) equipped with epifluorescence illumination and a 63 oil immersion plan-neofluar objective, and photographed with HP5 Ilford films.

Biotinylation

Cells were metabolically labeled with [S]methionine for 2 h in MEM, supplemented with nonessential amino acids and 150 µCi[S]methionine/ml. After the labeling the cells were rinsed twice with TBS and once with borate buffer, pH 9.0 (10 mM sodium borate, 154 mM NaCl, 12 mM KCl, 2.25 mM CaCl). Biotinylation with NHS-SS-Biotin (sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate, Pierce, 0.5 mg/ml in borate buffer), was carried out twice for 15 min (1.5 ml/100 mm plate). After two washes with borate buffer, free biotin was blocked with 50 mM NHCl/PBS for 10 min. The cells were rinsed twice again with borate buffer and solubilized for 1 h in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.2% bovine serum albumin, 75 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml antipapain, 5 µg/ml pepstatin, 5 µg/ml leupeptin). The cell lysate was centrifuged for 30 min at 15,000 g and the supernatant was rocked overnight with 2 µl monoclonal antibody 5F10. 50 µl of protein A-Sepharose CL-4B were added to the mixture, and the incubation was carried on for 8 additional h before washing the beads six times with lysis buffer. To recover the immunoprecipitated biotinylated antigens, the beads were boiled twice for 3 min with 20 µl of 10% SDS, diluted with lysis buffer (1000 µl/tube), and centrifuged 2 min at 15,000 g. An aliquot of the supernatant (one-half of the recovered material) was taken as the immunoprecipitated, non-avidin-precipitated material. The remainder of the recovered material was incubated overnight with 50-µl avidin-agarose beads (50% aqueous slurry, 1-2 mg of avidin/ml of gel, Pierce). The beads were washed as described above and boiled in SDS-PAGE sample buffer. Immunoprecipitates and avidin precipitates were analyzed by gel electrophoresis and autoradiography.

Purification of the ATPase

The Ca-ATPase was purified by affinity chromatography on calmodulin-Sepharose, according to the procedure described by Niggli et al.(1979).

Ca Efflux Experiments

Cells were plated on 12-well plates (Costar Corp., Cambridge, MA) and let recover for 2-3 days at 37 °C until they reached a density of 2 to 5 10 cells/well. The medium was replaced by S-MEM medium (Life Technologies, Inc.), a modification of MEM with lower calcium content. After two washes with S-MEM, the cells were incubated in the same medium for 2-4 h at 37 °C in the presence of 1.4 10 cpm Ca/well (7 10 cpm/ml). The 12-well plates were transferred to room temperature, and the cells were quickly washed with S-MEM containing 1 mM CaCl. After the addition of S-MEM, 1 mM CaCl, 1 mM ATP, and 10% FCS, aliquots were withdrawn from the supernatant and counted. The radioactivity remaining in the pellet was measured after solubilizing the cells in 10 mM Tris-HCl, pH 8.0, and 1% SDS. The percentage of Ca released per unit of time was calculated from the total radioactivity in the cells before the induction of Ca efflux.

Cell Growth Assay

Cells were plated in a 24-well plate at a density of 2.4-2.6 10 cells/well. They were trypsinized and counted at regular intervals. Their integrity was checked by incubating them with trypan blue (Sigma). The cells were plated in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 35 mg/liter proline in the presence of different concentrations of FCS. The growth curves obtained with other media were qualitatively identical.


RESULTS

Generation and Selection of CHO Stable Cell Lines Expressing the PMCA Pump

The primary screening yielded about 30 MSX-resistant clones, 20 of which were analyzed for the presence of the PMCA4CI cDNA by Southern blotting. Four clones giving strong positive signals were subjected to additional rounds of selection at a higher concentration of MSX. Fig. 1shows a Southern blot with three clones: two isolated at 150 µM MSX (CHO-9-150 and CHO-10-150, lanes 2 and 4, respectively) and one (CHO-10-25, lane 3), at lower MSX concentration. Lanes 2-4 hybridized strongly with a EcoRI-EcoRI 1200-bp human PMCA4CI cDNA probe; the radioactive band was absent from nontransfected CHO cell (Fig. 1, lane 1). A calibration with known amounts of DNA (Fig. 1, lanes 5 and 6) indicated that the cells of the CHO-9-150 clone contained 10 copies of PMCA4CI-cDNA in their genome, that of clones CHO-10-150 and CHO-10-25, four to six copies. Additional clones (CHO-1 and CHO-3, not shown) were positive in dot-blot hybridization experiments, but showed a complex banding pattern on Southern blotting, indicating rearrangement in the coding sequence of PMCA4CI during the transfection.


Figure 1: Southern blot of genomic DNA. Genomic DNA was prepared from control CHO cells and the stable transfected cells, digested with EcoRI, and transferred to Nytran filters. The filters were hybridized with a random primed labeled 1200-bp EcoRI-EcoRI PMCA4CI-specific DNA fragment under high stringency conditions (Sambrook et al., 1989). 5 µg of genomic DNA from CHO cells (lane 1), CHO-9-150 (lane 2), CHO-10-25 (lane 3), and CHO-10-150 cells (lane 4). To estimate the copy number of PMCA4CI cDNA integrated in the cells, the amount corresponding to four (lane 5) or eight (lane 6) copies of PMCA4CI/cell was separated, transferred, and hybridized in parallel. The autoradiogram was exposed for 40 h at -20 °C. The densitographic scanning of the 1200-bp radioactive band yielded the following values. Lane 2, 530,000 ± 30,000 scanning units; lane 3, 290,000 ± 20,000 scanning units; lane 4, 300,000 ± 30,000 scanning units; lane 5, 220,000 ± 10,000 scanning units; and lane 6, 480,000 ± 20,000 scanning units.



Western Blotting Analysis of the Positive Clones

Membrane proteins from the three Southern blot-positive clones were prepared and analyzed with antibodies specific for the PMCA4CI pump. A C-terminal PMCA4CI-specific antibody yielded a strongly positive band at the expected molecular mass range (Fig. 2A, lanes 3-5), which migrated as the PMCA protein from erythrocytes (Fig. 2A, lane 1). The band at 60 kDa resulted from an unspecific reaction of the serum, since it was also present in control CHO cells (Fig. 2A, lane 2). The overexpressed pump was also recognized by the 5F-10 monoclonal antibody (Fig. 2A, lanes 6-7, CHO-9-150 and CHO-10-25 gave similar signals, not shown). It is important to emphasize that the amount of PMCA4CI pump present in the crude membrane protein preparations of the stable transfected cells was at least eight times higher than in the membrane of erythrocytes. The level of expression was highest in CHO-10-150 cells (Fig. 2A, compare lanes 3 and 4 with lane 5).


Figure 2: Detection of the PMCA4CI protein by Western blotting and stability of the overexpressing cell lines. A, crude membrane proteins were separated by SDS-PAGE according to Laemmli (1979) and transferred to nitrocellulose membranes. 10 µg of membrane proteins from normal CHO cells (lanes 2 and 6), CHO-9-150 (lane 3), CHO-10-25 (lane 4), and CHO-10-150 cells (lanes 5 and 7) were loaded on the gels. As a control 60-70 µg of erythrocyte membrane proteins were used (lane 1). The nitrocellulose sheets were incubated with the polyclonal antibody 94.2 (specific for the C-terminal portion of PMCA4CI, D. Guerini unpublished observation) (lanes 1-5) or with monoclonal antibody 5F-10 (Adamo et al., 1992b) (lanes 6 and 7). B, the stability of the overexpression was tested on 30 µg of total cell protein. The cells were washed in TBS before denaturing them in SDS-loading buffer. The PMCA4CI protein was detected with the polyclonal antibody 94.2. The CHO-10-150 cells were passed 40-50 times in the presence of MSX (the selection drug) (lane 1), 40 times with MSX followed by four passages without MSX (lane 2), or 40-50 times without MSX (lane 3). Nontransfected CHO cells are shown in lane 4. 50-60 µg of erythrocytes ghosts were used as a control (lane 5).



To test the stability of the expression level, the stable transfected cells were maintained for 50 passages (up to 6 months) in tissue culture with and without addition of the selection drug MSX. The amount of overexpressed pump was assessed by Western blotting. Culturing the cells for a short time without selection didn't significantly affect the amount of expressed pump (Fig. 2B, lanes 1 and 2), but after 30-40 passages a substantial decrease became evident (Fig. 2B, lane 3). The expression level of the pump in cells maintained in the selection media for as long as 6 months remained constant.

Localization of the Overexpressed Protein

The overexpressed pump was found to be targeted to the plasma membrane of COS cells (Zvaritch et al., 1992) but other experiments indicated that a significant fraction of it was apparently retained in the endoplasmic reticulum (Adamo et al., 1992a). The latter result may have been due to overexpression, causing a saturation of the sorting machinery and accumulation of the pump in the endoplasmic reticulum. Immunocytochemistry was therefore performed with the stable transfected CHO cells. Strong signals were detected for the CHO-9-150 cells (Fig. 3a, D), the staining being concentrated on the cell surface. No reticular staining was ever observed, indicating that most of the expressed pump was correctly delivered to the plasma membrane. This was also true for the stable cell lines CHO-10-25 and CHO-10-150 (not shown). Not all the cells showed the same intensity of staining, but a reasonable number (more than 50-60%) had a similarly strong signal. The differences with the remainder of the cells could have been due to the incomplete permeabilization of the cells, which was necessary due to the cytoplasmic localization of the epitope. Inhomogeneity of staining was also observed by others with stable transfected CHO cells (Hussain et al., 1992).


Figure 3: Localization of the overexpressed pump protein. a, immunocytochemistry was performed as outlined under ``Experimental Procedures.`` The experiments shown in the figure have been carried out with non transfected CHO cells (A, B) and with CHO-9-150 cells (C, D). (Identical results were obtained with the CHO-10-25 and CHO-10-150 cells). A and C, phase contrast photograph; B and D, immunofluorescence after incubating the permeabilized cells with the polyclonal antibody 94.2. b, surface labeling of the CHO cells. Cells were labeled with [S]Met, treated with NHS-SS-biotin as described under ''Experimental Procedures`` prior to immunoprecipitation with the 5F-10 monoclonal antibody. Half of the immunoprecipitated sample was separated on the gel (lanes 1 and 3). The other half was precipitated with avidin beads before loading into the gel (lanes 2 and 4). The experiment was performed with CHO-10-150 cells (lanes 1 and 2) and nontransfected CHO cells (lanes 3 and 4). The dried gel was exposed at -70 °C for 2-3 days. Exposure of up to 7 days failed to reveal radioactive bands in lane 4.



Biotinylation experiments (Fig. 3b) confirmed that the PMCA4CI protein was localized in the plasma membrane. After immunoprecipitation a large amount of the pump protein was bound by the streptavidin beads, indicating that the protein had become labeled in the intact cells (Fig. 3b, lane 2). The monoclonal antibody 5F10 (recognizing PMCA from different species, Borke et al., 1989) was used for these experiments to detect also the endogenous PMCA protein in nontransfected CHO cells (Fig. 3b, lane 3). In spite of the much longer exposure time of the autoradiogram, the amount of biotinylated endogenous PMCA was evidently too low to be detected with the protocol used (Fig. 3b, lane 4). The experiments thus supported the conclusion (Fig. 3a) that the largest fraction of the overexpressed PMCA4CI pump was correctly targeted to the plasma membrane.

Formation of the Phosphorylated Intermediate by the Overexpressed Pump

The formation of the phosphorylated intermediate of the reaction cycle was studied on microsomes or crude membrane preparations from CHO and CHO-10-150 cells. The relative content of plasma membrane was estimated by measuring the endogenous Na/K-ATPase activity (see ``Experimental Procedures''). The membranes used for the phosphoenzyme assay (preparations similar to the ones used for the experiment in Fig. 4A) didn't show any significant difference between membranes from CHO cells (380 ± 45 nmol P/mg proteinh) and CHO-10-150 cell line (410 ± 40 nmol P/mg proteinh). The experiment was performed in the presence of Ca/La to optimize the detection of the PMCA pump intermediate (Fig. 4A, lanes 1 and 4). The intermediate formed by membranes from CHO-10-150 was 10-15 more abundant than that of membranes from nontransfected cells (Fig. 4A, compare lanes 1 and 4). Densitometric scanning of gels similar to that shown in Fig. 4A resulted in a value of 17,000 scan units for the 135-kDa band in lane 1 and of 370,000 scan units for the corresponding band in lane 4. As expected, the intermediate was sensitive to NHOH (Fig. 4A, lanes 3 and 6) and was not seen in the presence of EGTA (Fig. 4A, lanes 2 and 5). The radioactive band comigrated with the corresponding pump band of erythrocytes (Fig. 4, lane 7), i.e. it indeed corresponded to the phosphoenzyme intermediate of the PMCA pump. Interestingly, the amount of intermediate formed by the endogenous SERCA pump (the radioactive band at 100 kDa) was reproducibly lower in membrane preparations of CHO-10-150 cells than in control cells (Fig. 4A, compares lanes 1 and 4). Experiments with different membrane preparations were performed to test if the ``artifactual'' enrichment of a given membrane fractions was responsible for the observation: however, in all cases the amount of SERCA intermediate in cells overexpressing the PMCA4CI pump was lower than in control cells. This was found to be true for all three stable transfected cell lines (not shown). To rule out possible artifacts related to the ATP concentration used, experiments were performed with different concentrations of the latter (Fig. 4B). A total membrane fraction was used in this case (see ``Experimental Procedures''), since the difference in the amounts of the SERCA phosphoenzyme between transfected and nontransfected cells was particularly evident in it. The results of Fig. 4B (lanes 1-6) demonstrate that the difference in the amount of the SERCA intermediate (the radioactive band at 100 kDa) between transfected and nontransfected cells was unrelated to the ATP concentration. Since La inhibits slightly the formation of the SERCA intermediate (Carafoli and Guerini, 1993), the phosphorylation reaction was also performed in the absence of La. Under these conditions the plasma membrane intermediate was not visible (Fig. 4B, lanes 7 and 8), but the amount of the SERCA intermediate in CHO-10-150 cells (Fig. 4B, lane 8) was still lower than in control cells (Fig. 4B, lane 7). It was important to demonstrate that the 100-kDa band was indeed the SERCA pump. Addition of La in the assay buffer resulted in the appearance of the PMCA-specific phosphoenzyme (Fig. 4B, lane 10). A weaker band at the same molecular mass was also present in the lane corresponding to the CHO cells (Fig. 4B, lane 9, compare also Fig. 4A, lane 1 and Fig. 4B, lane 1). Under the same conditions (+La), the 100-kDa band became weaker (Fig. 4B, lanes 9 and 10), which is a property of the phosphoenzyme intermediate of the SERCA pump. The 100-kDa band was sensitive to thapsigargin, a well known inhibitor of the SERCA pump, whereas the 135-kDa (PMCA) band was not (Fig. 4C, lanes 1 and 2). Additionally proofs that the 100-kDa band corresponded to the phosphoenzyme intermediate of the SERCA were obtained by immunoprecipitation experiments (Fig. 4C, lane 3-5). Immunoprecipitation with a SERCA 2b-specific antibody resulted in the almost quantitative recovery of the 100-kDa radioactive protein (Fig. 4C, lane 5). The PMCA-specific monoclonal antibody precipitated only the 135-kDa protein (Fig. 4C, lane 4). No bands around 100 kDa were ever observed. Since the 5F-10 monoclonal antibody recognized an epitope present in the C-terminal end of the big intracellular loop (amino acids 724-758) of all the PMCA isoforms (Adamo et al., 1992b), proteolytic fragments bigger than 90 kDa should have been recognized by it. The lower amount of the PMCA phosphoenzyme intermediate present before immunoprecipitation (Fig. 4C, lane 3) was due its low solubility in MEN buffer after the trichloroacetic acid precipitation. Addition of dithiothreitol and urea improved the solubilization, but no immunocomplexes could be formed under these conditions. Similar experiments were performed with nontransfected CHO cells, giving essentially the same results (not shown).


Figure 4: Activity measurements on the pump overexpressed in the CHO cells. Formation of the phosphoenzyme intermediate. A, 20-25 µg of microsomal proteins from control CHO cells (lanes 1-3) and from CHO-10-150 cells (lanes 4-6), and 70 µg of erythrocyte membrane proteins (lane 7) were phosphorylated with 0.4 µM [-P]ATP (200-300 Ci/mmol) as described under ''Experimental Procedures.`` The phosphorylation reaction was performed in the presence of 100 µM Ca and 100 µM La (lanes 1, 4, and 7) or in the presence of 1 mM EGTA (lanes 2 and 5). The samples in lanes 3 and 6 were phosphorylated like those of lanes 1 and 4 before incubating them for 30 min at room temperature with 200 mM NHOH. The proteins were separated by acidic gels after trichloroacetic acid precipitation (Sarkadi et al., 1987). The dried gels were exposed for 48 h at -70 °C. B, 25-30 µg of total crude membrane proteins from control CHO cells (lanes 1-3, 7, and 9) and from CHO-10-150 cells (lanes 4-6, 8, and 10) were phosphorylated under different conditions. Phosphorylation in lanes 1-6 was performed at different concentrations of ATP in the presence of 100 µM Ca and 100 µM La. The following ATP concentrations were used: lanes 1 and 4, 0.2 µM [-P]ATP (500 Ci/mmol); lanes 2 and 5, 0.5 µM [-P]ATP (200 Ci/mmol); lanes 3 and 6, 2 µM [-P]ATP (50 Ci/mmol). Phosphorylation in lanes 7 and 8 were done in the presence of 0.3 µM [-P]ATP (300 Ci/mmol) and 100 µM Ca. Under these conditions (absence of La) the phosphoenzyme of the PMCA pump was not visible. The scanning of the autoradiogram yielded the following results: lane 7, 130,000 ± 20,000 scanning units for the 100-kDa band; lane 8, 32,000 ± 4000 scanning units. Phosphorylation in lanes 9 and 10 was done in the presence of 0.3 µM [-P]ATP (300 Ci/mmol), 100 µM LaCl, and 100 µM CaCl. Thapsigargin sensitivity and immunoprecipitation of the phosphorylated intermediates. C, 25-30 µg of CHO-10-150 cells membranes were phosphorylated in the presence of 100 µM LaCl, 100 µM CaCl, and 0.3 µM [-P]ATP (300 Ci/mmol) (lanes 1 and 2). Prior to the phosphorylation reaction, the membranes were treated with (lane 1) or without (lane 2) 2 µM thapsigargin, 10 min at 37 °C in the presence of 100 µM ATP. The membranes were sedimented, washed twice in 20 mM MOPS-KOH, pH 6.8, and 100 mM KCl before the phosphorylation reaction. The immunoprecipitation experiment (lanes 3-5) was performed as described under ''Experimental Procedures.`` Lane 3, membrane proteins after the phosphorylation reaction and solubilization in MEN buffer but before immunoprecipitation (corresponding to 20 µg of membrane protein); lane 4, phosphorylated proteins immunoprecipitated by the 5F-10 (PMCA specific) antibody (corresponding to 100-130 µg of membrane proteins); lane 5, phosphorylated bands immunoprecipitated with the polyclonal antibody against the SERCA2b pump (corresponding to 100-130 µg of membrane proteins). D, Ca uptake by microsomes. Ca uptake was measured at a free Ca concentration of 2.2 µM as described under ''Experimental Procedures.`` Microsomes obtained from the CHO-9-150 () and control CHO cells () were used. Each point is the average of at least three measurements. In the case of the CHO-9-150 cells the uptake was studied in the presence () and in the absence of ATP (). The Ca dependence of the Ca uptake is shown in the inset. The free Ca concentration was determined according to the program of Fabiato and Fabiato (1979). The uptake was measured 10 min after the addition of ATP. 100% of the Ca transport was the maximal rate obtained at 2.2 µM Ca., CHO-9-150 cells; &cjs2123;, CHO-10-150 cells.



Ca Uptake by Microsomes Obtained from the Stable Transfected CHO Cells

Fig. 4D shows the uptake of Ca by microsomes obtained from CHO-9-150 and control cells. The fractionation scheme described under ``Experimental Procedures'' produced a fraction that contained, in addition to vesicles of the endoplasmic reticulum, a significant amount of inside/out plasma membrane vesicles. The specific activity of the Na/K-ATPase in these membranes was 1-2.2 µmol of P released per mg proteinh. No significant difference between control and stable transfected cells was observed. In both cases the uptake was linear for the first 20-25 min, after which saturation (in the case of the CHO-9-150 cell at 200 nmol of Ca/mg of microsomal protein) was reached. The uptake by CHO-9-150 cell microsomes, 8 nmol of Ca/mgmin was 8-10 times higher than that of nontransfected CHO cells. The uptake by microsomes from CHO-10-150 cells was even higher and amounted to 14.8 nmol of Ca/mg of microsomal proteinmin. This result was in good agreement with those of the phosphorylation experiments (see above), were a 15-fold increase of ATPase activity was also seen.

The Ca dependence of the uptake process was also determined. Fig. 4D (inset) shows that in all cases the K for Ca was about 0.3 µM, in good agreement with the value obtained for the purified pump (Niggli et al., 1981)

Ca Release from the Cells

The efflux of Ca was studied in nontransfected CHO cells and from CHO-10-25 and CHO-10-150 cells. Cells were passively loaded with Ca by incubating them for some hours at 37 °C, and the release was started by the addition of ATP and serum; ATP has been found to induce a transient increase of Ca in CHO cells, which normally lasts for 30-50 s (Pijuan et al., 1993). Fig. 5shows that under these conditions higher amounts of Ca were released by the CHO-10-25 and CHO-10-150 cells 10 and 40 s after the addition of ATP (14-17% of the total) as compared to nontransfected CHO cells (7-8%). The increased efflux lasted about 60 s, the difference with control cells disappearing at later times. Experiments on passive Ca accumulation or on passive Ca release, i.e. cells were treated as described above, but after transferring them to the release medium (see ``Experimental Procedures''), no ATP and no serum were added, showed that cells overexpressing the PMCA pump behaved as control CHO cells (not shown).


Figure 5: Ca release from the cells. Cells were plated in 12-well plates and loaded with Ca (see ''Experimental Procedures``). After washing the cells, the efflux from them was started by the addition of ATP and FCS. Aliquots of the reaction medium were withdrawn and counted. The values reported in the figure are the average of eight measurements and represent the amount of Ca released after the time indicated. 100% refers to the total amount of Ca in the cells at the beginning of the efflux measurements. , CHO; , CHO-10-25; , CHO-10-150.



Growth of the Stable Transfected Cells

It was noticed during routine manipulation of the cells that those expressing high levels of PMCA were not growing as fast as control cells or transfected cells which were overexpressing lower amounts of the PMCA pump. Different growth conditions were thus analyzed. Fig. 6A shows that nontransfected and transfected cells were unable to grow at a concentration of FCS below 2%. In the presence of 5 or 10% FCS, nontransfected CHO cells grew reproducibly faster than the CHO-9-150 and CHO-10-150 cells. The semilogarithmic plot of Fig. 6A shows that CHO-10-150 and CHO-9-150 cells had a growth delay in the first 12-24 h. This behavior persisted if the cells were grown in different media, provided that adequate amounts of FCS were present. The growth delay was not seen in MSX-resistant clones which were not expressing the plasma membrane ATPase. In the inset of Fig. 6B the duplication time was calculated from the curves between 40 and 140 h after plating; the values were very similar for all cell lines, indicating that once the initial lag was over, the stable transfected cells behaved like nontransfected CHO cells. Thus cells overexpressing the PMCA4CI pump apparently needed a longer ``recovery'' time after trypsinization and diluting in fresh medium.


Figure 6: Growth curve of the cells. A, the growth curve of control CHO cell (circles), CHO-10-150 cells (squares), and CHO-9-150 cells (triangles) was determined at four different FCS concentrations. (filled circles, triangles, and squares, 1%; diagonally slashed open circles, triangles, and squares, 2%: dotted circles, triangles, and squares, 5%; and open circles, triangles, and squares, 10%). The values reported are the average of two to three independent measurements. B, some of the data of A (left: curve with 10% FCS; right, curve with 5% FCS) are represented in a semilogarithmic diagram. The duplication times of the cells are given in the insets and were calculated between 40 and 140 h.



Purification of the Pump from the CHO Cells

The overexpressed plasma membrane Ca-ATPase was purified to essential homogeneity from CHO-10-150 cells by affinity chromatography on calmodulin-Sepharose 4B (Fig. 7, lane 2). The preparation showed a major band at 135 kDa and minor contaminants at 100 and 60 kDa (Fig. 7, lane 2). Some of the latter were also present in a similar preparation from nontransfected CHO cells (Fig. 7, lane 1). In the latter no detectable band at 135 kDa was detected, again showing that the endogenous plasma membrane pump is exceedingly low in CHO cells.


Figure 7: Purification of the PMCA4CI pump from CHO-10-150 cells. Membrane proteins (control CHO and CHO-10-150 cells) were solubilized and incubated with calmodulin-Sepharose 4B, essentially as described previously (Niggli et al., 1979). Aliquots were separated by SDS-PAGE and visualized by silver staining (Merryl et al., 1981). Fractions were eluted from calmodulin-Sepharose with EDTA. Lane 1, control CHO cells; lane 2, CHO-10-150 cells. Molecular weight markers are shown in lane 3.




DISCUSSION

All three stable CHO cell clones described expressed high levels of the PMCA4CI. Numerous attempts were made to obtain cell clones expressing still higher levels of PMCA, but the upper limit was apparently that of the CHO-10-150 clone, whose level of expression was at least 15 times higher than that of the parental CHO cells; similar levels of pump expression had been previously obtained by transfecting CHO cells with the SERCA pump cDNA (Hussain et al., 1992), although a different selection system had been used. The three clones were isolated independently, i.e. they resulted from the incorporation of the cDNA at different sites as indicated also by the fact that no correlation was found between the copies of PMCA4CI cDNA incorporated and the amount of expressed pump. The experiments presented here and those on the SERCA pump (Hussain et al., 1992) thus demonstrate that CHO cells can tolerate high amounts of active Ca pumps. Stable transfected cells produced more PMCA than transiently transfected ones (generally three to six times PMCA activity over the background is observed in COS cells (Heim et al., 1992; Enyedi et al., 1993)). An advantage of the stable transfection system is that all the cells express similarly high amounts of protein, whereas after transient transfection only a smaller proportion of the cells (5-10%, Zvaritch et al., 1992) expressed it. In addition, cells stable transfected with the SERCA pump showed homogenous distribution of the expressed protein in the membranes, at variance with transiently transfected COS(1) cells, where only a small fraction of the membranes contained high amounts of the pump (Hussain et al., 1992); this resulted in a fast rising concentration of luminal Ca in the vesicles and in the inhibition of the pump activity. Although the duplication time of cells overexpressing the PMCA pump was not different from that of the parental cells, all clones had slower growth recovery after trypsinization and plating. This was not related to the presence of glutamine synthetase in their genomes, since clones of MSX-resistant CHO cells transfected with glutamine synthetase had normal growth pattern.

Trypsinized cells loose all the attachment points to the plastic bottles. After replating, they attach to the surface and begin to crawl, a process in which Ca plays an important role (Stossel, 1993). It is also known that addition of FCS containing a variety of hormones and growth factors activates different signaling pathways which are responsible for the reshaping of the cells (Stossel, 1989, 1993). Cells overexpressing the PMCA protein evidently extruded higher amounts of Ca and thus lowered it in the cytosol at a faster rate. Possibly, this disturbed the FCS mediated reshaping of the cells.

The overexpressed pump was correctly delivered to the plasma membrane. This was in agreement with earlier results by Heim et al. (1992) and Zvaritch et al.(1992), but at variance with those by Adamo et al. (1992a). The very high level of pump expression in the latter study may have overloaded the protein targeting system, leading to the trapping of part of the overexpressed pump in the endoplasmic reticulum. In the experiments reported here the enrichment of the PMCA pump in the stable CHO cell lines was about 15-fold, whereas in the transiently transfected COS cells, the overexpression factor (assuming a transfection efficiency of 10% and a total overexpression of six times) was probably much higher.

An interesting, but intriguing, property of the stable transfected cells was their lower amount of active SERCA pump. Whether the latter was specifically down-regulated at the level of expression or whether the observed decrease of activity was related to other unknown mechanisms cannot be decided at the moment. However, it may be added that the decrease was observed under different experimental conditions and was independent of the method used to prepare the membranes. The regulation of the expression of the SERCA genes is an open problem; non-muscle tissues express mostly the SERCA2 and SERCA3 genes (Bobe et al., 1994; Wuytack et al., 1994), and indications have been presented that the SERCA3 gene can be up-regulated in platelets of hypertensive rats (Bobe et al., 1994). Experiments with CHO cells transfected with the SERCA DNA have shown that they were able to grow in the presence of higher concentration of thapsigargin than nontransfected cells (Gutheil et al., 1994). When exposed for long times to increasing concentrations of thapsigargin, they expressed higher amount of the SERCA pump (and also of the P-glycoprotein). This indicated that cells can regulate the expression of the SERCA pump in response to external influences. It would be tempting to propose that the factor regulating the pump expression processes is Ca itself. It would be interesting to see whether a similar phenomenon occurs in cells overexpressing the other system exporting Ca from the cytosol, i.e. Na/Ca exchanger.

Attempts to determine the in vivo membrane topology of the PMCA pump in red blood cells were hampered by the low amount of endogenous pump protein (between 0.1 and 0.01% of the total membrane protein). It was nevertheless possible to experimentally demonstrate the existence of the first predicted cytosolic loop between transmembrane domains 1 and 2 (Feschenko et al., 1992). The stable transfected CHO cells overexpressed the PMCA pump in a form which apparently was topologically correct, e.g. the protein could be labeled from the extracellular side with high efficiency. These cells will thus be a useful tool in experiments on the membrane topology of the PMCA. It may be added that CHO cells can be adapted to grow in suspension cultures (Cockett et al., 1990), i.e. it would in principle be possible to scale up of the production the PMCA4CI isoform.


FOOTNOTES

*
The work has been made possible by the financial contribution of the Swiss National Science Foundation Grant 31-30858.91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Biochemie III, ETH-Zentrum, Universitätstr. 16, CH-8092 Zürich, Switzerland. Tel.: 41-1-632-30-11; Fax: 41-1-632-12-13.

The abbreviations used are: PMCA, plasma membrane Ca ATPase; FCS, fetal calf serum; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; SERCA, sarcoplasmic/endoplasmic reticulum Ca ATPase; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; bp, base pair(s); CHO, Chinese hamster ovary; MSX, methionine sulfoximide; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; MEM, minimal essential medium; S-MEM, minimal essential medium modified for suspension cultures.


ACKNOWLEDGEMENTS

We thank Dr. J. T. Penniston (Rochester, MN) for the gift of the 5F10 monoclonal antibody, Dr. F. Wuytack (Leuven, Belgium) for the polyclonal antibody against the SERCA2b, and Thees Breyhan (ETH, Zürich, Switzerland) for the help in performing some of the experiments. We would also like to thank Celltech Ltd. for providing the pEE14 vector.


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