of chemistry no. 16, in u. s. a. expression of an ...we have used a gene transfer system to...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 16, Issue of June 5, pp. 7726-7733,1988 Printed in U. S. A.

Expression of an Ouabain-resistant Na,K-ATPase in CV-1 Cells after Transfection with a cDNA Encoding the Rat Na,K-ATPase a1 Subunit*

(Received for publication, October 13, 1987)

Janet Rettig EmanuelS, John Schulzj, Xiao-Mai Zhouj, Rachel B. Kent711 , David HousmanV, Lewis Cantleyg, and Robert Levenson*** From the $Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, the §Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, and the llCenter for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K- ATPase a1 subunit gene and ouabain-resistant Na,K- ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase a1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2al) conferred resistance to 100 PM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase a1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive “Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K- ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATP- ase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat a1 subunit gene is directly responsible for ouabain- resistant Na,K-ATPase activity in transfected CV- 1 cells.

The cardiac glycoside ouabain is a specific inhibitor of the Na,K-ATPase (l), the enzyme primarily responsible for the active transport of Na+ and K’ in most animal cells. Cell lines derived from different species vary considerably with respect to ouabain sensitivity. Rodent cells are commonly resistant to ouabain concentrations of at least 100 pM while primate cells are generally sensitive to concentrations of ouabain of 10 nM (2-4). For this reason, resistance to ouabain has long been used as a selection strategy in somatic cell genetics (3). Because ouabain cytotoxicity is attributable in all cases to

*This work was supported in part by Grants CA-26712 to the Massachusetts Institute of Technology Center for Cancer Research (R. Hynes, Principal Investigator), GM-36133 (to L. C.), and CA- 38992 from the National Cancer Institute and grants from the March of Dimes and the American Heart Association (to R. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 Supported by Public Health Service Grant CA-07919 from the National Cancer Institute.

** Established Investigator of the American Heart Association. To whom correspondence should be addressed.

inhibition of Na’ and K’ transport, the variation in ouabain sensitivity has been ascribed to species-specific differences in the structure of the Na,K-ATPase (5). However, the complex subunit structure of the enzyme (6) and the existence of alternative isoforms of the Na,K-ATPase (7) have left the molecular basis for the differences in ouabain sensitivity between rodent and primate cells uncertain. Furthermore, the identification of a murine gene which can confer ouabain resistance to primate cells but does not encode either the a or /3 subunit of the Na,K-ATPase (8, 9) has made it difficult to delineate those rodent sequences which are directly responsible for ouabain resistance.

The Na,K-ATPase has been shown to consist of two subunits. The a subunit contains the catalytic site for ATP hydrolysis (6). The function of the @ subunit has not yet been established. Ouabain is believed to bind to the a subunit of the enzyme (10). Two biochemically distinct forms of the Na,K-ATPase a subunit (a and a+) have been identified in the rat (7, 11). These differ both in sensitivity to ouabain and in tissue distribution. The a form, a polypeptide of M, 112,573 (12), is the predominant species found in kidney and is relatively resistant to ouabain (7). The a+ form, a polypeptide of M, 111,736 (12), is considerably more sensitive to ouabain (7) and has been detected in brain (7), adipose tissue ( l l ) , and skeletal muscle (11). The tissue distribution and ouabain sensitivity of a third a subunit isoform (aIII; M, 111,727) (12) has not been determined as yet.

Recent work has shown that the Na,K-ATPase a subunit isoforms are encoded by a multigene family (13, 14). Three separate a subunit genes have been localized to different chromosomes in the mouse (14). The a1 or a subunit gene (Atpa-l) is located on chromosome 3; the a2 or a111 subunit gene (Atpa-2) maps to chromosome 7; the a3 or a+ subunit gene (Atpa-3) is located on chromosome 1. By using somatic cell hybrids, Kozak et al. (15) had previously assigned the gene responsible for ouabain resistance to mouse chromosome 3. The assignment of the Na,K-ATPase a1 subunit gene to chromosome 3 (14) suggests that this concordance reflects the encoding of a ouabain-resistant a subunit by the Atpa-1 locus on mouse chromosome 3. The direct transferance of ouabain resistance to African green monkey CV-1 cells by a murine Na,K-ATPase a1 subunit cDNA (16) is consistent with this hypothesis.

These observations form the basis of a gene transfer system we have used to assess the biochemical activity of the rat Na,K-ATPase a1 subunit. A cDNA clone encoding the entire rat a1 subunit was inserted into the expression vector pSV2neo. This construct transferred resistance to 100 p M

7726

Expression of Rat Na,K-ATPase a1 Subunit cDNA 7727

ouabain to CV-1 cells. Hybridization analysis indicated that transfected clones contained both rat-specific and endogenous Na,K-ATP a1 subunit DNA and mRNA sequences. Trans- fected clones possessed two classes of ouabain-inhibitable Na,K-ATPase activity. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. The transfer of ouabain resistance by rat Na,K-ATPase a1 subunit cDNA suggests that this subunit contains a region sufficient to determine the differential sensitivity between rodent and primate cells to ouabain. Our results suggest a direct correlation between expression of the introduced rat Na,K-ATPase a1 subunit gene and ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.

MATERIALS AND METHODS

Construction of Expression Vector-A cDNA clone containing the entire coding region of the rat Na,K-ATPase a1 subunit was constructed in pUC19 by in vitro recombination of two partial, overlap- ping clones (Ral-a and Ral-b) (17), which shared a common BamHI restriction site. The cDNA fragment was isolated from pUC19 by NcoI partial digestion and complete HindIII digestion. HindIII linkers (Collaborative Research; Waltham, Mass.) were added to the cDNA, and the segment was inserted between the unique Hind11 site and HindIII-linked SmaI site of pSV2neo (kindly provided by K. Kabnick, MIT) to yield the expression vector pSV2a1. The a1 subunit cDNA insert commences at the translation start site and ends at a BamHI site 126 bp' downstream from the translation stop site (12). The cDNA insert lacks a polyadenylation signal sequence. Details con- cerning the mouse Na,K-ATPase a1 subunit cDNA expression vector pSV2a3.6 have been described previously (16). The cDNA insert of pSV2a3.6 contains the complete coding sequence for the murine a1 subunit inserted in the expression vector pSV2. The cDNA insert also contains approximately 100 bp of 5'-untranslated sequence and 300 bp of 3"untranslated sequence.

DNA Transfection and Ouabain-resistant Phenotype-African green monkey CV-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Armour), or in a medium supplemented with 13% fetal calf serum. Transfec- tions were performed essentially as described previously (16). CV-1 cells were exposed to a calcium phosphate precipitate of plasmid DNA (10 pg of plasmid DNA per approximately lo6 cells) or to calcium phosphate without DNA. Forty eight h later, cultures were split 1 to 4. Medium containing ouabain (1 p ~ ) or G418 (0.5 mg/ml) was added 4 h later. Colonies of transfected cells were picked at 4 weeks and grown into cell lines which were maintained in DMEM containing 1 p~ ouabain and supplemented with 10% fetal calf serum. To assess the extent of ouabain resistance in transfectants, 5 X lo5 cells from each of the cell lines, as well as control NIH 3T3 and CV-1 cells were plated in T-25 flasks in medium supplemented with various concentrations of ouabain (1, 10, 100 p ~ ) . Cells were fed 3 times per week to maintain a constant drug concentration in the medium. Ten days later, the number of cells in each culture was determined.

DNA Hybridization Analysis-High molecular weight DNA was prepared from the various cell lines as described previously (18). DNA samples were digested with HindIII, fractionated by agarose gel electrophoresis, and transferred to Zetabind filters (AMF) essentially as described by Southern (19). Blots were prewashed according to the manufacturer's instructions and then prehybridized, hybridized, and washed as described previously (18). Radiolabeled DNA probes were synthesized using the Klenow fragment of DNA polymerase I with random hexadeoxyribonucleotides (Amersham Corp.) by the method of Feinberg and Vogelstein (20). Blots were washed to a final strin- gency of 15 mM NaC1, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 65 "C and exposed to Kodak XRP film at -80 "C with an intensifying screen.

RNA Hybridization Analysis-Total cellular RNA was isolated by the guanidine isothiocyanate method of Chirgwin et al. (21). RNA

' The abbreviations used are: bp, base pairs; DMEM, Dulbecco's modified Eagle's medium; HEPES, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraa- cetic acid; kb, kilobases.

samples were denatured by heating at 65 "C for 10 min in 50% (vol/ vol) formamide and fractionated by electrophoresis through a 1% agarose-containing formaldehyde gel (18). The RNA was transferred to Zetabind, prewashed, and then prehybridized and hybridized as described previously (18, 22). For reprobing, blots were washed 2 times at 95 "C in 0.1 X SSCPE (15 mM NaCl, 1.5 mM sodium citrate, 1.3 mM KH,P04, 100 p~ EDTA), 0.1% sodium dodecyl sulfate.

ffiRb+ Uptake Measurements-ffiRb+ uptake measurements were carried out essentially as described by English et al. (9). CV-1 cells and ouabain-resistant transfectants cultured in the presence of 1 p~ ouabain were washed once in ouabain-free medium, harvested by trypsinization, resuspended at a density of 1.5 X lo6 to 3 X lo6 cells/ ml in DMEM supplemented with 5 mM HEPES, pH 7.4, and appor- tioned into 1-ml aliquots. Each aliquot was incubated in the presence of ouabain for 30-40 min at room temperature and then cooled on ice. Before isotope addition, cells were warmed to 37 "C for 10 min with gentle shaking. The assays were initiated by the addition of 2-5 pCi of ffiRb+ buffered in DMEM/5 mM HEPES, pH 7.4. Aliquots of 200 p1 were removed at intervals and centrifuged through 800 p1 of ice-cold DMEM/5 mM HEPES, pH 7.4, and 300 p1 of dinonyl phthal- ate/silicone oil (1:l) for 2 min in an Eppendorf microcentrifuge. Aqueous and oil layers were removed by aspiration, and the cell pellets were excised and counted for ffiRb+ as described previously (9). Nonspecifically bound ffiRb' (determined by the ffiRb+ associated with the cells at 37 "C within seconds after addition of the tracer) was subtracted from all data. All determinations were carried out 3 min after addition of isotope.

Preparation of Rat Kidney Cortical Cells-Rat kidney cortical cells were prepared according to the procedure of Stanton et al. (23). Rats were killed and kidneys excised into ice cold Hanks' buffered salt solution (GIBCO). Cortical tissue was dissected, minced, and incubated for 90 min at 37 "C in 2-4 volumes of incubation buffer (18 volumes of Hanks' buffered salt solution, 2 volumes of 100 mM Tris, 160 mM HEPES, pH 7.4, 0.1 volume of 5 mM glucose, 0.1% (w/v) collagenase) through which a mixture of 95% air, 5% CO, was bubbled. The resulting solution was sequentially filtered through sieves of pore size 180, 75, and 53 pm. The final filtrate was centrifuged at 1000 X g for 10 min. The resulting pellet of cortical cells was resuspended and washed twice in cold Hanks' buffered salt solution. Cells were lysed by hypotonic shock and assayed for ouabain-sensitive Na,K- ATPase activity as described below. Protein concentration of the lysate was determined by the method of Lowry et al. (24).

Na,K-ATPase Assay-CV-1 and transfectant cells were washed as described above and then depleted of Na+ by two 10-min incubations at 30 "C in 200 packed cell volumes of Na+-free Ringer's solution (2.5 mM H3P04, 1 mM MgSO,, 1 mM CaCl,, 136 mM tetramethylammonium chloride, 2 mM glucose, 1% (w/v) bovine serum albumin, adjusted to pH 7.4 with Tris base). After Na+ depletion, cell lysates were prepared by vortexing cells in deionized water (approximately 7 X IO6 cells/ml), freezing in Dry Ice/acetone, and thawing at room temperature. Measurement of Na+-dependent NADH-linked ATPase activity was performed by the dual cuvette spectrophotometric method described by Lytton (25) except that tetramethylammonium chloride was used instead of choline chloride. Cell lysate (0.1 ml) was added to 2.2 ml of prewarmed (37 'C) Na+-free 1.1 X coupled assay buffer (27.8 mM HEPES, 27.8 mM Tris, 2.8 mM Tris-ATP (vanadate-free), 27.7 mM KCl, 1.1 mM EGTA, 0.1 mM EDTA, 8 mM MgCl,, 10 units/ ml pyruvate kinase and lactate dehydrogenase, 7.2 mM phosphoen- olpyruvate (tricyclohexylamine salt), pH 7.4). An aliquot of the resulting suspension (0.9 ml) was added to each of two matched cuvettes and the final volume brought to 1.0 ml by the addition of 0.1 ml of either 1.36 M tetramethylammonium chloride (reference cuvette) or 1.36 M NaCl (experimental cuvette). ATPase activity was monitored until linearity was obtained (5-10 min). At this point, 10 pl of 100 X ouabain in dimethyl sulfoxide was added to each cuvette (final ouabain concentration of 10 nM-1.0 mM) and ATPase activity monitored for an additional 5-10 min.

Data Analysis-All data were subjected to nonlinear least squares analysis. Ouabain inhibition of =Rb' uptake and Na,K-ATPase activity in CV-1 and rat kidney cortex cells was fit by a simple one-site model expressed in Equation 1,

% Inhibition = (Zmax X [oua])/(K + [oua]) (1)

where Imax is the maximum percent inhibition, K is the apparent ouabain inhibition constant, and [oua] is the concentration of ouabain. I,,, and K were allowed to vary to obtain the best fit.

Ouabain-inhibitable ATPase activity and =Rb' uptake of CV-1

7728 Expression of Rat Na,K-ATPase a1 Subunit cDNA

transfectants did not conform to a simple hyperbolic saturation curve but were closely approximated by the two independent-site model expressed in Equation 2,

% Inhibition = (Zmaxl x [oua])/(K1 + [oua]) (2)

where Zm1 is the maximal inhibition of the high affinity component, Kl is the apparent inhibition constant of the high affinity component, Zmar2 is the maximal velocity of the low affinity component, and K2 is the apparent inhibition constant of the low affinity component. ZmaXl and Zmu2 were allowed to vary, while Kl and K2 were either varied or fixed at the value obtained from fitting the CV-1 (KJ and rat kidney (K2) ATPase data to a one-site model as described in Equation 1. Equation 2 fit the data equally well whether Kl and K, were fixed at the predetermined values or allowed to vary.

+ (1-2 X [oual)/(KZ + [oual)

RESULTS

Transfection of Rat a1 Subunit cDNA Confers Ouabain Resistance to CV-1 Cells-To determine whether a cDNA encoding the rat a1 subunit was capable of conferring ouabain resistance to monkey CV-1 cells, a 3.2-kb cDNA fragment containing the entire coding region of the rat a1 subunit was introduced into the eukaryotic expression vector pSV2neo. Insertion of the cDNA in the sense orientation generated plasmid pSV2al. A schematic representation of the pSV2al construct is shown in Fig. 1. This plasmid was introduced into ouabain-sensitive CV-1 cells by the calcium phosphate coprecipitation procedure (26). Transfected cells were selected for the ability to proliferate in 1 FM ouabain, a concentration of drug which is cytotoxic to CV-1 cells (8, 16). The results of this experiment are shown in Fig. 2 and Table I (Experiment 2). Transfection of pSV2al into CV-1 cells gave approximately 120 ouabain-resistant colonies/106 cells/pg DNA (Fig. 20). Transfection of CV-1 cells with plasmid pSV2a3.6, an expression vector containing a cDNA insert encoding the mouse a1 subunit (16) yielded approximately 230 ouabain- resistant colonies (Fig. 2E). No ouabain-resistant colonies were observed in control groups that were transfected with pSV2neo (Fig. 2B) or mock-transfected (Fig. 2 A ) . CV-1 cells transfected with pSV2neo and selected with G418 gave approximately 125 drug-resistant colonies (Fig. 2C).

In three separate experiments, transfection of pSV2al into CV-1 cells gave an average of 263 ouabain-resistant colonies/ lo6 cells/pg DNA (Table 1). A clone, pSV2al.R, containing

Pvu IT

\ -v

FIG. 1. Schematic representation of rat Na,K-ATPase crl subunit cDNA clone and insertion into the vector pSV2neo. A cDNA clone encoding the entire rat crl subunit was constructed and introduced into the eukaryotic expression vector pSV2neo to generate plasmidpSV2al. This plasmid contains the SV40 origin and promoter (SV40 ori), the SV40 t antigen splice site (intron) and the SV40 poly A addition site (PA tail site). The arrow below the stippled region indicates the direction of transcription.

the rat a1 subunit cDNA inserted into the vector in the antisense (3’ to 5‘ orientation) yielded no ouabain-resistant colonies. No colonies were observed in control groups that were transfected with pSV2neo DNA or mock-transfected. We next measured the survival of five independently isolated clones at various concentrations of ouabain (data not shown). The survival of the five transfectant clones at each ouabain concentration tested (up to 100 PM), was the same as that of control NIH 3T3 cells, suggesting that these clones were true transfectants for rodent ouabain resistance. CV-1 cells did not survive at any of the ouabain concentrations used in this experiment.

Genomic DNA Analysis of Transfected Cells-To confirm that the ouabain-resistant phenotype was conferred to CV-1 cells by the introduction of the rat a1 subunit cDNA construct, we analyzed genomic DNA from individual transfectant clones by Southern blotting. Genomic DNA isolated from ouabain-resistant transfectants was digested with HindIII, an enzyme that liberates the 3.2-kb cDNA insert from pSV2al (Fig. 1). The blot was then hybridized to a probe made from the entire cDNA insert. The results are shown in Fig. 3. The probe detected a 3.2-kb DNA band representing the intact HindIII cDNA fragment of pSV2al in all transfectants. The probe also detected DNA fragments 5.0,5.2, and 6.2 kb in size in CV-1 cells and in each transfected clone; these represent the endogenous copy of the monkey a1 subunit gene. Addi- tional bands were also detected by the cDNA probe (lanes CV-1, A1.5, E1.5). The 7.2-kb band observed in the CV-1 lane and the 8.0-kb band seen in the A1.5 lane most likely represent partial digestion products. When normalized to account for loading differences, the level of hybridization to endogenous monkey sequences in the transfectants was approximately equal to the level of hybridization observed in CV-1 cells, indicating that amplification of endogenous monkey sequences does not contribute to the ouabain-resistant phenotype of the transfectants. It is interesting to note that clone EL5 possesses many intensely hybridizing DNA fragments. These fragments may originate from integration of multiple copies of pSV2al followed by rearrangement of the expression vector. When a 1.35-kb fragment of the pSV2neo cloning vector was used as a hybridization probe on this blot, a similar pattern of bands emerged (data not shown). These results indicate that the ouabain-resistant phenotype of the transfected clones is correlated to the presence of the rat a1 subunit cDNA fragment.

mRNA Expression in Transfected Clones-The rat a1 subunit cDNA inserted between the HindIII and HindIII-linked SmaI site of pSV2neo is transcribed into a hybrid mRNA containing the cDNA sequence followed by an SV40 sequence serving as a 3”untranslated region. This situation is advan- tageous as it allows a distinction between the endogenous a1 subunit mRNA transcribed from the monkey CV-1 gene and the mRNA transcribed from the transfected cDNA construct (approximately 800 bp larger). We used the rat a1 subunit 3.2-kb cDNA as a hybridization probe to screen Northern blots containing RNA isolated from CV-1 cells and individual ouabain-resistant transfectants. As shown in Fig. 4A, the endogenous a1 subunit mRNA (approximately 4.5 kb) was visualized in all cell lines tested including the untransfected ouabain-sensitive control. A hybrid mRNA approximately 5.3 kb in size was detected in transfectant clones A1.4, A1.5, A1.6, B1.4, D1.5, and E1.5. Clone C1.5 lacked the 5.3-kb mRNA but contained an RNA of >10 kb. This RNA species was also present in clone E1.5, as were several additional RNAs which probed with varying intensities. When the same blot was hybridized to a 1.35-kb DNA fragment derived from the region


A B C

D E

FIG. 2. Transfection of rodent a1 subunit cDNA clones into CV-1 cells. CV-1 cells were transfected with a calcium phosphate precipitate of the following plasmids; pSV2neo (B, C), pSV2al (Dl, pSV2a3.6 (E) , mock- transfected (A) . Transfected cells were plated in medium containing M ouabain (A, B, D, E ) or 500 pg/ml G418 (C). Four weeks after transfection colonies were fixed and stained with Giemsa.

TABLE I Efficiency of transfection of cDNA clones into CV-I cells

DNA transfection of CV-1 cells was performed essentially as described previously (16). CV-1 cells were exposed to a calcium phosphate precipitate of plasmid pSV2a1, pSV2al.R, pSV2a3.6, or pSV2ne0, or to calcium phosphate without DNA. Forty-eight h later cells were subcultured a t a dilution of 1:4, and ouabain was added to the cells 4 h later to a final concentration of 1 p ~ . Four weeks after transfection, the colonies were fixed and stained with Giemsa. ND, not determined.

DNA transfected ColoniesJpg DNAJ10’ cells

EXD. 1 EXD. 2 EXD. 3

pSV2al” 400 120 275

pSV2a3.6‘ ND 230 ND pSV2al.Rb 0 ND ND

pSV2neo 0 0 ND None 0 0 0

a Rat a1 subunit cDNA construct. bRat a1 subunit cDNA inserted into pSV2neo in the 3’ to 5’

direction. Mouse a1 subunit cDNA construct (15).

of pSV2neo serving as the 3”untranslated region, the 5.3-kb band in the transfectants hybridized to the probe, whereas the endogenous 4.5-kb band did not (Fig. 4B). This conclusion is supported by the fact that the CV-1 cell and rat brain 4.5- kb a1 subunit mRNA bands failed to hybridize to the vector- derived probe. The high molecular weight bands present in clones C1.5 and E1.5 also hybridized to the vector probe.

- > -’ “ “ ‘ D e s y 2 w ~ Q Z c o w n ~

23 - 9.4 - 6 6-

44-

2.0 - 2.2 -

FIG. 3. Southern analysis of genomic DNA from cells transfected with pSV2al. Clones A1-E1 were originally selected in 1 pM ouabain. Each clone was then subcloned and grown in the presence of 1 p~ ouabain (A1.6), 10 pM ouabain (A1.5, C1.5, 01.5, E1.5), or 100 p~ ouabain (AI.4, B1.4). Genomic DNA (10 pg) from CV-1 cells and transfectants was digested with HindIII. DNA fragments were electrophoresed through a 1% agarose gel and transferred to a Zeta- bind filter. The filter was hybridized to 1.5 X 10’ cpm of radiolabeled a1 subunit cDNA. Molecular weight markers are shown at the left.

These results are consistent with the idea that the 5.3-kb band is a hybrid mRNA containing both the rat a1 subunit and SV40 sequences. I t seems likely that the high molecular weight bands seen in clones C1.5 and E1.5 represent RNA transcribed from rearranged copies of the transfected cDNA. No significant differences in the endogenous a1 subunit mRNA expression levels were detected between the ouabain- resistant transfectants and ouabain-sensitive CV-1 cells, in-

7730

A.

28s -

18s -

B.

28S-

18s-

Expression of Rat Na,K-ATPase a1 Subunit cDNA

.,-

(ur

FIG. 4. RNA blot analysis of cells transfected with pSV2al. Total cellular RNA from CV-1 cells (20 pg), the ouabain-resistant clones described in Fig. 3 (20 pg), and rat brain (5 pg) was electrophoresed through a 1% formaldehyde-agarose gel and then transferred to a reusable Zetabind filter. A, hybridization of the filter to 1.5 X lo7 cpm of radiolabeled a1 subunit cDNA. B, the same filter as in A, reprobed with 1.5 X IO' cpm of a radiolabeled 1.35-kb HindIII- BamHI fragment derived from pSV2neo.

dicating that the ouabain resistance of the transfectants was not due to a significant increase in the expression level of the monkey a1 subunit gene.

Expression of Ouabain-resistant =Rb+ Uptake in Transfec- tant Clones-To further analyze Na,K-ATPase a1 subunit gene expression we measured the ouabain sensitivity of mon- ovalent cation uptake in transfectant clones grown in ouabain and in parental CV-1 cells. "Rb+ was used to monitor K+ flux (9). As shown in Fig. 5, CV-1 cells exhibited a rate of =Rb+ uptake of 15 nmol/min/lO' cells (0). Ouabain inhibited this rate of uptake by 80% with an apparent Ki between and

M, indicating that 80% of %Rb+ uptake under these conditions was mediated by the CV-1 cell Na,K-ATPase. Transfectant clones A1.4 (+) and B1.4 (H) exhibited lower maximal initial uptake rates than CV-1 cells (6-7 nmol/min/ IOfi cells), but quantitative conclusions are not warranted by the data because the cells were not preloaded with Na+. The ouabain-sensitive =Rb+ uptake of the transfectants was 100- fold more resistant to ouabain than that of CV-1 cells. Ap- proximately 70% of "Rb+ uptake in the transfectant clones was ouabain-inhibitable with an apparent Ki (ouabain) of 30 p ~ . This value is similar to that reported for the "Rb+ uptake activity of the rat renal Na,K-ATPase (11, 27). In transfectants, the =Rb+ uptake catalyzed by the endogenous CV-1 Na,K-ATPase cannot be assessed under these conditions since ouabain (present during growth of the cells) remains associated with the endogenous ATPase throughout the course of the experiment.

o-/. . 7 . 1 4

o 10-7 10-6 10-4 10-2

Ouabain (M)

FIG. 5. Ouabain-resistant "Rb+ uptake in CV-1 cells transfected with pSV2al. CV-1 cells and ouabain-resistant transfectants were grown and harvested, and HRRb+ uptake was assayed as described under "Materials and Methods." Each uptake measurement was initiated a t 37 "C by the addition of 20 pl of DMEM/HEPES containing 2-5 pCi of RFRb+ to 2.0 ml of cell suspension containing the concentration of ouabain indicated on the abscissa. Aliquots of the cell suspension were removed and uptake determined at the time of and 3 min after isotope addition. Uptake was linear for 5 min. Values represent the mean * S.E. from two separate experiments each done in triplicate (six individual measurements). 0, CV-1 cells; *, transfectant clone A1.4; W, transfectant clone B1.4.

0 10-710-610-5 10-310-2

Ouabain (M)

FIG. 6. Removal of strophanthidin reveals high affinity '"Rb+ uptake in transfected cell lines. Ouabain-resistant transfectants were removed from ouabain and grown in medium containing 25 p~ strophanthidin. Before initiating MRb' uptake measurements, cells were resuspended in drug-free medium and incubated a t 37 "C for 7-10 min to allow dissociation of strophanthidin. Cells were then suspended in medium containing the concentration of ouabain indicated on the abscissa. Each point represents the mean * S.E. of two separate uptake experiments each performed in triplicate. 0, transfectant clone A1.4 (rat a1 subunit cDNA recipient); *, transfectant clone B1.4 (rat a1 subunit cDNA recipient), W transfectant clone D6.1 (mouse a1 subunit cDNA recipient).

To determine whether transfectant clones express more than one component of ouabain-sensitive "Rb' uptake (more than one class of Na,K-ATPase), %Rb+ uptake was measured in cells which were grown in the presence of strophanthidin, a cardiac aglycone analog of ouabain, and then washed free of the drug before assays were performed. Strophanthidin, unlike ouabain, rapidly dissociates from CV-1 cells so that the activity of the endogenous enzyme can be detected (30). The rate of %Rb+ uptake by transfectant clones grown in the presence of strophanthidin is shown in Fig. 6. In clones A1.4, B1.4, and D6.1, "'Rb+ uptake exhibited biphasic ouabain


sensitivity. Clones A1.4 and B1.4 had a total sGRb’ uptake rate under steady-state conditions of approximately 6.5 nmol/ min/106 cells (0, +), while clone D6.1 ( a transfectant har- boring mouse d subunit cDNA) (16) had an “jRb+ uptake rate of 5.5 nmol/min/106 cells (W). It should be noted that these rates are not strictly comparable to those determined in the experiment depicted in Fig. 5. This is because during the incubation period prior to initiating =Rb+ uptake measurements, cells washed free of strophanthidin, as opposed t o those bearing persistently bound ouabain, have a greater number of active sodium pumps which extrude proportion- ately more Na’ and produce a lower steady-state intracellular Na’ concentration during the uptake assay. Cells grown in strophanthidin will thus exhibit less “Rb’ uptake per unit pump than cells grown in ouabain. Quantitation in this type of experiment is also complicated by the fact that only approximately 70% of the strophanthidin is actually removed by the washing procedure.

As shown in Table 11,26% of total *‘Rb+ uptake was highly ouabain sensitive in clones B1.4 and D6.1, while 17% of total uptake was highly ouabain-sensitive in clone A1.4. The apparent ouabain dissociation constants for the transfectants were derived from the data in Fig. 6 and are presented in Table I1 (the ouabain dissociation constant for CV-1 cells was determined from the data of Fig. 5). The high affinity component of ouabain-sensitive %Rb’ uptake in transfectants exhibited the same K, app for ouabain (between 1 and 3 x M; Kl of Table 11) as the =Rb+ uptake of CV-1 cells. A second, lower affinity component of ouabain-sensitive “Rb’ uptake was observed in all three transfectant clones with a K; Bpp

between and M ouabain (Kz of Table 11). These data suggest that expression of the rodent Na,K-ATPase a1 subunit gene is responsible for the appearance of the low affinity component of ouabain-sensitive =Rb+ uptake present in transfectant clones.

Ouabain-resistant Na,K-ATPase Actiuity in Transfec- tants-Because both the maximal apparent initial Rb’ uptake rate and the relative proportions of high and low ouabain affinity uptake are influenced by variations in cytoplasmic Na+, we measured ouabain-sensitive, Na+-dependent ATPase activity in cell lysates prepared from rat kidney cortex, CV-1 cells, and transfectant clones. These assays, conducted in the presence of saturating Na’ and K’ provide a more reliable quantitative estimate of both total Na,K-ATPase activity in the cell and the proportion of the activity contributed by high and low ouabain affinity enzymes.

The results, normalized for each cell type and fit by nonlinear least squares analysis, are presented in Fig. 7. Maximal ouabain-sensitive Na,K-ATPase activity in lysates prepared

TABLE I1 Calculated kinetic parameters for ouobain-inhibitable %Rb’ uptake in

CV-1 and transfected cell lines Data were analyzed by the nonlinear least squares procedure as

described under “Materials and Methods.” K , and K2 are the apparent ouabain dissociation constants for the high and low affinity components of ouabain-sensitive =Rb+ uptake. Clones A1.4 and B1.4 were transfected with the vector pSV2al (rat a1 subunit cDNA construct). Clone D6.1 was transfected with vector pSV2a3.6 (mouse a1 subunit cDNA construct).

Fraction due to high affinity

component

Cell line K, K*

cv-1 3 X 10-~ A1.4 2 X 10-7 2.4 X 10-6

B1.4 3 x 10-~ 4.7 X 10-6

0.17 0.26

D6.1 1 x 10” 8.3 X 10-5 0.26

10-8 10-6 10-4 10-2

0 UABAIN (M)

10-6 10-4 10-2

OUABAIN(M)

OUABAIN(M)

FIG. 7. Ouabain-sensitive Na,K-ATPase activity in transfected CV-1 cells expressing the rodent a1 subunit. Lysates from rat kidney cortex, CV-1 cells, and ouabain-resistant transfectants were prepared and assayed for ouabain-inhibitable Na,K-AT- Pase activity as described under “Materials and Methods.” Symbols represent the normalized values f S.E. from two to four separate experiments performed in duplicate. A, CV-1 cells (0) and rat kidney cortex (0). The values were best fit with the one-site model expressed in Equation 1. B, transfectant B1.4 (O), rat kidney cortex (- - -), and CV-1 cells (- - -1. C, transfectant A1.4 (O), rat kidney cortex (- - -), and CV-1 cells (- - -). For transfectants, the data was best fit with the two-site model expressed in Equation 2. The best fit was obtained with a ratio of high to low affinity sites of 1:l (8) and a ratio of 1:2 (C). The best fit is represented by the line drawn through the open circles. The best fits to the CV-1 data (dotted lines) and rat kidney data (closed circles) are included ( B and C ) to emphasize that Na,K-ATPase activity in transfectants is a biphasic combination of low and high affinity components.

from CV-1 and transfected cells was approximately 7-8 nmol/ min/106 cells. Assuming a stoichiometry of 1 ATP/2 Rb’, this value is in good agreement with the Rb’ V,,, which we have observed in Na+-loaded transfectants (31). The maximal ouabain-sensitive Na,K-ATPase activity in a rat kidney cortex cell lysate was approximately 1.5 rmol/min/mg protein. Ly-

7732 Expression of Rat Na,K-ATPase a1 Subunit cDNA

sates prepared from CV-1 cells and rat kidney cortex cells (Fig. 7 A ) exhibited a single component of ouabain-sensitive, Na+-dependent ATPase activity which was closely approximated by the one-site model expressed in Equation 1 under “Materials and Methods.” Nonlinear least squares fit of Equa- tion l to the data produced an apparent Ki (ouabain) of 2 X 1 O “ j M for the CV-1 cell ATPase activity and 1.6 X M for the ATPase activity of rat kidney cortex cells.

Data obtained from transfectants were not well fit by the one-site model of Equation 1, suggesting that the transfectants possessed more than one component of ouabain-sensitive, Na+-dependent ATPase. Reasoning that the transfectant data might be adequately modeled by a simple combination of the CV-1 and rat kidney cortex ouabain-sensitivity curves, we fit the transfectant data with Equation 2 (“Materials and Methods”) in which ICl was fixed at the value derived for CV- 1 (2 X M), K2 fixed at the value derived for rat kidney (1.6 X M), and the relative proportions of the two sites were allowed to vary. Such analysis produced an optimal fit to the data curves of Fig. 7, B and C which was not improved by allowing the Kc values to vary (ix. the assigned value gave the minimum sum of the squares). In transfectant clone B1.4 (Fig. 7B), the low affinity site contributed approximately one- half the total Na,K-ATPase activity, while in transfectant clone A1.4 (Fig. 7C) the low affinity site accounted for approximately 65% of the total activity. It should be noted that the apparent K, values determined in cell lysates were higher than those obtained in “Rb+ uptake assays (Table 11). This is because K+ (25 mM) present in the ATPase assay solution acts as a competitive inhibitor of ouabain binding. These results suggest that the low affinity component of Na+-dependent, ouabain-sensitive Na,K-ATPase activity in transfected CV-1 cells is derived from expression of the rat a1 subunit gene.

DISCUSSION

This study demonstrates that a cDNA construct encoding the rat Na,K-ATPase a1 subunit confers ouabain resistance to ouabain-sensitive CV-1 cells. We show for the first time that permanent cell lines derived by transfection of rat a1 subunit cDNA express a component of Na+-dependent ATP- ase activity with a ouabain sensitivity similar to that of rat kidney Na,K-ATPase. These results suggest that the ouabain affinity of the Na,K-ATPase is determined by the a and not the p subunit of the enzyme.

A comparative analysis shows that the extent of ouabain resistance in transfectants containing rat or mouse a1 subunit cDNA sequences is essentially the same (100 I.IM) and is similar to that of NIH 3T3 cells (28). Acquisition of the ouabain-resistant phenotype by CV-1 cells is correlated with the presence of the rat Na,K-ATPase a1 subunit cDNA fragment, the expression of rat specific a1 subunit mRNA transcripts, and the appearance of an enzymatic activity with the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Hybridization analysis has shown that the ouabain-resistant phenotype of transfectants does not result from amplification of the endogenous monkey gene or overexpression of endogenous a1 subunit mRNA. Further- more, since ouabain resistant colonies were never obtained when CV-1 cells were mock transfected or transfected with the pSV2neo vector alone, it is extremely unlikely that a mutation in the endogenous monkey a1 subunit gene could have conferred ouabain resistance in this protocol. The complete correlation observed between expression of rodent Na,K- ATPase activity and the high degree of ouabain resistance exhibited by transfected cells strongly suggests that ouabain

resistance results from the integration and expression of the rat Na,K-ATPase a1 subunit gene.

The Na,K-ATPase has been purified and extensively stud- ied at the biochemical level. However, the analysis of structure function relationships for the Na,K-ATPase has been difficult, primarily because reconstitution of functional enzyme from purified subunits has not been achieved (6). The expression system we have developed offers a strategy for addressing this issue. The appearance of ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells provides the first evidence that the expressed rat a1 subunit is assembled into an active enzyme, presumably in conjunction with the monkey /3 subunit. It will clearly be of interest to determine the subunit composition of hybrid Na,K-ATPase molecules produced in this system. A number of lines of evidence indicate that the molar ratio of a lp subunits in the holoenzyme is 1:l (6). However, evidence supporting both a@ and (a@)* structures for the active enzyme have been reported (6). Thus hybrid Na,K-ATPase molecules containing (rat al)l(monkey p)l, (rat al),(monkey ,f3)2, or (rat al)l(monkey al),(monkey /3)* subunit combinations are possible. The development of species-specific a1 subunit antibodies should permit identification of the a1 subunit polypeptides present within different hybrid Na,K- ATPase molecules.

It is interesting to note that, although the acquisition of the ouabain resistant phenotype was correlated with the appearance of a Na,K-ATPase activity with ouabain sensitivity similar to that of the rat kidney enzyme, the total cellular Na,K-ATPase activity in transfected cells did not signifi- cantly increase. Several potential explanations could account for these results. One possibility is that the availability of the p subunit limits the total number of Na,K-ATPase molecules which can be produced. If this were true, then the rat and monkey a subunits would compete for available /3 subunit polypeptides. Alternatively, the cell may employ other regu- latory mechanisms to insure that the total pumping activity per cell remains relatively constant.

The transfer of ouabain resistance by Na,K-ATPase a1 subunit cDNA is consistent with the view that this subunit contains a region sufficient to determine the species-specific variation in ouabain sensitivity. This does not exclude the possibility that the products of other cellular genes may modify the sensitivity of the Na,K-ATPase (9, 30). The differences in ouabain sensitivity between rodent and primate Na,K-ATPase a1 subunits can thus be directly attributed to primary sequence of the a subunit polypeptides. However, comparison of the human (29) and rat (12, 17) a1 subunits reveals that the two polypeptides are approximately 97% homologous. How do the DNA sequences which encode each a1 subunit lead to differences in the ouabain sensitivity of the Na,K-ATPase? The construction of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive al subunit cDNAs should permit identification of the sites within the a1 subunit which are responsible for differential ouabain sensitivity.

The ability to assess the biological activity of the Na,K- ATPase a1 subunit in a transfection protocol raises interesting questions regarding the ouabain sensitivity of other rodent a subunit isoforms. Three genes encoding distinct a subunit isoforms have been described in the mouse (14). Sweadner (7) has presented evidence that the a+ (a3) form of the Na,K- ATPase from rat brain is more sensitive to ouabain than the

( d ) form of the ATPase. However, the basis for the differential ouabain sensitivity between ATPase isoforms has not been explained. The isolation of cDNA clones for the three rat Na,K-ATPase a subunit isoforms (12, 17) should


now make it feasible to analyze the relationship of a subunit isoforms to the phenotype of ouabain resistance. Initial experiments, involving hybridization of cDNAs specific for each of the three a subunit isoforms to genomic DNA from monkey cells made ouabain-resistant by the transfer of metaphase mouse chromosomes, implied that expression of the rodent a2 and a3 subunit genes was unlikely to confer resistance to high levels of ouabain (28). The expression system we have developed should be extremely useful in evaluating the relative ouabain sensitivity of the various rodent a subunit isoforms. The identification of sites within the a subunit which interact with ouabain and/or are responsible for differential ouabain sensitivity should also provide a valuable tool in the study of Na,K-ATPase structure and function.

Acknowledgment-We are grateful to Dr. Karen Kabnick for valuable suggestions regarding construction of the rat a1 subunit cDNA expression vector.

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