Expression and Functional Role of the gamma  Subunit of the Na,K-ATPase in Mammalian Cells*

Alex G. TherienDagger §, Steven J. D. Karlish, and Rhoda BlosteinDagger parallel

From the Dagger  Department of Biochemistry, McGill University, Montreal, Quebec H3G 1A4, Canada and the  Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The functional role of the gamma  subunit of the Na,K-ATPase was studied using rat gamma  cDNA-transfected HEK-293 cells and an antiserum (gamma C33) specific for gamma . Although the sequence for gamma  was verified and shown to be larger (7237 Da) than first reported, it still comprises a single initiator methionine despite the expression of a gamma C33-reactive doublet on immunoblots. Kinetic analysis of the enzyme of transfected compared with control cells and of gamma C33-treated kidney pumps shows that gamma  regulates the apparent affinity for ATP. Thus, gamma -transfected cells have a decreased K'ATP as shown in measurements of (i) K'ATP of Na,K-ATPase activity and (ii) K+ inhibition of Na-ATPase at 1 µM ATP. Consistent with the behavior of gamma -transfected cells, gamma C33 pretreatment increases K'ATP of the kidney enzyme and K+ inhibition (1 µM ATP) of both kidney and gamma -transfected cells. These results are consistent with previous findings that an antiserum raised against the pig gamma  subunit stabilizes the E2(K) form of the enzyme (Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628-32634). Overall, our data demonstrate that gamma  is a tissue (kidney)-specific regulator of the Na,K-ATPase that can increase the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-gamma antiserum.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Na,K-ATPase is the sodium pump protein responsible for maintaining the electrochemical gradient present across the membranes of most animal cells (1). It consists of at least two subunits, alpha  and beta , each of which exists as one of several isoforms (alpha 1, alpha 2, alpha 3, and alpha 4 and beta 1, beta 2, and beta 3; for review, see Ref. 2). The alpha  subunit, also known as the catalytic subunit, contains the binding sites for the enzyme's nucleotide and cation substrates, as well as the catalytic and regulatory (calcium-dependent and cAMP-dependent protein kinase C and A, respectively) phosphorylation sites. The role of the beta  subunit is less clear, but it is required for normal processing and expression of the enzyme and may have a role in regulating the interaction of cations with the alpha  subunit (3). The different isoforms of the pump are expressed in a tissue- and development-specific fashion and are believed to be distinct in both function and modes of regulation (2).

A small single-transmembrane protein called the gamma  subunit was originally believed to be a third subunit of the pump. It was discovered by Forbush et al. (4) in 1978 and later cloned in rat, mouse, cow, sheep (5), human (6), and Xenopus laevis (7); it has sequence homology to a family of channel-inducing peptides (8-10). Although its function has remained elusive, experiments in Xenopus oocytes have shown that the gamma  subunit alters the K+ affinity of the pump in a voltage- and Na+-dependent fashion (7) and may induce cation channel activity (11). Our recent Western blot analysis using an anti-gamma antiserum indicated that gamma  protein is detected only in the kidney medulla but not in other tissues tested (red blood cells, heart, brain, and kidney glomerulus) including cultured cell lines derived from cells of the kidney tubule. We showed that the antibodies bound to the cytoplasmic tail of gamma  and stabilized the E2 form of the enzyme, presumably by disrupting alpha -gamma interactions (12).

In this report we show that expression of gamma  in cells devoid of this protein results in a significant increase in apparent affinity for ATP and that the gamma -transfected cells resemble the alpha 1beta 1gamma kidney enzyme in that this effect is abrogated by antiserum raised against a 10-residue peptide of the C terminus of the gamma  subunit.

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Antibodies-- gamma C33 is a rabbit polyclonal antiserum raised against a peptide representing the C-terminal 10 amino acids of the gamma  subunit. In the experiments reported herein, gamma C33 was used, and a control nonimmune serum was obtained from the same rabbit prior to immunization. The peptide, KHRQVNEDEL, was synthesized at the Alberta Peptide Institute, University of Alberta, and used either as the free peptide for competition studies or linked to keyhole limpet hemocyanin and emulsified with Freund's adjuvant before injection into rabbits. Antibody 6H is a mouse monoclonal antibody specific for the alpha 1 isoform of the Na,K-ATPase, and was a generous gift from Dr. Michael Caplan, Yale University. Horseradish peroxidase-labeled secondary antibodies (donkey anti-rabbit) were purchased from BIO/CAN Scientific.

5'-RACE and pREP4-gamma Synthesis-- 5'-Rapid amplification of cDNA ends (5'-RACE)1 was carried out using CLONTECH Marathon-ready cDNA from rat kidney following the manufacturer's instructions. Appropriate primers (see below) were synthesized, and the gamma  subunit gene sequence was amplified by polymerase chain reaction. The 5'-end primer contained a site for HindIII endonuclease (boldface), a Kozak sequence (underlined; see Ref. 13), and the first 24 bases of the gamma  subunit gene as determined by 5'-RACE (GGGGGGGAAGCTTGCCGCCACCATGACAGAGCTGTCAGCTAACCAT). The 3'-end primer contained a BamHI endonuclease site (boldface) and bases complementary to the last 24 bases of the gamma  subunit gene as determined by Mercer et al. (5) (GGGGGGGATCCGTCACAGCTCATCTTCATTGACCT). The resulting DNA was then cleaved with these endonucleases and ligated into the corresponding sites of pREP4 vector (Invitrogen) to make pREP4-gamma . Sequencing of the recombinant plasmid was carried out using a Pharmacia T7 sequencing kit. pREP4 and pREP4-gamma DNA used for transfections were purified using Qiagen affinity columns according to the manufacturer's instructions.

Transfections, Tissue Culture, and Membrane Preparations-- HEK-293 cells at 50% confluency in a 14-cm culture plate were transfected with pREP4 or pREP4-gamma using FuGENE 6 reagent (Roche Molecular Biochemicals) and following the manufacturer's instructions. Cells were selected for 10 days, divided among 5 × 14 cm plates, and allowed to grow to confluency (about 3 weeks) in Dulbecco's modified Eagle's medium containing 10% newborn calf serum and 400 µg/ml hygromycin B. Cellular membranes from transfected cells and from rat kidney medulla were prepared by the procedure described elsewhere (12).

Western Blots-- Western blot analysis and densitometry measurements were carried out as described previously (14) with the following modifications. 10% polyacrylamide gels were run on a Protean II gel electrophoresis apparatus (Bio-Rad), transferred to polyvinylidene difluoride membranes (Millipore), and blotted with 6H antibodies and gamma C33 antiserum, both at dilutions of 1:10,000.

Enzyme Assays-- Na,K-ATPase and Na-ATPase assays were carried out at 37 °C in a final volume of 100 µl as described previously (12). For Na,K-ATPase assays, final concentrations of reactants were: 100 mM NaCl, 10 mM KCl, 40 mM choline chloride, 4 mM MgSO4, 1 mM EDTA, 30 mM Tris-HCl (pH 7.4), and varying concentrations of ATP as indicated. Na,K-ATPase activities shown represent the ATPase activities inhibited by 5 mM ouabain and ranged from 1500 to 4500, 130 to 180, and 110 to 130 nmol Pi/mg/min for kidney, HEK-pREP4, and HEK-pREP4-gamma membranes, respectively. For Na-ATPase assays, final concentrations of reactants were: 20 mM NaCl, 20 mM choline chloride, 2 mM MgSO4, 1 mM EDTA, 5 mM EGTA, 20 mM histidine-Tris (pH 7.4), and 1 µM ATP. To determine effects of K+ on Na-ATPase, choline chloride was replaced by the indicated concentrations of KCl. For assays of effects of anti-gamma , membranes were preincubated for 1 h at 4 °C in the presence of immune (gamma C33) or nonimmune (preimmune) sera at a ratio of 1:100. For experiments of K inhibition of Na-ATPase, the sera were dialyzed for 48 h at 4 °C against three changes of 1000 volumes of 5 mM imidazole (pH 7.4). When present, the 10-mer peptide was used in the antiserum preincubation at a concentration of 20 µM. K'ATP values were calculated by analyzing ATP activation curves using the Michaelis-Menten formulation. All experiments shown are representative of at least three separate experiments, and each data point shown is the mean ± S.E. of the difference between triplicate determinations carried out in the absence and presence of ouabain.

    RESULTS
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ABSTRACT
INTRODUCTION
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DISCUSSION
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We showed previously that the gamma  subunit protein is expressed in a tissue-specific manner. Of the various rat tissues analyzed by Western blotting (kidney medulla, kidney glomerulus, red blood cells, heart, and axolemma), gamma  was detected only in the kidney medulla (12). In more recent experiments (not shown) this analysis has been extended to additional tissues of the rat, namely the lung, small intestine, stomach, and spleen. The gamma  protein could not be detected in these tissues except for a trace amount in the spleen (relative to alpha , amounting to <= 2% of that present in the kidney medulla). The kidney-specific presence of gamma  also holds true with mouse tissues (kidney, axolemma, and heart) analyzed similarly.2

Expression of the gamma  Subunit in Mammalian Cells-- Our earlier evidence of a modulatory role for the gamma  subunit on the conformational equilibrium of the Na,K-ATPase reaction was inferred from studies of the effects of an anti-gamma antiserum on enzymatic activity. To evaluate directly the functional role of gamma , it was essential to transfect cDNA encoding gamma  into mammalian cells devoid of gamma . An additional goal of such experiments was to establish the basis for the existence of gamma  as a doublet in the rat (5) as it is in the Xenopus kidney (7). Accordingly, we first used 5'-RACE to ascertain that the previously reported cDNA of the rat gamma  subunit comprised the full-length sequence and, if not, whether the doublet in Western blots is secondary to the presence of an additional start codon in the mRNA for the gamma  subunit as is the case for Xenopus kidney (7). The resulting sequence, shown in Fig. 1, confirmed the presence of a single initiator methionine. However, the gamma  cDNA thus obtained encodes a protein of 66 rather than 58 residues, as originally reported (5), and corresponds to the sequence subsequently revised by Minor et al. (11). The calculated molecular mass is 7237 Da. The dichotomy may be the result of either a cloning artifact or, possibly, an isoform variant.3


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Fig. 1.   5'-Untranslated and coding regions of rat gamma  subunit cDNA and deduced amino acid sequence. The nucleotide sequence was determined by 5'-RACE analysis as described under "Experimental Procedures."

Efforts to express gamma  in HeLa and HEK cells using a standard stable transfection system resulted in levels of expression that, compared with the kidney, were considered too low (gamma :alpha  <=  0.1) given the relatively modest effects of anti-gamma on the kidney enzyme. In an effort to increase the level of expression, we used the plasmid pREP4 that combines the advantages of "classical" transient and stable expression systems. In addition to a hygromycin resistance gene, this plasmid contains an origin of replication that allows it to remain expressed episomally for several weeks in the nuclei of primate and canine cells. Thus, hygromycin can be used to select for cells that contain multiple copies of the gene (rather than just one). Accordingly, we subcloned the gene for gamma  (revised sequence shown in Fig. 1) in pREP4 and transfected HEK-293 cells with both recombinant and wild type plasmids. Membranes were made from the transfected HEK-pREP4-gamma and control HEK-pREP4 cells, and the amount of gamma  subunit protein relative to alpha  subunit protein was estimated by a comparison with kidney membranes using Western blot analyses of both the gamma  and alpha  subunits.

The blots shown in Fig. 2 indicate that the gamma  doublet is present in both kidney and HEK-pREP4-gamma membranes but not in control HEK-pREP4 membranes. The densities of the gamma  subunit doublet and alpha  subunit band of HEK-pREP4-gamma were compared with those of the kidney using several dilutions and varying times of exposure to film. We determined that pREP4-gamma membranes contain 34 ± 12% (S.E.) of the amount of gamma  present in the kidney after normalizing for alpha 1 densities. Assuming that the gamma :alpha ratio of kidney is 1:1 (7, 15, 16), this indicates that the stoichiometry of the gamma :alpha proteins in HEK-pREP4-gamma  approx  1:3. That this ratio reflects gamma  associated with alpha  was confirmed in Western blots of immunoprecipitates using the antibody 6H (not shown).


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Fig. 2.   Western blot analysis of rat kidney, HEK-pREP4-gamma , and HEK-pREP4 membranes. Immunoblotting was carried out as described under "Experimental Procedures." Lane 1, 2.0 µg of rat kidney membranes; lane 2, 30 µg of HEK-pREP4-gamma membranes; lane 3, 30 µg of HEK-pREP4 membranes.

Functional Effects of gamma -- We showed earlier that binding of antibodies raised to the gamma  polypeptide doublet associated with the pig kidney Na,K-ATPase binds to the cytoplasmic tail of the gamma  subunit (12). This binding was associated with partial inhibition of the Na,K-ATPase activity. Moreover, inhibition varied as a function of conditions that affect the rate-limiting step(s) during steady-state hydrolysis, for example varying pH. Thus, the inhibition (approx 30%) observed under conditions of optimal concentrations of substrates and at pH 7.4 decreased as the pH level increased and increased as pH decreased. We concluded that the antiserum caused a shift in the E1 left-right-arrow  E2(K) equilibrium toward E2(K).

To maximize the inhibitory effect of the antiserum, particularly for tests of the effect of gamma  in the transfected cells in which the gamma :alpha ratio is lower than in the kidney medulla, we tested the prediction that inhibition would be greater at suboptimal ATP concentrations, under which conditions the E2(K) right-arrow E1 sequence becomes even more rate-limiting (17). For these experiments, a 10-residue peptide representing the C terminus of the gamma  subunit was synthesized and used for the production of gamma C33 antisera, allowing confirmation of the specificity of the anti-gamma effects and providing free 10-mer peptide for competition studies. Fig. 3A shows a representative experiment on the effects of anti-gamma (serum gamma C33) on Na,K-ATPase activity of renal enzyme at near saturating (1 mM) and subsaturating (10 µM) concentrations of ATP. As predicted, inhibition increases as the ATP concentration is lowered, from 36 ± 4% inhibition at 1 mM ATP to 70 ± 11% at 10 µM ATP (averages of several experiments). In addition, the presence of excess amounts of free peptide corresponding to the C terminus of gamma  during the preincubation reversed completely the inhibition observed at both ATP concentrations; no effect on the activity of nonimmune serum-treated enzyme was observed. Fig. 3B is a Lineweaver-Burk plot of a representative experiment showing the effect of ATP concentration on activity. It shows that pretreatment of the enzyme with antiserum gamma C33 caused a 1.8-fold increase in K'ATP (for values, see inset in Fig. 4). Vmax for gamma C33-treated enzyme was 78 ± 7% that for nonimmune serum-treated enzyme. The critical implication of this result is that anti-gamma reverses an increase in affinity effected by the gamma  subunit. This hypothesis was tested in HEK-pREP4-gamma cells and HEK-pREP4 cells.


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Fig. 3.   Effect of gamma C33 antiserum and gamma C33-reactive peptide on ATP affinity of renal pumps. A, rat renal membranes were assayed for Na,K-ATPase activity at 100 µM or 1 mM ATP after preincubation in the presence of gamma C33 antiserum or nonimmune rabbit serum and in the absence or presence of peptide representing the C-terminal 10 amino acids of the gamma  subunit (used to generate gamma C33). Differences between nonimmune and immune serum-treated enzyme are significant (p < 0.01 using Student's t test). , nonimmune serum; , nonimmune serum + peptide; square , gamma C33; ,  gamma C33 + peptide. B, rat renal membranes were assayed for Na,K-ATPase activity (v) at different ATP concentrations after preincubation in the presence of gamma C33 (open circles) or nonimmune serum (filled circles). Lineweaver-Burk plots of a representative experiment are shown.


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Fig. 4.   Effect of gamma C33 on ATP affinity of HEK-pREP4 and HEK-pREP4-gamma pumps. Membranes isolated from HEK-pREP4-gamma (filled circles) and HEK-pREP4 (open circles) were assayed for Na,K-ATPase activity (v) at varying ATP concentrations. Lineweaver-Burk plots of a representative experiment are shown, with Vmax values of the two membrane preparations normalized to 1.0. Inset, a table summarizing K'ATP values for gamma C33-treated and nonimmune serum-treated kidney enzymes and HEK-pREP4-gamma and HEK-pREP4 enzymes. IS, gamma C33; NIS, nonimmune serum. Differences between nonimmune and immune serum-treated renal enzyme (p < 0.01), as well as between pREP4- and pREP4-gamma -transfected cells (p < 0.02), are significant using Student's t test.

We first compared the effect of gamma C33 on HEK-pREP4-gamma , HEK pREP4 cells, and kidney enzymes, all assayed at 10 µM ATP. The experiment (not shown) indicated that gamma C33 caused 33 ± 2 and 82 ± 15% inhibition of HEK-pREP4-gamma and kidney enzymes, respectively, and had no effect on the activity of HEK-pREP4 cells. This inhibition is consistent with the aforementioned relative amounts of gamma  in kidney versus HEK-pREP4-gamma cells. Experiments were then carried out to determine whether the gamma  subunit has any effect on K'ATP. The plots shown in Fig. 4 indicate that the HEK-pREP4-gamma enzyme has a significantly higher affinity for ATP compared with control HEK-pREP4 enzyme (for K'ATP values, see inset). The gamma -mediated-1.3-fold decrease in K'ATP in these cells, although modest, is in fact similar to the effect of gamma  in the kidney membranes, taking into account the lower alpha :gamma ratio in the transfected cells (approximately one-third that of kidney membranes). This being the case, we used a more sensitive assay of ATP affinity to magnify the effect of gamma  and to determine whether anti-gamma antiserum can reverse its effects. This assay takes advantage of the fact that K+ inhibits Na-ATPase activity at a very low (1 µM) ATP concentration under which condition the (low affinity) ATP-activated K+ deocclusion reaction becomes rate-limiting. Accordingly, this inhibition decreases as the affinity for ATP at its low affinity binding site increases (18). As shown in Fig. 5A, K+ is less effective at inhibiting the Na-ATPase activity of pumps of gamma -transfected membranes than of control membranes. Experiments were then carried out to test and compare K+ inhibition, and the effect of anti-gamma thereupon, of the enzyme of the kidney medulla, HEK-pREP4-gamma , and HEK-pREP4. Fig. 5B shows the percentage inhibition at 0.2 mM KCl of these pumps in the presence of nonimmune versus immune serum. Whereas preincubation of kidney and pREP4-gamma pumps with gamma C33 effected 2.1- and 1.5-fold increases in K+ inhibition, respectively, no gamma C33-mediated change was detected for HEK-pREP4 pumps.


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Fig. 5.   K+ inhibition of Na-ATPase activity of rat kidney, HEK-pREP4, and HEK-pREP4-gamma pumps and the effect of gamma C33. Membranes were assayed for Na-ATPase activity in varying concentrations of KCl as described under "Experimental Procedures." A, K+ inhibition profile of pREP4 (open circles)- and pREP4-gamma (filled circles)-transfected cells. B, inhibition of Na-ATPase activity in the presence of 0.2 mM KCl after preincubation with gamma C33 or nonimmune serum. Differences between nonimmune and immune serum-treated kidney (p < 0.01) and HEK-pREP4-gamma enzymes (p < 0.02) are significant using Student's t test. , nonimmune serum; square , gamma C33.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The successful transfection of the gamma  subunit into mammalian cells with sodium pumps devoid of this subunit has enabled the direct analysis of the functional role of this Na,K-ATPase-associated protein. Although the gamma  subunit does not appear to be necessary for normal Na,K-ATPase activity (7, 15, 19), its role as a modulator of function is consistent with its appearance in a tissue (kidney)-specific manner.

Recently, Béguin et al. (7) have shown that the rat gamma  subunit lowers the affinity of the pump for K+ in cRNA-injected Xenopus oocytes, at least in the absence of Na+. A gamma -mediated decrease in K'ATP could explain this increase in K'0.5 for K+ because, as a first approximation, ATP and K+ affinities are inversely related (20). However, that result may be confounded by the use of cRNA synthesized using the original sequence for rat gamma  (5). In a recent report, the human gamma  subunit was shown to induce cation channel activity in Xenopus oocytes (11), consistent with several reports of other channel-inducing membrane peptides (8, 9, 21). These proteins have homology with the gamma  subunit but are generally larger, and some contain possible protein kinases C and A phosphorylation sites at their C-terminal ends that are not present in the gamma  subunit (5, 8-10). Although we have no information regarding such a role in our transfected cells, it should be pointed out that the sequence of the putative human gamma  subunit reported in the aforementioned study contains 30 extra amino acids at its N terminus (11) that are absent in rat gamma  (cf. Fig. 1). Whether the rat gamma  subunit also has a channel function and/or this extended N terminus confers a particular functional role in forming channels in Xenopus oocytes remains to be determined.

The N-terminal sequence of the rat gamma  subunit reported here and by Minor et al. (11) is different from the one originally reported (5). That it is the correct sequence is substantiated by the following observations. First, the gamma  subunit doublet present in membranes of transfected cells corresponds in size to that of kidney membranes (Fig. 2). Second, the presence of a lysine residue at position 13 (Fig. 1) where a glutamine was originally reported (5) is in accordance with the finding that the upper band of the rat gamma  subunit is cleaved by trypsin (treatment of intact right-side-out microsomes (12)). Third, preliminary results using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy indicate that the pig kidney gamma  subunit has a length of between 64 and 67 residues,4 consistent with a length of 66 residues reported here and in Ref. 11.

The presence of two distinct bands of gamma  has been the subject of some controversy. Whereas Mercer et al. first showed that a single RNA species could yield two protein products evidenced on Western blots using an artificial translation system (5), Béguin et al. (7) showed that in X. laevis the two bands were secondary to the presence of two distinct start codons (7). Our results with 5'-RACE analysis preclude the presence of distinct ATG codons for the rat protein, indicating instead that post-translational modifications are involved, because transfection of HEK-293 cells with a gene containing single start and stop codons yielded two bands of similar mobilities to those of the kidney gamma  subunit. In addition, preliminary mass spectroscopy results are consistent with the notion that the difference between the two bands is the result of post-translational modifications.4 The differences in the ratio of the densities of the upper to the lower band between gamma  subunits of kidney and transfected HEK membranes (see Fig. 2) suggest tissue-specific variations in post-translational modifications. Whether each band has some distinct role remains to be determined.

Overall, our results suggest an interaction between the Na,K-ATPase and the C-terminal tail of the gamma  subunit that regulates ATP affinity and that is reversible upon binding of antibodies to gamma . The finding that gamma  increases the apparent affinity for ATP in gamma -transfected cells is completely concurrent with the effect of anti-gamma on the alpha beta gamma pump of the kidney tubule. Moreover, under conditions in which K+ sensitivity of Na-ATPase at low ATP concentration is used as a sensitive marker of differences in ATP affinity, the reversal of the gamma  effect by anti-gamma is similar with the enzyme of gamma -transfected cells and the kidney medulla. These similarities underscore our earlier interpretation of the effect of gamma  from analysis of the effects of the anti-gamma antiserum. Whether the increased apparent affinity for ATP is, in fact, a true increase in affinity or a reflection of an alteration in conformational equilibrium toward E1 form(s) is unclear and requires further analysis, as does the question of whether the difference in ATP affinity can be evidenced in a change in apparent affinity for extracellular K+. Whatever the case, it is the change in ATP affinity that is likely to be of major physiological relevance.

The increase in apparent affinity for ATP effected by gamma  is approximately 2-fold, as evidenced in either the effect of anti-gamma on the kidney enzyme or of gamma  transfected into HEK cells, extrapolating the ratio of gamma :alpha in HEK-pREP-gamma to that of the kidney. Such a change in apparent affinity may be of critical physiological importance. Although other physiological functions may be served by the gamma  subunit (as suggested recently by Jones et al. (22)), an almost 2-fold shift in ATP affinity is a potentially important regulatory mechanism. The gamma  subunit may serve to preserve the pumping activity in cells or conditions in which the ATP level falls suddenly. Relevant to this notion is the observation that the renal outer medulla is highly prone to anoxia because it works on the brink of anoxia even in normal circumstances (23, 24). That the gamma  subunit effect is reversible upon addition of anti-gamma antibodies further underscores its physiological relevance. It may be hypothesized that, like the anti-gamma antibodies, some cytosolic factor binds to the gamma  subunit and disrupts its interactions with the enzyme. Mutational analysis of the C-terminal 10 amino acids that comprise the epitope reactive with anti-gamma may provide information on specific residues involved in alpha -gamma interactions.

    ACKNOWLEDGEMENTS

We thank Drs. Robert W. Mercer, Washington University, and David C. Clarke, University of Toronto, for invaluable suggestions and Mrs. Ania Wilczynska for technical assistance. The 6H antibody was a generous gift from Dr. Michael Caplan, Yale University. We also acknowledge the Alberta Peptide Institute for synthesis of the peptide used in generating antiserum gamma C33.

    FOOTNOTES

* This work was supported by grants from the Medical Research Council of Canada (MT-3876 to R. B.), the Quebec Heart and Stroke Foundation (to R. B.), and the Weizmann Institute Renal Research Fund (to S. J. D. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The necleotide sequence reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number AF129400.

§ Recipient of a predoctoral scholarship from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.

parallel To whom correspondence should be addressed: Montreal General Hospital, 1650 Cedar Ave., Rm. L11.132, Montreal, Quebec H3G 1A4, Canada. Tel.: 514-937-6011, Ext. 4501; Fax: 514-934-8332; E-mail: mirb{at}musica.mcgill.ca.

2 A. Therien, R. Daneman, and R. Blostein, unpublished observations.

3 R. W. Mercer, personal communication.

4 A. Shainskaya and S. J. D. Karlish, unpublished observations.

    ABBREVIATIONS

The abbreviation used is: 5'-RACE, 5'-rapid amplification of cDNA ends.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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