Site-directed Mutagenesis of a Conserved, Extracellular Aspartic Acid ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 01989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 36, Issue of December 25, pp. 21902-21906, 1989 Printed in U.S.A .

Site-directed Mutagenesis of a Conserved, Extracellular Aspartic Acid Residue Affects the Ouabain Sensitivity of Sheep Na,K-ATPase* (Received for publication, July 21, 1989)

Elmer M. Price$, Debbie A. Rice, and Jerry B. Lingrel From the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524

Site-specific mutagenesis was used to study the function of a conserved, extracellular aspartic acid residue from the sheep Na,K-ATPase a subunit. This amino acid, Asp- 121, is the penultimate residue of the first extracellular domain of the a! subunit. The border residues of this particular extracellular loop of thea! subunit have been shown to be determinants of ouabain sensitivity(Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to determine if Asp-121 isinvolved in ouabain binding, five different amino acid substitutions at this position were generated. Four of the five mutant a! subunits, containing either Asn, Ala, Glu, or Ser in place of Asp-121, conferred ouabain resistance to HeLa cells when expressed in those cells. Cloned sublines of cells selected in ouabain were characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. The cells expressing the mutant Na,K-ATPase a subunit containing either Asn, Ala, Glu, or Ser in place of Asp121 contained a component of Na,K-ATPase activity that was nearly 100-times more resistant to ouabain than the endogenous HeLa (human) or sheep enzyme. Apparently, conservative (Glu for Asp), isosteric (Asn for Asp), and nonconservative (Ala or Ser for Asp) substitutions all significantly decreased ouabain sensitivity. These data suggest that Asp-121 of the sheep Na,K-ATPase a subunit participates inthe binding interaction between the enzyme and ouabain.

ably contains the binding sitesfor Na+, K’, and ATP, is the larger of the two subunits witha M , of 112,177 (sheep enzyme, Shull et al., 1985). The p subunit (polypeptide M , = 34,937, sheep enzyme; Shull et al., 198613) is aglycoprotein whose function has yet to be determined.ComplimentaryDNAs encoding the a subunit have been isolated from numerous sources such as sheep (Shullet al., 1985), human (Kawakami et al., 1986a), porcine (Ovchinnikov et al., 1986), rat (Shull et al., 1986a), avian(Takeyasu et al., 1988), Xenopus laevis (Verrey et al., 1989), Torpedo californica (Kawakami et al., 1985), and Drosophila melanogaster (Lebovitz et al., 1989). Cloning (Shull et al., 1986a) and biochemical (Sweadner, 1979) dataindicatethat multipleisoforms of thesubunit exist, and these have been designated al, a2, and a3. The complimentary DNAs encoding the (3 subunit have alsobeen isolated from many sources (Shull et al., 1986b; Kawakami et al., 198613; Noguchi et al., 1986; Ovchinnikov et al., 1986; Young et al., 1987; Brown et al., 1987; Takeyasu et al., 1987; Verrey et al., 1989). A more complete review of the molecular biology of Na,K-ATPase will be published elsewhere (Lingrel et al., 1990). In addition to its multifacetedrole in cellular homeostasis, Na,K-ATPase is the targetenzyme for a class of drugs known ascardiac glycosides (Repke, 1963; Hansen, 1984). These drugs, such as digoxin, digitoxin, and ouabain, bindavidly to and subsequently inhibitmostforms of the enzyme. The resulting decrease inNa,K-ATPaseactivitycausesanincrease in intracellular Na’ levels, which is thought to cause an increase in intracellular Ca2+ concentration viaa Na+/ Ca’+ exchange protein (Thomas, 1981; Smith, 1988). In the Na,K-ATPase is a ubiquitous plasma membrane-derived failing heart, this increase in Ca2+ levels results in an increase enzyme which establishes and maintains an electrochemical gradient ofNa’ andK+ions across the cell membrane in the strength of myocardial contraction (Smith, 1988). For (Schwartz et al., 1975; Jorgensen, 1982). This is achieved by this reason, cardiac glycosides have been used extensively in the Na,K-ATPase-mediated active transport of Na+ out of the clinical management of congestive heart failure (Purdy drugs are often and K+ into the cell, a process which utilizes the hydrolysis and Boucek, 1988), despite the fact that these toxic to the patient (Smithet al., 1984). of ATP as the energysource. This ion gradient servesa Due to the pharmacological importance of these types of number of cellular functions such as providing the electrochemical energy required for active transport of certain sol- drugs, there exists considerable interest in the characterizautes (Ullrich, 1979), maintenance of cell volume (MacKnight tion of the cardiac glycoside binding site on Na,K-ATPase. and Leaf, 1977) and restorationof the membrane potential in Initial efforts entailed utilizing traditional biochemical approaches toprobe the binding sitefor these compounds. Most electricallyexcitable membranes(Thomas, 1972). The enzyme consists of both an cy and subunit and it is unclear as labeling studies using affinity and photoaffinityanalogues of towhetherthe activeenzyme existsasone a andone p cardiac glycosides resulted in the covalentmodification of the subunit or as an ( a p ) dimer (Askari, 1987). The cy subunit, a subunit of the enzyme (Ruoho and Kyte, 1974; Forbush et which has been designated the catalytic subunit as it presum-al., 1987; Rogers andLazdunski, 1979; Rossi et al., 1980; Jorgensen et al., 1982; Deffo et al., 1983; Goeldner et al., 1983). * This work wassupported by National Institutes of Health Grants When a photoprobe was used whose reactive moiety was far R 0 1 HL28573 and PO1 HL22619. The costs of publication of this removed from the body of the cardiac glycoside, both the cy article were defrayed in part by the payment of page charges. This and p subunits were labeled (Hall and Ruoho, 1980). Although article must therefore be hereby marked “aduertisement” in accordthese data indicate that most, if not all, of the cardiac glycoance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of National Institutes of Health Postdoctoral Fellow- side binding site resides on the a subunit, it is evident that the binding site iscomplex and may involve numerous extraship HL07806-02.

21902

Mutagenesis Site-directed cellular domains of the enzyme. A recent study (Price and Lingrel, 1988) using chimeras between sheep and rat Na,KATPase a subunits and site-specific mutagenesis has identified two amino acids that are responsible for the large difference in ouabain sensitivity observed between the rat Na,KATPase and most other forms of the enzyme (Repke et al., 1965; Periyasamy et al., 1983). These residues are the border amino acids of the first extracellular domain of the a subunit. Presumably,this region of theNa,K-ATPase molecule is involved in ouabain binding. In this report, we present evidence indicating thatAsp-121, which is conserved in all Na,KATPases of known sequence and islocated in the first extracellular domain of the a subunit,is involved inouabain binding. EXPERIMENTALPROCEDURES

Materials-Cell culture supplies were purchased from Gibco and Fisher. Molecular biology reagents were from New England Biolabs, Amersham Corp., Pharmacia LKB Biotechnology Inc., or Promega. Electroporationequipment was from Bio-Rad.Ouabain was purchased from Calbiochem. Pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, ATP,and NADH were from Sigma. Other reagents and supplies were of the highestquality available. The eukaryotic expression vector used in this study, pKC4, andthe expression vectors containing either the rat or sheep Na,K-ATPase a1 subunit cDNA have been described elsewhere (Price and Lingrel, 1988). Site-directed Mutagenesis-Asp-121 of the sheep Na,K-ATPase a1 subunit was changed to Asn, Ala, Glu, Ser, or Lys using site-specific mutagenesis (Kunkel, 1985). The DNA constructsas well as the methodology have been previously described (Priceand Lingrel, 1988). The mutagenesis efficiency was usually 50% or greater. Cell Culture and Transfection-HeLa cells were maintained as monolayers in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum and antibiotics. The medium was replaced every 3 daysand thecells were split via mild trypsinization when confluent. HeLa cells were transfected via electroporation essentially as described previously (Price and Lingrel, 1988). Briefly, the cells were harvested via trypsinization and washed once with culture media and once with phosphate-buffered sucrose (7 mM NaP04, pH7.4,272 mM sucrose). The cells were resuspended in phosphate-buffered sucrose at a density of 2.5 X lo6 cells/ml. The expression vector containing the mutant of interest was linearized with the restriction endonuclease ScaI, phenol/chloroform-extracted, ethanol-precipitated, and resuspended in 400 p1 of phosphate-buffered sucrose. Cells (400 pl) were incubated with the DNA for 2 min and electroporated at 370 volts, 25 microfarads. The cells were allowed to incubate at ambient temperature for 10 min and then placed into a 100-mm tissue culture dish containing 10ml of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Two days later, the medium was replaced and ouabain was added to a final concentration of 0.3 p ~ The medium was replaced every 2 or 3 days, as was the ouabain. After about 3 weeks, isolated colonies were picked using cloning cylinders and expanded into stable sublines. These ouabain-resistant cells were maintained in 0.1 pM ouabain. Ouabain-inhibitable cell growth was measured exactly as described previously (Price and Lingrel, 1988). Preparation of Crude Plasma Membranes from HeLa Cells-Crude membranes containing Na,K-ATPase were isolated from wild-type HeLa cells andtheouabain-resistanttransfectants essentially as described (Price and Lingrel, 1988) with some modifications. Cells were grown in 850-cm’ roller bottles and were harvested either by scraping with a rubberpoliceman or by mild trypsinization. The latter method consistently gave higher yields of Na,K-ATPase activity and was used for the assays described later. The yields ranged from 500 to 800 pgof total protein, determined by the Coomassie Blue dye binding assay (Bradford, 1976), using bovine serum albumin as the standard. The preparation was stored at -70 “C and was assayed within 1 week. Na,K-ATPase Activity Assay-Ouabain-inhibitable Na,K-ATPase activity was measured as described (Price and Lingrel, 1988) with some changes. Activity was quantitated using the spectrophotometric enzyme-coupled assay (Schwartz et al., 1969), mually using 10 pg of crude membrane protein for each data point. The sample was incubated with the desired concentration of ouabain for 30 rnin at 37 “C in a cuvette containing 25 mM histidine, 5 mM MgCl,, 100 mM NaCl,

of Na,K-ATPase

21903

10 mM KC1,l mM phosphoenolpyruvate, 5mM ATP, 0.43 mM NADH, 11 p~ sodium dodecyl sulfate, =5 units of pyruvate kinase, and -7 units of lactate dehydrogenase, pH 7.4. The enzyme activity was measuredduring the last5 min of this incubation. The activity obtained in the presence of 7.8 mM ouabain was subtracted from that activityobtained at each ouabain concentrationto yield residual Na,K-ATPase activity at each data point. The ouabain-inhibitable Na,K-ATPase activity accounted for 50-80% of the total ATPase activity in the samples, usually 25-85 pmol of ATP hydrolyzed per mg protein/h. No difference in enzyme activity was observed when a sample (10 pg) was preincubated with 4 pg of sodium dodecyl sulfate for 20 min prior to the assay, indicating that the membrane preparation was not made up of sealed vesicles. Northern Analysis-RNA was isolated from wild-type HeLa cells and HeLa transfectants, electrophoresed, and blotted onto Nytran (Schleicher & Schuell) as described previously (Price and Lingrel, 1988). The blots were probed with an approximately 430-base pair DNA fragment derived from the 3”untranslated region of pKC4, the expression vector used in this study. This probe contains sequences of the small t intron and the poly(A) addition signal, both derived from SV40 sequences. Such a probe should only hybridize to RNA expressed by the vector and should not hybridize to any endogenous HeLaRNA. In order to account for anydiscrepancies in sample loading, the blots were also probed with an actin-specific probe. RESULTSANDDISCUSSION

There existsconflicting reports in the literature pertaining of transmembranedomains of theNa,Ktothenumber ATPase a subunit,with some models describingsix such domains(Kawakami et al., 1986a), others showingseven (Ovchinnikov et al., 1986) and others indicating eight (Shull et al., 1985). There is, however, good agreement in the membrane organization of the amino-terminal half of the a subunit, which appears to have two extracellular domains. The border residues of the first extracellularloop of the a subunit have been shown to be involved in mediating the ouabain sensitivity of the enzyme (Price and Lingrel, 1988). When uncharged aminoacids are presentat each border, the enzyme has a high affinity for ouabain while placing charged residues a t thesepositionsresultsinthegeneration of a ouabainresistant enzyme. Based on this observation, it is logical to assume that the first extracellular domain of the Na,K-ATPase a subunit is part of theouabainbindingsite on the enzyme. Critical amino acids which play a functional role in Na,K-ATPaseactivityshouldnot diverge throughoutthe course of evolution. Of the 12 amino acids which make up this loop, two (Pro-118 and Asp-121 in the sheep sequence) are completelyconserved inallNa,K-ATPases of known sequence (Fig. 1).There is amino aciddiversity at the re. maining positions, although manyof the differences are conservative. In order to determine the functionalrole, if any, played by the conserved Asp-121 of the sheep a1 subunit, site-specific mutagenesis was used to make conservative and nonconser111 112 113 114 115 116 117 118119120121

Sheep a l , Human a l : Porcine a 1 Rat a l : Rata2: Rat a3: Avian a l : Xenopus a : Torpedo a : Drosophila a :

122

Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn Arg-Ser

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-

-

-

-

-Met -Asp Gly - - Asp-Asp Thr-Ser-Val-Met - Gly - - - Met - - - Val - - Val-Asp-Asn - Ser - Ser Leu

-

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-

-

Pro

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-

- Ser - - Ser-Gly - Asn-Ser - - - Ala -

Asp

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FIG. 1. Amino acid sequence comparisons of the first extracellular loop from the Na,K-ATPaseCY subunit. The upper amino acid sequence is the firstextracellular loop from the sheep, human or porcine a1 subunit, which have identical sequences in this region. The numbering is taken from the sheep sequence. The comparisons between other a subunits is shown below the numbered sequence. Identical amino acids are indicated with a dash (-) and when amino acid differences occur, the substituted residue is shown. The references for each sequence are stated in the text. -

Ala-Asp

-

21904

Site-directed Mutagenesis of Na,K-ATPase

vative amino acid replacements at this position. Five such mutant N subunits were generated: D121N (Asp a t position 121 replaced with a Asn), D121A (Asp to Ala), D121E (Asp to Glu), D121S (Asp to Ser), and D121K (Asp to Lys). The mutant D121N represents an isosteric substitution sinceAsn has nearly the same structure andhydrogen bonding characteristics asAsp, but lacks the negative charge (Knowles, 1987). D121A is a mutant thatwas designedto eliminate the negative charge but preserve the secondary structure in this region of the extracellularloop since bothAsp and Ala are predicted to becompatible with an N helix (Chou and Fasman, 1974). D121E is a conservative amino acid replacement as Glu is also negatively charged, but has a longer sidechain than Asp. D121S places a hydroxyl group near where the negative charge of Asp-121 is in the wild-type enzyme. D121K is a nonconservative amino acid replacement which substitutes a negatively charged amino acid with a positively charged one. Each of these mutantswere transfected into HeLa cells and assayed for their ability to confer ouabain resistance to the normally ouabain-sensitive cells. HeLa cells are human in origin and contain a form of Na,K-ATPase that is,like most other formsof the enzyme except rodent CUI, inhibited by low concentrations of ouabain. After transfection, resistant cells were selected in 0.3 PM ouabain, a concentration that never gave rise to resistant cells in control mock- or nontransfected cells. Four of the five mutant CY subunits generated numerous ouabain-resistant colonies when transfected into HeLa cells (Fig. 2). Expression of a mutant N subunit containing either Asn, Ala, Glu, or Ser instead of Asp a t amino acid position 121 is apparently sufficient to overcome the usually inhibitory effect of ouabain. As described previously, expression of the rat a1 subunit cDNA in HeLa cells also generates resistant cells (Fig. 2), while transfection with the sheep a1 subunit cDNA does not (data not shown; Price and Lingrel, 1988). Isolated colonies from each transfection were picked using cloning cylinders and maintained as stable sublines.Cells transfected withD121K were not able tosurvive the selection step in 0.3 P M ouabain. Experiments are currentlyunderway to determine if the D121K mutation results in an inactive enzyme. Northern analysis was performed on RNA isolated from

each subline to verify that each mutant was actually being expressed in the resistant cells. The probe, derived from the 3”untranslated region of the expressionvector containing SV40-derived sequences, should only hybridize to RNA that is transcribedoff the vector and not to any endogenous RNA. As shown in Fig. 3A, each of the sublines transfected with mutants D121N, D121A, D121E, or D121S, or the rat a1 cDNA express a single RNA species that hybridizes to the vector-specific probe.The size of this RNA isidentical to that seen when similar Northerns are probed with Na,K-ATPase CY subunit cDNA (Price and Lingrel,1988). No detectable hybridization is observed in RNAisolated from wild-type HeLa cells. Hybridization of the same blot with an actindifferences in signal specific probe (Fig. 3B) indicates that the intensities seenbetween samples is due to differences in levels of RNA expression and not toloading artifact. Each subline was also characterized in terms of ouabaininhibitable cell growth (Fig. 4). The transfectants were able to proliferate in levels of ouabain which were toxic to wildtype HeLa cells. There was a correlation between the level of expression of each mutant, determinedby Northern analysis (Fig. 3A), and the degree of ouabain-resistance, determined by cell growth. Mutant D121A, which had the lowest level of RNA expression, also had the lowest Iso for ouabain-inhibitM). Mutants D121N and able cell growth (about 5 X D121S, both had the highest levels of RNA expression as well M. Thesecorreasthehighest Iso of approximately 5 X lations were made with data from subline 1 of each transfectant and are not meant include to rat a1 transfectants. These results indicate that the concentration of ouabain required to kill aparticular transfectantis partially a function of the level of expression of the mutant a subunit. The above data indicate that determining the Is” for ouabain-inhibitable cell growth may be a function of both levels of mutant enzyme as well as the affinity of that mutant for ouabain. In order to directly study the interaction between the mutant enzymes and ouabain, ouabain-inhibitable Na,KATPase activity was measured in membranes prepared from each subline (Fig. 5). Na,K-ATPase isolated from untransfected HeLa cells exhibits an apparent Iso for ouabain inhim

1

2

1

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1

2

1

2

B.

FIG. 2. Ouabain-resistant HeLa cell colonies. Approximately 1 X lofi cells were electroporated with 20 pg of linearized vector as

descrihed under“Experimental Procedures.” Resistant cells were selected in 0.3 p~ ouahain for about 20 days, after which time the colonies were stained with crystal violet. Rat n l refers to cells transfected with the rat Na,K-ATPase n l cDNA.D121N, D121A, DIZlE, D121S, and D121K aredifferentsheepNa,K-ATPase nl suhunit mutants which are described in the text.

FIG.3. Northern analysis of RNA isolated from ouabainresistant HeLa cell transfectants. RNA was isolated from two sublines each (designated 1 and 2) of cells transfected with D121N, D121A, D121E, and D121S, as well as from wild-type ( W T ) HeLa cells and rat n l cDNA transfected cells. 20 pg of RNA was run on each lane. A , hybridization pattern observedwhen the RNA was probed with a DNA fragment that was derived from the 3”untranslated region of the expression vector pKC4. Shown is the autoradiogram following a 4-day exposure. The arrow indicates the location of the 28 S ribosomal RNA. R, same as A, hut the blot was probed with a 1.1-kilohaseDNA fragment from the rat p-actincDNA. Shown is the autoradiogramfollowing a 1-day exposure. The arrow indicates the location of the 18 S ribosomal RNA.

Site-directed Mutagenesis of Na,K-ATPase

21905

I

100 times more resistant than the HeLa or sheep enzyme. The proportion of low ouabain affinity Na,K-ATPase observed in the rata1 cDNA transfectants is inconsistent with the levels of mRNA expression (Fig.3). This may be explained by the fact that the amount of endogenous,high ouabain affinity Na,K-ATPase may not be constant between the different transfectants. The active enzyme in the transfectants is presumably a hybrid molecule, containing the a subunit encoded by the expression vector and the endogenous HeLa p subunit. The presence of such a hybrid Na,K-ATPase has been experimentally verified in mouse cells transfected with avian subunits (Takeyasuet al., 1987). Ouabain. The experiments presented here indicate that Asp-121 of FIG. 4. Ouabain-inhibitable cell growth. Theconcentration sheepNa,K-ATPaseis involved inouabain binding. Four dependence of ouabain-inhibitable cell growth of wild-type HeLa cells different amino acid substitutions at this position were genand HeLa transfectants was determined as described under “Experimental Procedures.” Duplicatedata pointswere taken at each ouabainerated that, when expressed in HeLa cells, generate a relaconcentration and the experiment was repeated at least twice. The tively ouabain-insensitive Na,K-ATPase. One of the substidata shown here are the averages from duplicate data points, using tutions (D121N) was isosteric, changing Asp to Asn, which subline 1 from each transfectant. Error bars have been omitted for should preserve the structure and hydrogen bonding characthe sake of clarity, but the percenterror was rarely greater than 5%. teristics of the region while removing the negative charge. The data are presented as percent cell growth at a particular ouabain concentration relative to the growth observed in the absence of The second replacement (D121A) was with an Ala residue, secondary strucouabain. 0, wild-type HeLa; 0, D121A; 0, D121E; V, D121S; A, which should maintain essentially the same ture, while removing both the charge and hydrogen bonding D121N; 0, rat a1 cDNA transfectants. characteristics of the original Asp. The third replacement (D121E) was a conservative one, changing Asp-121 to a Glu, I I which is also negatively charged but hasa side chain which is one methylene grouplonger than that of Asp. The fourth mutation (Dl2lS) changed Asp-121 to Ser, which has a hydroxyl group which mayparticipate inhydrogen bonding, but is uncharged. All of these mutants resulted in active an Na,KATPase with a reduced sensitivity for ouabain (Fig. 5). Interestingly, each mutant had an identical 150 for ouabain-inhibitable Na,K-ATPase activity. In terms of ouabain binding, these data indicate that several amino acids including a different acidicresidue (glutamate) or a structurallysimilar residue (asparagine) cannot effectively substitute for an asOuabain. M partate at position 121 of the sheep Na,K-ATPasea subunit. FIG. 5. Ouabain-inhibitableNa,K-ATPase activity. The con- This suggests an involvement of the carboxyl group of Asp121 in ouabain binding and the necessity for precisepositioncentration dependence of ouabain-inhibitable Na,K-ATPase activity in wild-type HeLa cells and HeLa transfectants was determined as ing of this group. The carboxylate of Asp-121 may be interdescribed under“Experimental Procedures.”Duplicatemeasureacting with another extracellular region of Na,K-ATPase in ments were taken at each ouabain concentration. Error bars have a manner which helps maintain the integrity of the binding been omitted for the sake of clarity, but the percent errorwas rarely greater than 10%. The data are presented as percent activitymeas- site. Alternatively, this carboxyl group may directly interact ured at a particular ouabain concentration relative to the total Na,K- with the ouabain molecule, perhaps with the lactone moiety ATPase activity in the absence of ouabain. The symbols are thesame of the inhibitor which has been postulated to associate with as in Fig. 4. The actual measured specific Na,K-ATPase activity (as an anionic site on theenzyme (Thomas et al., 1974). micromoles of ATP hydrolyzed per mg protein/h) of each preparation was: 44 t 5 (wild-type HeLa); 27 t 3 (D121A); 71 t 3 (D121E); 46 t 10 (Dl2lS);84 6 (D121N); 24 t 3 (rat a1 transfectants).

REFERENCES

Askari, A. (1987) J. Bioenerg. Biomembr. 19,359-374 Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 bition of about 8 X lo-’ M. This is approximately the same as Brown, T. A., Horowitz, B., Miller, R. P., McDonough, A.A., and Farley, R. A. (1987) Biochim. Biophys. Acta 912, 244-253 the apparent1.50 for ouabain inhibitionof sheep kidney Na,KChou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 222-245 ATPase (Wallick et al., 1980).Cells that express the a1 Deffo, T., Fullerton, D. S., Kihara, M., McParland, R. H., Becker, R. subunit from the ouabain-resistant rat Na,K-ATPase contain R., Simat, B. M., From, A.H., Ahmed, K., and Schimerlik, M. I. two populationsof enzyme activity, onehaving a highaffinity (1983) Biochemistry 22,6303-6309 for ouabain and one exhibiting a low affinity. The 150 for the Forbush, B., 111, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676 high affinity formcorresponds to the endogenous HeLa Na,KGoeldner, M. P., Hirth, C. G., Rossi, B., Ponzio, G., and Lazdunski, ATPase, while the low affinity IsO (-5 X M ouabain) M. (1983) Biochemistry 22, 4685-4690 corresponds to the observed 160for the rat enzyme (Wallick Hall, C., and Ruoho, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, et al., 1980). Preparations from cells expressing either D121N, 4529-4533 D121A, D121E, or D121S also had biphasic ouabain inhibitionHansen, 0. (1984) Pharmacol. Reu. 36, 143-163 curves. Again, similar to rat a1 subunit transfectants, these Jorgensen, P. L. (1982) Biochim. Biophys. Acta 694,27-68 transfectants all contained ahighaffinity form of Na,K- Jorgensen, P. L., Karlish, S. J. D., and Gitler, C. (1982) J . Biol. Chem. 257, 7435-7442 ATPase corresponding to the endogenous HeLa enzyme. The K., Noguchi, S., Noda, M., Takahashi, H., Ohta, T., lower affinity form of the enzyme in thecase of the transfec- Kawakami, Kawamura, M., Nojima, H., Nagano, K., Hirose, T., Inayama, S., tants, while not as ouabain-insensitive as the rat enzyme, Hayashida, H., Miyata, T., and Numa, S. (1985) Nature 316,733exhibited an ISO of approximately 5 X lou6M, which is nearly 736

21906

Mutagenesis Site-directed

of Na,K-ATPase

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