K+-independent active transport of Na+ by the (Na+ and K+ ...

Communication K+-independent Active Transport of Na' by the (Na' and K+)stimulated Adenosine Triphosphatase* (Received for publication, December 1, 1980, and in revised form, January 26, 1981)

Michael Forgac andGilbert Chin$ From the Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138

The (Na' and K+)-stimulatedadenosine triphosphatase ((Na+,K+)-ATPase)from canine kidney reconstituted into phospholipid vesicles showed an ATP-dependent, ouabain-inhibited uptake of "Na+ in the absence of added K+. This transport occurred against a Na+ concentration gradient, was not affected by increasing the K+ concentration to 10 p~ (four times the endogenous level), and could not be explained in terms of Nai', s Na&,,exchange. K+-independent transport occurred with a stoichiometry of 0.5 mol of Na+ per mol of ATP hydrolyzed as compared with 2.9 mol ofNa+ per mol of ATP for K+-dependenttransport.

five successive acetoneprecipitationsfromether (101, v/v) (6). Purifiedlipidwas stored as asuspension (140 mg/ml) in water containing 5 mM 2-mercaptoethanol a t -70 "C under nitrogen. Tns/ ATP (vanadate-free)and cholicacidwere obtained fromSigma Chemical Co. and the cholic acid was crystallized from 95% ethanol. ?'NaCl, Na"C1, and "RbCI were purchased from New England Nuclear. Reconstitution of the (Na+,K')-ATPase was carried out essentially by the procedureof Goldin (4) except thatphospholipid was addedas an aqueoussuspension at 4 "C and the reconstitution buffer contained 0.25 M sucrose, 50 m~ NaC1, 30 mM imidazole (pH 6.8). 10 mM 2mercaptoethanol, and 1 m~ EDTA. ATPase activity was measured by release of 32P,from [y"P]ATP (4) and transport activity was measured by separation of vesicles from the external solution on a Sephadex G-50 (coarse) column (0.7 X 22 cm, 1.0 ml/min) (4). Protein concentrations were measured by the method of Lowry et al. (7) in the presence of 1%sodium dodecyl sulfate. The amountof K' present was determined by atomic absorption spectroscopy and the amount of NH: present was determined by amino acid analysis. RESULTS AND DISCUSSION

In order to measure active transportof Na+ by the (Na')ATPase without interference from ATP-stimulated Na; Naft exchange (8), it was necessary to pre-equilibrate the vesicles with "Na+. From the data in Fig. 1, it can be concluded that Na' is completely equilibrated after incubation for 3 days a t 37 "C (C1- reaches the same equilibrium level but more rapidly). Addition of M g + and ATP to vesicles equilibrated by the above procedure resulted in an increase in the amount of The (Na',K+)-ATPase' functions in vivo to couple the 22 Na' trapped (Fig. 2). This Na' uptake was dependent on active transport of Na+ and K' across theplasma membrane ATP and inhibitedby ouabain which had been added at the of animal cells to ATP hydrolysis (for review, see Ref. 1). In start of theequilibration period(Fig. 2). SinceNa+ was the absenceof K', a ouabain-inhibited (Na+)-ATPase activityinitially present at the same concentration on both sides of is observed (2) and one might ask whether Na+ transport is the membrane, this uptake represents net transport of Na+ coupled to this Na+-stimulated ATP hydrolysis. Glynn and against a concentration gradient. Measurement of (Na')Karlish (3) have observed an ATP-dependent, ouabain-inATPase activity under the same conditions gave a stoichihibited Na' efflux from erythrocyte ghosts resealed right-side- ometry of 0.52 (k 0.04)mol of Na' per mol of ATP hydrolyzed out and suspended in medialacking Na' and K'. They have (4 determinations). When reconstituted vesicles were equilisuggested that this efflux represents the transport mode as- brated with "Na' and 20 mM KCl, ATP-dependent uptake of sociated with the (Na')-ATPase.However,since Na+ flux 22 Na' occurred with a stoichiometry of 2.9 (k 0.1) mol of Na' occurs down a concentration gradientin this system, the result per mol of ATP hydrolyzed (2 determinations). When "Rb' could be explainedas an ATP-induced leakage of Na+ through transport was measured under the sameconditions, an ATPthe enzyme. In an attempt to determine whether Na' can be dependent 86Rb+efflux was observed with a stoichiometry of pumped against a concentration gradient in the absence of 1.67 (& 0.10) mol of "Na' per mol of "Rb+ (2 determinations). K', we have examinedNa' transport by the (Na',K+)-ATPase This value is in agreement with the stoichiometry observed reconstituted into artificial phospholipid vesicles by the proby Goldin (4) in reconstituted vesicles and by Sen and Post cedure of Goldin (4). Our results indicate that uphill transport (9) in red blood cells. Thus, reconstituted vesicles were still of Na' can occur in a K'- independent process. capable of normal ATP-dependent Na+ and K' transport after incubation for 3 days a t 37 "C. MATERIALS AND METHODS T o determine whether the observed Na' uptake could be (Na',K+)-ATPase was isolated from canine kidney by the proceexplained in termsof coupled Na'/K' transport at theendogdure of Jorgensen (5). Phosphatidylcholine was purified from fresh enous level of K' (2.5 p ~ )Na+ , transportwas measured at 5.0 egg yolk by chloroform/methanol extraction (2:1, v/v) followed by and 10.0 ~ L MK' (Table I). The amount of Na+ uptake is seen in this concentration range. The * This work was supported by National Institutes of Health Grant to be independent ofK' HL 08893 and National Science Foundation Grant PCM 78-04364. concentration of NH; ion (a K' analog) was determined b y The costs of publication of this article were defrayed in part by the payment of page charges.Thisarticlemustthereforebehereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ National Science Foundation Predoctoral Fellow. The abbreviations used are: (Na',K')-ATPase, (Na+ and K + ) stimulatedadenosinetriphosphatase;(Na+)-ATPase,(Na+)-stimulated adenosine triphosphatase; NaL $ Na:,, exchange, ATP-stimulated exchange of an equal numberof internal and externalNa' ions.

'

(Na')-ATPase activity of reconstituted vesicles a t 23 "C ranged from 1.2 to 2.2 pmol of ATP/h/mg of protein while (Na',K')-ATPase activity of vesicles equilibrated with 20 mM KC1 ranged from 10 to 22 pmol of ATP/h/mg of protein. ATPase activity was 30 to 40% inhibited by externallyaddedouabain(although equilibrationwith ouabain caused greater than95% inhibition), indicating that 30 to 40R of the ATPase was unreconstituted. The stoichiometries given were corrected for this ouabain-inhibitable activity.

3645

K'- independent Na' Transport by the (Na',K+)-ATPase

3646

TABLE I Dependence of Nu+ uptake on K' concentration Vesicles (0.25 mg/ml of protein) were equilibrated in reconstitution buffer containing 10 p C i / d of **Na+and the indicated concentrations of KC1 for 3 days at 37 "C. After equilibration, 2 mM ATP and 5 m MgC12 were added and "Na+ transport was measured at 23 "C as described under "Materials and Methods." Values are expressed as a percentage of the uptake observed at 2.5 p~ KC1 (each value is an average of the percentage uptake measured at 1.0and 2.0 h).

1.4

[KClL

56 control "Na'

uptake

(PM)

2.5 5.0 10.0 1

I

0

10

40

40

80

100

rim. (hrrl FIG. 1. Passive permeability of reconstituted vesicles to "Na+ and 38Cl- at 37 "C. Reconstituted vesicles (0.20 mg/ml of protein) were incubated at 37 "C in reconstitution buffer containing 10 p C i / d of **NaCI (0) or 5 &/ml of Na"C1 (8). At the indicated times, 20-4 aliquots were removed and analyzed for trapped isotope as described under "Materials and Methods."

1.4

1.2

c

4 1

I

20

40

I

I

1

I

40

80

100

120

rim.(min)

FIG. 2. Na+transport by the (Na+)-ATPase.Reconstituted vesicles (0.20 mg/ml of protein) were equilibrated for 3 days at 37 "C in reconstitution buffer containing 10 pCi/ml of"NaC1 either in the absence (0,A) or the presence (0) of 200 p~ ouabain. After equilibration, either 5 m MgC12 (A)or 2 m ATP and 5 m MgC12 (0, 0) were added, vesicles were incubated at 23 "C, and 50 p1 aliquots were removed at the indicated times and analyzed for trapped '*Na+ as described under "Materials and Methods."

amino acid analysis to be 1.4 PM and measurement ofNa' transportat 2 and 4 times this concentration gave rates relative to that observed at the endogenous level of 1.03 and 1.13, respectively. Finally, in order to explain the observed Na' uptake on the basis of a 3 Na+/2 K+ exchange (which would require leakage of K' back into the vesicles), a passive

diffusion rate of 8000 times the measured value wouldbe required (data not shown). Our results indicate that the (Na',K')-ATPase is capable of K'-independent transport of Na' against a concentration gradient. The stoichiometry of 0.5 mol of Na' per mol of ATP hydrolyzed (as compared with 2.9 mol of Na' per mol of ATP hydrolyzed for K+ -dependent transport), can be accounted for in a number of ways. One possibility is that Na' ions (either 2 or 3) bind to thesites which normally bind K'. Thus, for every 2 ATP molecules hydrolyzed, hydrolysis of 1 is coupled to a 3 for 2 Na' exchange, while hydrolysis of the other accompanies a 3 for 3 exchange. An alternate explanation is that 3 NaC ions move in one direction and that those binding sites return empty (as is suggested to occur when Na' and K+ are absent from the extracellular side of the membrane (3)).However, to account for the observed stoichiometry, this model would involve coupling of only 1 out of every 6 ATP molecules hydrolyzed to Na' transport. At present, we cannot distinguish between these possibilities. Acknowledgments-We thank Guido Guidotti (in whose laboratory this work was done) for his constant encouragement and advice, Stanley Goldin (Dept. of Pharmacology, Harvard Medical School) for his useful suggestions concerning the reconstitution procedure, Robert Quirk (Dept. of Geological Sciences, Harvard University) for his kind instruction in the use of his atomic absorption apparatus, and Michael Ho for his assistance in carrying out the amino acid analyses. REFERENCES 1. Robinson, J. D., and Flashner, M. S. (1979) Biochim. Biophys. Acta 549, 145-176 2. Neufeld, A. H., and Levy, H. M. (1969) J.Biol. Chem. 244,64936497 3. Glynn, I. M., and Karlish, S. J . D. (1976) J.Physiol. 256,465-496 4. Goldin, S. M. (1977) J . Biol. Chem. 252,5630-5642 5. Jorgensen, P. L. (1974) Biochim. Biophys. Acta 356,36-52 6. Folch, J. M., Lees, M., and Sloane Stanley, G. H. (1957) J. Biol. Chem. 266,497-509 7. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J . Bid. Chem. 193, 265-275 8. Garrahan, P. J., and Glynn, I. M. (1967) J.Physiol. 192, 159-174 9. Sen, A. K.,and Post, R. L. (1964) J.Biol. Chem. 239,345-352