Energetics of sodium efflux from Escherichia coli

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 229, No. 1, February 15, pp. 98-103, 1984

Energetics of Sodium Efflux from Escherichia MIGUEL Department

of Biological

G. BORBOLLA

Chemistry,

University

AND

of Maryland

BARRY

co/i’

P. ROSEN’

School of Medicine,

Received August 5, 1983, and in revised form September

Baltimore,

Maryland

21201

23, 1983

When energy-starved cells of Escherichia coli were passively loaded with ‘%a+, efflux of sodium could be initiated by addition of a source of metabolic energy. Conditions were established where the source of energy was phosphate bond energy, an electrochemical proton gradient, or both. Only an electrochemical proton gradient was required for efflux from intact cells. These results are consistent with secondary exchange of Na+ for H+ catalyzed by a sodium/proton antiporter.

Living cells ubiquitously maintain cytosolic sodium ion concentration lower than that of the extracellular milieu. Prokaryotic organisms have evolved a variety of transport mechanisms for the extrusion of sodium against a concentration gradient. Streptococcus faecalis uses an ATP-driven primary sodium pump reminiscent of the eucaryotic cation-translocating ATPases (l-3). In Vibrio alginolyticus sodium extrusion has been postulated to be coupled directly to the respiratory chain (4). In everted membrane vesicles of Klebsiella aerogenes, Veillonella alcalescens, and Acidam.inococcus fermentans, sodium transport has been shown to be coupled to decarboxylation of acids (57), although the significance of the in vitro studies to the sodium metabolism of these organisms is not clear. In Halobacterium halo&m the protein halorhodopsin has been postulated to catalyze the direct coupling of sodium extrusion to light(S), although more recent studies suggest that Cl- and not Na+ may be the species transported (9).

postulated the presence of a bacterial sodium/proton antiporter. In our laboratory (11,12) and in others (13,14) sodium/proton antiporter activity has been observed in both everted and right-side-out membrane vesicles of E. coli. Similar activities have been reported in membrane vesicles prepared from cells of H. halobium (15), Bacillus alcalophilus (16), Salmonella typhimurium (17), and S. faecalis (2). In each case the electrochemical proton gradient was shown to be suficient for in vitro antiporter function, leading to the hypothesis that sodium efflux from intact cells is coupled to the electrochemical proton gradient through secondary exchange catalyzed by the antiporter. The work of Harold and co-workers has challenged the validity of this conclusion (l-3). Although membrane vesicles from S. faecalis exhibited secondary sodium/ proton exchange (2), sodium transport in intact cells was ATP-dependent; an electrochemical proton gradient was neither necessary nor sufficient (1). Indeed, vesicles protected from proteolysis during preparation catalyzed primary ATP-dependent sodium uptake and not antiporter activity (3). It seems clear in this organism that a primary pump exists for the extrusion of sodium and that the secondary exchange seen in vesicles was an artifact of preparation.

West and Mitchell (10) found that sodium increased the proton permeability of the membrane of Escherichia coli and first ’ This work was supported by Grant PCM U-15769 from the National Science Foundation. ‘To whom request for reprints should be sent. 0003-9861/84 Copyright All rights

$3.00

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

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Thus, in vitro studies using membrane vesicles may be inadequate to determine in vivo mechanisms. No matter how carefully vesicles are prepared, they are still artifacts. Their value for biochemical investigations is unquestionable, but only by complementation with results with intact cells can unambiguous conclusions about the physiological form of membrane systems be drawn. In this report we describe the in vivo energetics of sodium extrusion from cells of E. coli. The results confirm our earlier conclusions (11, 12) based on the use of everted membrane vesicles: sodium efflux is coupled to the electrochemical proton gradient; ATP is not required. Existence of a sodium/proton antiporter is the most reasonable conclusion. MATERIALS

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FIG. 1. Extrusion of =Na+ against a concentration gradient. Starved cells were loaded either with 10 mM NaCl and 130 mM KC1 ((A), right ordinate) or 140 mM ((a), left ordinate) NaCl, as described under Materials and Methods. The cells were equilibrated with carrier-free “?Ja’ for 42 min, at which time glucose was added to 10 mM to initiate efflux.

METHODS

Bacterial strains and growth umditions. Cultures were grown in L broth (18). For the experiment described in Fig. 5 the cells were grown in a basal salts medium (19) supplemented with 0.5% glycerol, 0.1 mg/ml of arginine, and 2.5 &g/ml thiamine. E. coli K12 strain AN120 (uncA401, argE3, thi-1, rpsL) (20) was kindly provided by F. Gibson (Australian National University). Starvation of the cells. Cells were depleted of endogenous energy reserves by the method of Berger (21). Overnight cultures (50 ml) were centrifuged at room temperature, suspended in a buffer consisting of 10 mM Tris, pH 7.5, containing 140 mM NaCl and 1 mM MgClz (buffer A) plus 5 mM 2,4-dinitrophenol. In part of the experiment described in Fig. 1 140 mM NaCl was replaced with 10 mM NaCl and 130 mM KCl. Cells were incubated for 2 h in the above medium with shaking at 37°C. The starved cells were centrifuged and washed three times with buffer A minus dinitrophenol. Cells at this point were essentially depleted of ATP and exhibited no endogenous respiration (22). The procedure simultaneously produced sodiumloaded cells. Transport assays. Starved cells (4 mg protein) were suspended in 0.2 ml of buffer A. Carrier-free %a+ (1 &i) was added (final specific activity, 35.7 mCi/ mol), and the suspension was allowed to passively equilibrate at room temperature. As seen in Fig. 1, the equilibration process was complete within 40 min. Efflux was initiated by dilution of sodium-loaded cells LOO-fold (0.05 ml into 5.0 ml) into room-temperature buffer A in which 140 mM choline chloride (or, as noted, other salts) replaced NaCl. Equivalent results were obtained if energy sources was added to the dilution buffer or to the suspension after dilution.

Transport was measured by filtration of a portion of the cells (0.02 ml of undiluted or 1 ml of diluted cells)through 0.45Frn pore size nitrocellulose filters. The filters were washed with 5 ml of the same buffer, dried, and radioactivity measured by liquid scintillation counting. Other methods. Protein was determined by the method of Lowry et aL (23) using bovine serum albumin as a standard. Respiration was measured using a Clark-type electrode. Chemicals. ‘ZNa+ (carrier free) was obtained from New England Nuclear Corporation. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP)3 was a gift from E. I. DuPont De Nemours Company. Other chemicals were reagent grade and purchased from commercial sources. RESULTS

Extrusion of sodium against a cmcentration gradient is energy dependent. Cells of E. coli were depleted of endogenous en-

ergy sources and during that process passively loaded with 10 or 140 mM NaCI. At that point 22Naf was added and allowed to equilibrate. The equilibration was complete by about 40 min (Fig. 1). The free sodium concentration inside and outside the cell at this point would be approximately the 3 Abbreviations used: FCCP, carbonyl cyanide-p trifluoromethoxyphenylhydrazone; PMS, phenazine methosulfate.

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loading concentration. The cells exhibited net extrusion of 22Na+ against a concentration gradient when glucose was given as an energy source (Fig. 1). Downhill sodium extrusion requires energy. We found previously that cells loaded with calcium underwent extensive redistribution of 45Ca2+upon loo-fold dilution of the cells into calcium-free buffer, suggesting that a significant fraction of the calcium was bound to the external surface of the cells (24). In contrast, when sodiumloaded cells were diluted into sodium-free medium nearly all of the radioactivity remained with the cells. The radioactivity was released by permeabilizing the cells with toluene, indicating that the sodium was in free solution in the cytosol (Fig. 2). In the absence of a source of energy, the rate of sodium efflux was slow. When glucose was added as an energy source, rapid downhill efflux of sodium from the cells occurred. The effect of 140 mM NaCl or LiCl on the rate of energy-dependent efflux of 22Na+was nearly equivalent to that of choline chloride (data not shown), suggesting that sodium/sodium exchange was much slower than net extrusion (data not shown). On the other hand, the rate of energy-dependent efflux of sodium down a concentration gradient was accelerated by the presence of KC1 (Fig. 3) or RbCl (data not shown) in place of choline chloride in the

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FIG. 2. Extrusion of wa+ down a concentration gradient. Starved cells were loaded with 140 mM NaCl and equilibrated with carrier-free ?‘J’ for 40 min and then diluted lOO-fold into Buffer A containing 140 mM choline chloride without an energy source (X), with 10 mM glucose (0) or 0.7% (v/v) tohrene (A).

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FIG. 3. Effect of potassium on %a+ efflux. Starved cells loaded with 140 mM NaCl and equilibrated with carrier-free %a’ were diluted loo-fold into sodiumfree buffer A containing either 140 mM choline chloride or KC1 with or without 10 mM glucose. (X), choline buffer without an energy source; (Cl), choline buffer with glucose; (A), KC1 buffer with glucose.

external medium. As discussed below, potassium may be acting as a counterion for sodium. Energetics of sodium extrusion. Strain AN120, which is defective in the H+-translocating ATPases, cannot interconvert phosphate bond energy with the electrochemical proton gradient, allowing conditions to be established in which the source of energy available for transport are both phosphate bond energy and an electrochemical gradient (during metabolism of glucose) or only phosphate bond energy (glucose metabolism in the presence of uncouplers or respiratory inhibitors) or only an electrochemical gradient of protons (during respiration of substrates of the electron transport chain) (21). Respiration of reduced PMS, which results in formation of an electrochemical proton gradient but not ATP, supported efflux of 22Nat (Fig. 4A), and efflux was inhibited by addition of the uncoupler FCCP. Efflux coupled to glucose was also inhibited by FCCP (Fig. 4B), even though ATP produced through the metabolism of glucose is unaffected by uncoupler in this strain (22). Respiration in broth-grown cells was only partially cyanide sensitive, and sodium efflux in these cells was only partially inhibited by cyanide (data not shown). Cells grown in minimal medium

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ENDOGENQUS bGLUCOSE + FCCP

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FIG. 4. Energetics of %a+ efflux. Starved cells were loaded with 140 mM NaCl and equilibrated with ?Ja’. (A) Cells were diluted into Buffer A containing 140 mM KC1 without an energy source (X), with 0.2 mM PMS and 20 mM Tris ascorbate (Cl) or with PMS, Tris ascorbate, and 10 PM FCCP (a). (B) Cells were diluted into Buffer A containing 140 mM choline chloride without an energy source (X), with 10 mM glucose (0) or with glucose and 10 &M FCCP (a).

with glycerol as a carbon source exhibited cyanide-sensitive respiration, and in these cells cyanide nearly completely inhibited sodium et&ix (Fig. 5). We have previously shown (22) that under these conditions cyanide has no effect on intracellular ATP pools.

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Sodium gradients have basically the same roles in procaryotes; sodium-solute cotransport systems are widespread. However, in these organisms the major generators are proton pumps, and the major currency of electrochemical energy is the H+ (25). This reliance on protons for energy metabolism provides the organism greater degrees of freedom in the design of devises for the establishment of sodium gradients. The diversity of these machines is amazing. At least four very different mechanisms have evolved, including a Na+- translocating ATPase (3), a Na+-coupled redox chain (4), Na+-coupled decarboxylases (5-‘7), and Na’/H+ antiporters (10-17). Note that the first three are primary sodium pumps, that is, they convert chemical directly to into electrochemical energy. The fourth, on the other hand, is a secondary porter, that is, it converts one form of electrochemical energy into another, in this case proton gradients into sodium gradients. Is there any correlation between the physiology of the cell and the method used for sodium extrusion? E. coli and K. aerogenes are both Enterobacteriaciae, yet the former has an antiporter and the latter a decarboxylase-coupled pump, as do A. fermentans and I? alcalescens, which are anaerobic gram-negative cocci. However, it is not known whether E. coli also has a sodium-coupled decarboxylase or whether

DISCUSSION

It is like that most if not all cells extrude sodium. Why they do so may be related not so much to toxicity of intracellular sodium as to the utility of the resulting electrochemical gradient of sodium. In higher eucaryotes the Na+/K’-ATPase is the main generator of electrochemical energy, which is used to drive cellular motors, e.g., secondary transport systems in which sodium is transported with or in exchange for another solute.

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FIG. 5. Effect of cyanide on %a+ efflux. Starved cells loaded with 140 mM NaCl and equilibrated with zNa+ were diluted loo-fold into buffer A containing 140 mM choline chloride without an energy source (X), with 10 mM glucose (0) or with 10 mM glucose and 10 mM NaCN (A).

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the others have antiporters. Vibrio are also gram-negative, but V; alginolyticus is a marine organism and so may have unique sodium handling requirements. The grampositive S. faecalis is the only procaryote known to use a Na+-ATPase. Sodium/proton antiporters are used by both halophiles (15) and nonhalophiles, alkalophiles (16), and nonalkalophiles. In fact, organisms in every kingdom use sodium/proton antiports, including true bacteria (E. coli (lo-14)), archeabacteria (H. halo&urn (15)), and eucaryotic mitochondria (26)). This may imply an early evolution of antiporters. Similarly, both eucaryotes and procaryotes use Na’-ATPases. Even though S. faecalis does not appear to use this enzyme as a major generator of electrochemical energy, it does extrude sodium as the counterion for potassium accumulation (27) and in this sense fulfills a function similar to that of the eucaryotic Na+/K+-ATPase. It is not known yet whether the redox or decarboxylase systems are physiologically important in the generation of sodium gradients. In short, at this time we know too little about either the physiology of the cell or the molecular mechanism of sodium extrusion to explain this diversity. So far the differences have been shown in vitro but not in vivo. Even though sodium/proton exchange in vesicles of E. coli has been clearly demonstrated (lo-14), the work of Harold and co-workers (l-3) pointed out the difficulty of extropolation back to the unbroken cell. It was important, therefore, to elucidate the mechanism of sodium extrusion in intact E. coli. The first step in approaching sodium transport on the molecular level was to determine whether it is a primary or secondary transport event. In S. faecalis sodium extrusion is primary, catalyzed by a Na+translocating ATPase (l-3). In intact cells of that organism sodium was pumped out of the cells as long as intracellular ATP pools were maintained (1). Efflux was independent of the magnitude of the electrochemical proton gradient. In intact E. coli we obtained opposite results: downhill efflux was independent of internal ATP and required a proton gradient. Clearly sodium

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extrusion from E. coli is secondary, with exchange of sodium and protons catalyzed by an antiporter. At the pH at which these experiments were performed there is no transmembrane pH gradient (28), thus the proton motive force is entirely in the form of a membrane potential. This implies electrophoretic exchange; the simplest stoichiometry which fulfills this net inward movement of positive charge is 2 H+:Na+. Extrusion both against (Fig. 1) and down (Fig. 2) a concentration gradient occurred, and both were energy requiring reactions. External K+ stimulated efflux (Fig. 3). Rb+ could replace K+ but neither external Na+, Li+, or choline+ could. A reciprocal stimulation of potassium uptake by sodium has been observed both in E. coli (29) and S. faecalis (27). It is unlikely that the two fluxes are coupled. Since intracellular Na+ is balanced by equal equivalents of nonpermeant anions, uptake of a permeant cation is required for charge compensation. Even though Nat exchanges for H+, protons cannot serve as the compensating cation without acidification of the cytosol. Thus there must be a constant recirculation of Hf. There are transport systems for K+ which act as routes for rapid entry (30); thus potassium can serve as a counterion for sodium extrusion. Sodium/sodium exchange was not found to occur at a rate comparable to net extrusion. Essentially no energy-independent exchange was observed when cells were loaded with 10 or 140 mM “Na+ and diluted into 140 mM unlabeled NaCl, LiCl, KCl, Tris-HCl, RbCl, or choline Cl (data not shown). Similar results were obtained when cells loaded with unlabeled NaCl or KC1 were diluted into buffers containing 22Na+ (the slow equilibration shown in Fig. 1 is representative of this body of data). Energy-dependent exchange was also investigated and was found not to be significant compared to net efflux. For example, when cells loaded with 140 mM ZNa’ were diluted into buffer containing 140 mM unlabeled NaCl, producing a lOO-fold isotopic dilution, the rate of efflux was not significantly different than dilution into choline chloride. In other experiments cells were loaded with 140 mM unlabeled NaCl

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and diluted into 2.8 mM =Na+. Lack of sodium/sodium exchange under conditions in which sodium/proton exchange occurs implies a mechanism in which there is obligatory binding of sodium and protons on opposite sides of the membrane. The work described here suggests that the intact cell uses a sodium/proton antiporter for extrusion of sodium, indicating that the antiporter found in everted membrane vesicles is not artifactual. Further studies on the mechanism of sodium extrusion in E. coli can reasonably be performed in vitro with membrane vesicles. REFERENCES 1. HEEFNER,

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