Proc. Nat. Acad. Sci. USAVol. 71, No. 4, pp. 1520-1524, April 1974

Role of Phosphate and Other Proton-Donating Anions inRespiration-Coupled Transport of Ca2+ by Mitochondria

(Ca2+ transport/electron transport/oxidative phosphorylation)

ALBERT L. LEHNINGER

Department of Physiological Chemistry, The Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205

Contributed by Albert L. Lehninger, January 17, 1974

ABSTRACT Measurements of extra oxygen consump-tion, "Ca2 + uptake, and the osmotic expansion of thematrix compartment show that not all permeant anionsare capable of supporting and accompanying the energy-dependent transport of Ca2+ from the medium into thematrix in respiring rat-liver mitochondria. Phosphate,arsenate, acetate, butyrate, j3-hydroxybutyrate, lactate,and bicarbonate + C02 supported Ca2+ uptake, whereasthe permeant anions, nitrate, thiocyanate, chlorate, andperchlorate, did not. The active anions share a commondenominator, the potential ability to donate a proton tothe mitochondrial matrix; the inactive anions lack thiscapacity. Phosphate and the other active permeant anionsmove into the matrix in response to the alkaline-insideelectrochemical gradient of protons generated across themitochondrial membrane by electron transport, thusforming a negative-inside anion gradient. It is postulatedthat the latter gradient is the immediate "pulling" forcefor the influx of Ca2+ on the electrogenic Ca2+ carrier inrespiring mitochondria under intracellular conditions.Since mitochondria in the cell are normally exposed to anexcess of phosphate (and the bicarbonate-CO2 system),particularly in state 4, inward transport of these proton-yielding anions probably precedes and is necessary forinward transport of Ca2 + and other cations under bio-logical conditions. These observations indicate that anegative-inside gradient of phosphate generated by elec-tron transport is a common step and provides the im-mediate motive power not only for (a) the inward trans-port of dicarboxylates and tricarboxylates and (b) theenergy-dependent exchange of external ADP3- for internalATP4- during oxidative phosphorylation, as has alreadybeen established, but also for (c) the inward transport ofCa2", K+, and other cations.

Mitochondria isolated from various vertebrate tissues havethe capacity to accumulate large amounts of Ca2+ in the innermatrix compartment by a transport process energeticallycoupled to electron transport (reviewed in refs. 1 and 2).A permeant anion, such as phosphate, enters the matrixwith Ca2+ (1-7). It has been generally held that Ca2+ trans-port is the primary process most directly coupled to electrontransport and that the permeant anion follows secondarily(1, 2, 6, 8-11). The alternative view, namely, that aniontransport is primary and Ca2+ transport secondary (3, 12,13), has not been given wide consideration. Indeed, relativelylittle attention has been given the capacity of various anions toaccompany or support the respiration-dependent accumula-tion of Ca2+ (4, 6, 7).In this paper it is shown that not all anions known to

penetrate through the membrane of intact mitochondriawill support and .accompany respiration-coupled entry ofCa2+ into the matrix. Phosphate, bicarbonate + C02, andthe anions of weak aliphatic acids are active, but a number

of other permeant anions, including nitrate and thiocyanate,are shown to be inactive. The active anions share a commonproperty: in each case the species actually penetrating themembrane can, directly or potentially, yield a proton to thematrix and can thus be "pulled" across the membrane andinto the matrix in response to the alkaline-inside protongradient generated by electron transport. These data arebrought together with other published observations into aunifying hypothesis in which respiration-driven transportof the proton-donating anion phosphate is postulated to be acommon first step under intracellular conditions, not only inoxidative phosphorylation and in transport of dicarboxylatesand tricarboxylates, but also in the inward transport of Ca2+and other cations.

EXPERIMENTAL DETAILS

Oxygen consumption was followed with a Clark oxygen elec-trode and light absorption changes at 700 nm in a Gilfordrecording spectrophotometer. Uptake of OCa2+ was deter-mined by measurement of the radioactivity remaining in themedium after rapid filtration of the mitochondrial suspen-sion through Millipore filters. Rat-liver mitochondria washed3 times with 0.25 M sucrose were used throughout. Otherdetails are given in the legends.

RESULI¶'Passive Entry of Variou Anions into Nonrespiring Mito-

chondria. In this paper the capacities of various anions toenter the matrix compartment of respiring mitochondriaas counterions with Ca2+ are compared. The relatively im-permeant anions used as controls in this study were isethionate(14) and chloride (15). The permeant anions used were phos-phate and arsenate, which pass through the membrane inexchange for hydroxyl ions via the phosphate-hydroxyl anti-porter (16), acetate, p-hydroxybutyrate, butyrate, and lactate,which actually do not penetrate as such but enter as the un-dissociated free acids (15, 16), the bicarbonate + CO2 system,of which dissolved CO2 is the penetrating species (17), andnitrate and thiocyanate (15), which pass the membrane incharged form, presumably because of a relatively high degreeof charge delocalization.

In order to establish more securely the basis for the differ-ence between the action of nitrate and thiocyanate, on one

hand, and phosphate, arsenate, bicarbonate, and acetate, onthe other, it was necessary to identify additional anions re-

sembling nitrate and thiocyanate in being able to penetratethe mitochondrial membrane readily as such, in the absenceof proton-conducting uncoupling agents. As is seen in Fig. 1,

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Phosphate and Mitochondrial Transport Processes 1521

FIG. 1. Penetration of some anions into nonrespiring mito-chondria. The test medium (2.0 ml) was i5omM potassium chlo-ride, ethionate, nitrate, thiocyanate, chlorate, or perchlorate, 2mM Tris * chloride pH 7.4, and 1 MuM rotenone. Valinomycin (1 Mg)was added where shown. Light absorption changes were rasuredat 700nm and 25°.

rat-liver mitochondria suspended in 120 mM potassium saltsof nitrate, thiocyanate, chlorate, and perchlorate were foundto swell rapidly on addition of valinomycin, whereas theanions chloride and isethionate failed to support swelling.Anions of weak aliphatic acids, such as acetate, butyrate,and fl-hydroxybutyrate, supported swelling only if a proton-conducting agent (FCCP) was present, in accordance withearlier observations (18). Moreover, in confirmation of theresults of Selwyn et al. (14), we have found that. Ca2+ readilyenters respiration-inhibited rat-liver mitochondria, presum-ably on a specific electrogenic uniporter or carrier (12, 14),down a concentration gradient with thiocyanate anion in theabsence of a proton-conducting uncoupler. In similar testswe have found that nitrate, chlorate, and perchlorate alsoenter nonrespiring mitochondria with CaI+ under these con-ditions. Thus by two different tests, entry with K+-valino-mycin or entry with Ca +, the anions thiocyanate, nitrate,chlorate, and perchlorate have been found to penetrate readilyinto the osmotically active matrix compartment of non-respiring mitochondria as such and not as protonated species.

Specificity of Anions in Supporting Stimulation of Respira-tion by Ca2+. The various permeant anions discussed abovewere then tested for their capacity to support respiration-coupled transport of low concentrations of Ca2+ into the mito-chondrial matrix, up a gradient of concentration, startingfrom an external concentration of 200 AM Ca2+ against aninternal concentration that is approximately 10-fold higher.This was shown by three different types of measurement.The first is a test of the capacity of the anion to support

respiratory stimulation by Ca2+ under conditions in which apermeant anion is required. Respiring rat-liver mitochondriain the absence of added permeant anions have the capacityto bind, largely to the membrane ["membrane loading" (12) ],about 80 ng-atoms of Ca2+ per mg of protein, accompaniedby a stoichiometric jump in oxygen uptake (4). After thisamount of Ca2+ is bound, the rate of respiration returns tothe original pre-Ca2+ state-4 rate. A second addition of 80ng-atoms of Ca2+ to the suspending medium at this pointis unable to stimulate respiration because the membrane-binding sites are fully occupied by Ca2+ and no permeantanions are available in the medium, as indicated by the oxygen

FIG. 2. Effect of delayed addition of various anions in support-ing Ca2+-dependent respiratory stimulation. The medium (2.0 ml)contained 120 mM KCl, 5 mM potassium succinate, 5 mM Tris-chloride (pH 7.4), and rat-liver mitochondria (5 mg of protein).Oxygen uptake was measured with the oxygen electrode. All addi-tions of Ca2+, made at points shown, were 200 MuM (80 nmol ofCa2+ per mg of protein). Phosphate (Pi) was added at 0.25 mM,arsenate (Asi) at 0.5 mM, and CO2 at 0.4 mM; acetate (Ac-) and,%hydroxybutyrate (BOH-) were added at 30 mM. Other anionswere added up to 40mM without response. In the set of traces atthe right the initial Ca2+-induced jump is not shown. The tem-perature was 250.

electrode traces in Fig. 2. If a delayed addition of the per-meant anion phosphate is now made, there is a prompt stim-ulation of oxygen uptake, stoichiometric with translocationof both the Ca2+ and phosphate into the matrix, which con-tinues until all the Ca2+ has been accumulated, and thenreturns to the original state4 rate, in confirmation of earlierstudies (see refs. 4 and 8). Delayed addition of the permeantanions arsenate, acetate, and ,8-hydroxybutyrate also yieldeddelayed oxygen jumps (Fig. 2); similar results (not shown)were obtained with butyrate and lactate. Moreover, as shownearlier (17), delayed addition of dissolved CO2 also evokes aburst of oxygen uptake in the presence of excess Ca2+. Addi-tion of the impermeant anions chloride or isethionate, after theexcess Ca2+, yielded no stimulation of oxygen uptake; how-ever, the mitochondria were still intact and responsive sincea subsequent addition of phosphate elicited the usual stoichio-metric burst of oxygen uptake. Most significant is the findingthat delayed additions of the permeant anions nitrate, thio-cyanate, chlorate, or perchlorate at concentrations up to 40mM completely failed to stimulate oxygen uptake whenadded in the presence of excess Ca2+, but in each case a sub-sequent addition of phosphate elicited the usual stoichio-metric oxygen uptake jump (Fig. 2).These experiments show that permeant anions fall into two

classes. One class, hereafter called Class A anions (nitrate,thiocyanate, chlorate, and perchlorate), failed to support

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1522 Biochemistry: Lehninger

TABLE 1. Effect of anions on respiration-dependent Ca2+accumulation and water uptake

Ca+ takenup after

second Ca2 +addition(ng-atoms Decrease inper mg of A700

Anion mM protein) (X 103)

None 0 <10Phosphate 0.25 71.4 300Arsenate 0.50 72.3 280Acetate 30 65.3 190Butyrate 30 62.2 160P-Hydroxybutyrate 30 69.1 180C02-HCOs- 0.4 62.8 120Nitrate 30 0 <10Thiocyanate 30 0 <10Chlorate 30 0 <10Perchlorate 30 0 <10Chloride 30 0 <10Isethionate 30 0 <10

The test system (2.5 ml; 250) contained 120 mM KC1, 5 mMpotassium succinate, 5.0 mM Tris* chloride, pH 7.4, and rat-livermitochondria (1.25 mg of protein). About 40 sec after addition ofthe mitochondria, 40 AM "6Ca2+ was added as the chloride, yield-ing the usual jump in oxygen uptake; 60 sec later another additionof 40 AM 41Ca2+ was made, yielding no respiratory stimulation.The anions shown were added 60 sec after the second addition of4"Ca2 . Samples of the medium were filtered 90 sec after additionof the anion and the uptake of 45Ca2+ from the medium deter-mined. Trhe absorbance changes at 700 nm were measured 2.0 minafter addition of the anion. The values for these changes havebeen multiplied by 103.

Ca2+-dependent oxygen uptake. The other, Class B anions[phosphate, arsenate, C02 + bicarbonate, acetate, butyrate,B-hydroxybutyrate, and lactate] all stimulated oxygen con-sumption in an amount equivalent to the excess Ca2+ added,as expected from earlier work (1, 4, 6, 7, 17, 18). Phosphate,arsenate, and bicarbonate were active at concentrations of0.25-2.0 mM, whereas 10 mM acetate, butyrate, and f3-hy-droxybutyrate and 30 mM lactate were required.

Measurement of Ca2+ Uptake Supported by Anions. In thesecond type of measurement used, the effect of differentanions on the uptake of *Ca2+ by the mitochondria was de-termined in experiments arranged as in Fig. 2. A first additionof isotopic Ca2+ (80 ng-atoms per mg of protein) was madein the absence of added permeant anion, to saturate the Ca2+_binding sites; over 98% of the added Ca2+ was removedfrom the medium. A second addition of the same amount ofisotopic Ca2+ was then made, which failed to produce stim-ulation of oxygen uptake, as in Fig. 2. Delayed additionsof various anions were then made, with the system monitoredwith the oxygen electrode. After return of the oxygen con-sumption to the pre-anion state4 rate, the reaction mediawere quickly filtered and the net uptake of *Ca2+ from themedium after the anion addition was determined. The result-ing data (Table 1) show that the impermeant anions chlorideand isethionate failed to support accumulation of Ca2+, asexpected. Again, the permeant anions fell into two classes.Phosphate, acetate, butyrate, ,3-hydroxybutyrate, arsenate,

and bicarbonate + CO2 all supported accumulation of 6Cafollowing the delayed addition of the anion, but nitrate, thio-cyanate, chlorate, and perchlorate did not.

Osmotic Swelling Induced by Entry of Anions with Ca2+.The third approach used to follow the entry of Ca2+ andpermeant anions into the matrix involved light-absorptionmeasurements of the osmotic swelling of respiring rat-livermitochondria subsequent to the delayed addition of variousanions in the presence of excess Ca2+ under conditions ar-ranged as in Fig. 2. The collected data are given in Table 1.The Class A anions, chlorate, nitrate, thiocyanate, and per-chlorate, as well as the impermeant chloride, failed to yieldosmotic swelling, whereas the Class B anions, phosphate,arsenate, acetate, g-hydroxybutyrate, butyrate, and C02,supported osmotic swelling, due to entry of water into thematrix accompanying the respiration-dependent entry ofCa2+ and the counteranion.

DISCUSSIONThe three types of measurement used show that anions per-meant through the mitochondrial membrane may be placedin two classes. Those incapable of supporting respiration-dependent net transport of Ca2+ into the mitochondrialmatrix, namely, nitrate, thiocyanate, chlorate, and perchlo-rate (Class A anions) and those capable of supporting respira-tion-coupled transport of Ca2+ up a gradient of concen-tration, namely, phosphate, acetate, butyrate, j3-hydroxy-butyrate, lactate, bicarbonate + CO2, and arsenate (ClassB anions). An explanation must now be sought for thefact that nitrate, thiocyanate, chlorate, and perchloratecan readily enter the inner matrix compartment with Ca2+(or K+ in the presence of valinomycin) when the cation en-ters down a concentration gradient in respiration-inhibitedmitochondria, but cannot support the respiration-dependententry of Ca2+ up a concentration gradient. One possible butunlikely explanation is that Class A anions can readilypenetrate the membrane of respiration-inhibited mitochondriabut are unable to pass through the membrane of respiringmitochondria. If this is the case, for which there is no otherevidence, then it is also necessary to assume that the ClassB anions must penetrate the membrane of both respiration-inhibited and respiring mitochondria equally.

There is, however, an alternative hypothesis that is farmore likely and is, in addition, consistent with a large bodyof recorded information. The permeant anions of Class B(phosphate, acetate, bicarbonate + C02, etc.) share a commonproperty: the penetrating species in each case can yield aproton, directly or indirectly, on passing into the mitochondrialmatrix, whereas the anions of Class A, which are very weakproton acceptors, pass through the membrane as anions andare unable to donate protons to the matrix. Many lines ofevidence now indicate that mitochondrial electron transportis capable of generating an alkaline-inside electrochemicalproton gradient across the mitochondrial membrane (19,20), although it is not certain whetfier this gradient arisesfrom vectorial arrangement of H +-yielding and H +.extractingreactions of electron transport, as postulated in the chemi-osmotic hypothesis (19), from vectorial hydrolysis of a high-energy chemical intermediate generated by electron trans-port, or from some other type of vectorial process. Mostevidence indicates that two H+ ions are transferred fromthe matrix to the medium by each of the three energy-con-serving sites of the respiratory chain (19, 21), thus rendering

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Phosphate and Mitochondrial Transport Processes 1523

TRANSPORT OFHl -YIELDING ANIONS

OUT IN

L °; H20AHACHA4A I7 H A

SEQUENCE OFTRANSPORT PROCESSESPHOSPHATE FIRST

t. I I2 I

H20-40--

H2POi-Co2+

OUT

"9l

Ca F I RST

H-20

HC3 2C 032C02-

H20

-HFHO_H20

OUT IN

FIG. 3. (Left) Inward transport of phosphate on the phosphate-hydroxide antiporter of weak acid anion A- as undissociated HA,and of HCO3- as C02, in response to the alkaline-inside H + gradient generated by electron transport. (Right) Anion-first and cation-first

transport of Ca2 + and phosphate. The anion-first sequence is postulated as more probable under intracellular conditions. For simplicity of

representation the vectorial equations are not fully balanced for proton movements and charges.

the matrix alkaline. Phosphate and other anions of Class Bmay be pulled electrophoretically into the alkaline matrixof respiring mitochondria because of their capacity to donateprotons (Fig. 3, left). Thus, H2PO4- may exchange for matrixhydroxide ions on the phosphate-hydroxide antiporter (seerefs. 15 and 18), a reaction formally equivalent to the entryof undissociated H3PO4, followed by loss of a proton to thealkaline matrix. Moreover, once the H2PO4r ion has gainedentry into the matrix it may on further dissociation yieldadditional protons to the alkaline matrix and thus furtheraid in generating a phosphate gradient. Arsenate maysimilarlyenter the alkaline matrix on the phosphate-hydroxide anti-porter, in exchange for internal hydroxide. Salts of weakacids, such as acetate, butyrate, 8-hydroxybutyrate, andlactate, are pulled into the alkaline matrix because theyactually pass the membrane as the corresponding free acids(15, 18), which then yield their protons to the excess OH-in the matrix (Fig. 3). Although bicarbonate itself cannotpass the membrane (17, 18), it is in equilibrium with dis-solved C02, which penetrates the membrane into the matrix,where it is hydrated by carbonic anhydrase to yield H2C08(17). The latter then loses protons to the alkaline matrixand becomes HCO3- (Fig. 3, left). Thus, all the anions ofClass B can be transported uphill into the mitochondrialmatrix by acid-base coupling to the H+ gradient generatedby electron transport. The permeant anions of Class A can-

not be transported into the matrix in this manner since theyare unable to yield protons to the alkaline matrix and evi-dently are unable to exchange across the membrane withhydroxide ions.The net effect of the entry of Class B anions into the matrix

of respiring mitochondria is to convert the alkaline-insideelectrochemical gradient of protons generated by electrontransport into a negative-inside gradient of the transportedanion (Fig. 3, left); this conversion results in discharge of theproton gradient, thus rendering the matrix compartment

nearly neutral with respect to pH. It is here postulated thatthe internal excess of anion generated in this way is the im-mediate "pulling" force for electrophoretic transport of Ca2+from the medium into the matrix, on the specific electrogenicCa2+ uniporter (12, 14), particularly under intracellularconditions.

This explanation also accounts for the observations ofBrierley and his colleagues on the relative effectiveness ofvarious anions in supporting the accumulation of K+ andother monovalent cations by respiring heart mitochondria(22, 23). They reported that phosphate, acetate, and propi-onate support respiration-dependent entry of K+, whereasnitrate did not. Moreover, it also accounts for the fact thatrespiration-dependent entry of the ornithine+ cation into rat-liver mitochondria is supported by phosphate, acetate, andbicarbonate, but not by other anions (24).The data reported here cannot reveal whether the entry of

the proton-yielding anion into the matrix actually precedesentry of the Ca +. In fact, Skulachev and his colleagues haveshown that very small amounts of lipid-soluble cations suchas tetrabutylammonium may enter respiring mitochondriain the absence of added phosphate, presumably in response

to the negative-inside gradient of H+ across the membrane(20). However, the sequence of events postulated here, withthe respiration-dependent entry of a proton-yielding anionsuch as phosphate preceding the electrophoretic entry ofcation (Fig. 3, upper right), is more likely to occur biologicallythan is the reverse sequence, i.e., entry of cation first in re-

sponse to the negative-inside alkaline electrochemical protongradient generated by electron transport, followed by sec-

ondary entry of the prQton-donating anion (Fig. 3, lowerright). Because mitochondria in the intact cell are at all timesbathed in a medium containing as major permeant anionsthe proton-yielding phosphate and bicarbonate-CO2 buffersystems, the total thermodynamic gradient generated across

the mitochondrial membrane during state-4 respiration in the

-*H2P°4-W Ca2+

IN

Proc. Nat. Acad. Sci. USA 71 (1974)

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Proc. Nat. Acad. Sci. USA 71 (1974)

AN ION ICMETABOL ITETRANSPORT

OXIDATIVEPHOSPHORYLATION

H+<

H20-

H2POi

Co2+

Oj-

-o H2P04

Co2+

H20401H-.-

H2POi eH

MO l te2-_

_ OH-

H2POjl

so M~alate2-

H+<H20O.{

HOH2PO4

ATP4-

I H2POib ADP3

FIG. 4. Utilization of the respiration-genprated negative-inside phosphate potential for three major energy-dependent mitochondrialtransport activities. (As in Fig. 3, the left side of the membrane is the "out" side.)

intact cell is largely a negative-inside potential caused by theinternal excess of phosphate (and possibly bicarbonate),with only a small H+ gradient across the membrane, in con-

sonance witfh the conclusion of Mitchell and Moyle (25) thatthe transmembrane potential makes up the major portionof the electrochemical gradient generated by electron trans-port. The observation that uptake of phosphate, arsenate,and acetate can cause mitochondrial acidification (6, 10, 26)and can also impose an energy load on electron transport(27-29) is fully consistent with the sequence proposed here.Moreover, the anion-first sequence proposed here is not in

conflict with the observation that interaction of Ca2+ withthe membrane and with the respiratory chain is very fastand precedes matrix pH changes (8-10). It is now well estab-lished that, in the absence of' phosphate, Ca2+ is bound tothe respiration-energized membrane (8-10, 12, 26); however,it is not transported into the matrix from its binding sites inthe membrane until, and, presumably after, a proton-yieldinganion enters the matrix in response to the alkaline-insideH+ gradient generated by electron transport.As shown in Fig. 4, a negative-inside gradient of phosphate

generated by electron transport can provide the immediatemotive power for (1) inward transport of various bivalentand univalent cations such as Ca2+ and K+, (2) inward trans-port of various anionic metabolites, such as dicarboxylates,which enter in exchange for phosphate, and tricarboxylates,which can exchange with internal dicarboxylates (see refs.16, 30-32), and (3) the energy-requiring exchange of ex-

ternal ADP'- with internal ATP4- during oxidative phos-phorylation (30, 31, 33), which is electrically equivalent toinward transport of a positive charge. The inward transport ofH2PO4- in response to the electrochemical proton gradientgenerated by electron transport is thus viewed as an obliga-tory first step, under intracellular conditions, for threemajor energy-dependent functions coupled to mitochondrialelectron transport: cation transport, anionic metabolitetransport, and oxidative phosphorylation.

This work was supported by grants from the U.S. Public HealthService (GM-05919) and the National Science Foundation(GB-36015).

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982.18. Chappell, J. B. & Haarhoff, K. N. (1967) in Biochemistry of

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CATIONTRANSPORT

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