Proc. Nati. Acad. Sci. USAVol. 73, No. 9, pp. 3141-3145, September 1976Biochemistry

Mechanism of uncoupling in mitochondria: Uncouplers asionophores for cycling cations and protons*

(classical uncouplers/uncoupler combinations/release of bound cations/electrogenic ionophores)

RALPH J. KESSLER, CHARLES A. TYSON, AND DAVID E. GREENInstitute for Enzyme Research, University of Wisconsin, Madison, Wisc. 53706

Contributed by David E. Green, July 6,1976

ABSTRACT Classical uncouplers such as 2,4-dinitrophenolhave been shown to be ionophores with the capability fortransporting monovalent or divalent cations with equal effi-ciency. The conditions appropriate for the maximal expressionof this ionophoric capability have been explored. Two criticalfactors are the polarity of the organic phase and the pH of theaqueous phase that is equilibrated with the organic phase. Thedemonstrated cationic ionophoric capability of uncouplers,taken in conjunction with the known ability of uncouplers tocycle protons across a membrane phase, provides the experi-menta basis for the thesis that uncoupling of electron flow fromATP synthesis via classical uncouplers involves the substitutionof one coupled process by another. Uncoupling thus reduces tothe replacement of one driven reaction (ATP synthesis) by thedriven reaction (cyclical transport) mediated by the uncou-pler.

Hotchkiss (1), in the early 1940s, discovered that reagents suchas 2,4-dinitrophenol blocked the link between electron transferand ATP synthesis in bacterial systems without affectingelectron transfer, and later Loomis and Lipmann (2) and Crosset al. (3) demonstrated the same phenomenon in mitochondrialsystems. This effect of 2,4-dinitrophenol could be duplicatedby a dozen variously substituted nitro- or halophenols, but notall such substituted derivatives were active (3). In the inter-vening years, the list of compounds that can replace 2,4-dini-trophenol in blocking the link between electron transfer andATP synthesis without affecting electron transfer has grownconsiderably, and these compounds represent a wide varietyof chemical structures (4-8). Somehow, the term uncoupler hasbecome common for describing the compounds that block thelink of electron transfer to ATP synthesis, and the term un-coupling is now usually applied to the process by which thisblock is achieved. This is unfortunate, for implicit in the termuncoupling is the notion that a coupled process is replaced byan uncoupled process, and this notion is undoubtedly incorrect.However, it is too late to eliminate this usage from the scientificliterature. We shall use the term uncoupling to denote thesubstitution of one coupled process by another mediated by theuncoupler, and the term uncoupler merely as the reagent thatexecutes uncoupling so defined.

Evidence will be presented that all uncouplers that duplicate

Abbreviations: A-23187, a carboxylic acid antibiotic (C29H37N306)containing a ketopyrrole sector linked to a benzoxazole carboxylic acidsector via a spiral ring; FCCP, carbonylcyanide p-trifluoromethoxy-phenylhydrazone; m-CICCP, carbonyl cyanide m-chlorophenylhy-drazone; S13, 5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicyl anilide;SF-6847, 3,5-di-t-butyl-4-hydroxybenzylidinemalononitrile; TTFB,4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole; X537A, a car-boxylic acid antibiotic (CnaHsoO) containing a salicylate sector linkedto a fused tetrahydrofuran-hydroxytetrahydropyran ring system viaa 7 carbon atom chain with an hydroxy and keto group; 1799; a,a'-bis(hexafluoracetonyl)acetone.* Part I of a series.

the action of 2,4-dinitrophenol (classical uncouplers) have acommon functional attribute, namely, the ionophoric capacityto transport cations across an organic phase or an equivalentmembrane phase. But it is first necessary to define preciselywhat a classical uncoupler can do, and to specify which mo-lecular species can be classified as classical uncouplers beforeevaluating the invariant correlation of this attribute with thefamily of classical uncouplers. There is now extensive docu-mentation in the literature for the view that classical uncouplersuniformly show five properties: (i) the complete release ofrespiratory control; (ii) the substitution of all coupled processes(ATP synthesis, energized, transhydrogenation, reverse electronflow, active transport of cations) by a coupled process mediatedby the uncoupler; (iii) the elimination of all protonic and cat-ionic gradients generated across the mitochondrial membrane;(iv) no discrimination in these actions between one couplingsite and another; and (v) no discrimination between coupledprocesses driven by electron transfer and coupled processesdriven by ATP hydrolysis (3-5; 9-13). We shall define as aclassical uncoupler any species that shows all these five prop-erties and exclude from this definition any species that showsone or more, but not all, of these properties (see footnotet fora listing of the reagents that we now classify as pseudo uncou-plers and not as classical uncouplers).The combination of two ionophores (valinomycin and nig-

ericin) in the presence of K+ can duplicate all the five propertiesof classical uncouplers listed above (9, 15, 16). We shall referto such ionophore combinations as uncoupling combinations,and distinguish between uncoupling by classical uncouplers and

t There is a considerable list of reagents that inhibit oxidative phos-phorylation or that mimic some, but not all, aspects of uncoupling.We shall consider all such reagents as pseudo uncouplers. Thesewould not be expected to show the unique functional attributes ofclassical uncouplers. The pseudo uncouplers fall into three categories:reagents that induce a transition that leads to the release of respiratorycontrol (Ca2+, mercurials, heavy metals, thyroxine, fatty acids); re-agents that provide only one component of an uncoupling combi-nation (picric acid, tetraphenylboron, tetraphenylphosphonium ion,valinomycin, hydroxyoctadecadienoates, nigericin, X537A, andA23187); and reagents that are site-specific in respect to their effectson oxidative phosphorylation (azide and biguanides). The reagentsthat induce a change in the coupling mode of the inner membraneare not directly the causative agents of uncoupling, but only themolecular vehicles for inducing the generation of the intrinsic io-nophores that are ultimately responsible for uncoupling via cyclicaltransport (14, 15). With respect to the second category of pseudouncouplers, it is the combination of two ionophoric species thatunderlies uncoupling and not either one alone of the two species (16,17). The site-specific reagents cannot be classified as bona fide un-couplers, since uncoupling by classical uncouplers is site-unspecific.It is of interest that the inhibitory effect of azide on respirationcoupled to ATP synthesis is in fact reversed by classical uncouplers,according to Wilson and Chance (18).

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uncoupling by ionophore combinations (see footnote* for alisting of the various ionophore pairs that can form such un-coupling combinations). The identity of action of uncouplersand uncoupler combinations has three very important conse-quences. First, it leads to the prediction that uncouplers mustbe cationic ionophores; second, it specifies that uncouplers, likeuncoupler combinations, must be inducing cyclical transportof a cation; and third; it specifies that uncouplers must combinein one molecule the same properties duplicated by the twoionophoric components of the uncoupler combination. Whatare these two properties? The neutral ionophore valinomycin,complexed with a cation (K+), forms a species that can moveelectrogenically with the electron, i.e., electron flow can driveactive transport of K+ mediated by valinomycin. The acidicionophore nigericin can either complex with K+ or form a co-valent link with a proton, but electron flow cannot drivetransport of K+ mediated by nigericin. Thus, the electrogenicneutral ionophore transports the K+ into the matrix space, andthe nonelectrogenic acidic ionophore transports K+ out of thematrix space. The latter enters with a proton and leaves witha K+. In this fashion, cyclical transport of K+ is consummatedby the valinomycin-nigericin combination. This is highly sug-gestive that classical uncouplers can exist in an electrogenicform that is equivalent to the valinomycin-K+ complex, andin a nonelectrogenic form that is equivalent to nigericin-K+or to the protonated form of the uncoupler.

EXPERIMENTALThe present communication is addressed specifically to theobjective of presenting evidence that classical uncouplers areuniformly cationic ionophores and that this capability is de-monstrable by three different criteria.

Ionophoric capability of uncouplersThere are two direct ways of demonstrating the ionophoricproperties of uncouplers; first, by the uncoupler-mediatedpartition of a cation between an aqueous and an organic phase,and second, by the uncoupler-mediated transport of a cationbetween two aqueous phases separated by an organic phase(24). Table 1 contains data from a partition study which showsthat a representative group of uncouplers can induce thetransfer of Na+, Rb+, Mg2+, Ca2+, and Mn2+ from the aqueousinto the organic phase. The organic phase was made up of amixture, by volume, of 55% toluene and 45% n-butanol; theaqueous phase was buffered to pH 8.3.The ionophoric properties of uncouplers, like those of other

ionophores, are profoundly affected by two variables-the.polarity of the organic phase (Fig. 1) and the pH of the aqueousphase (Fig. 2). The more polar the organic phase, and the morealkaline the aqueous phase, the greater is the proportion ofcation that partitions into the organic phase. When the pH ofthe aqueous phase is about 10, the full ionophoric potential ofuncouplers is usually expressed (Table 2) and at this pH witha suitable organic phase, all classical uncouplers tested show

Valinomycin can be replaced by a considerable number of neutralcyclic peptides or crown ethers that can transport Na+ or X+ elec-trogenically in the mitochondrial system [gramicidin A and B (19),monazomycin (19), the nactins (19), and avenaciolide (20)]. Similarly,nigericin can be replaced by acidic ionophores with nonelectrogenictransport activity for Na+ or K+ [hydroxyoctadecadienoates (21),monensins (19), A23187 (22), and X537A (23)]. Ionophore combi-nations of the valinomycin-nigericin genre are implicated in, andresponsible for, the uncoupled state of mitochondria in the orthodoxconfiguration. These ionophores have been isolated from mito-chondria by Blondin (21).

Table 1. lonophoric capability of uncouplers and of theelectrogenic components of uncoupler combinations as

determined by partition study

Concentration of cation in theorganic phase (ng-atoms)

Uncoupler Na+ Rb+ Ca2+ Mg2+ Mn2+t

2,4-Dinitrophenol 130 166 196 185 16FCCP 410 474 250 4 8m-ClCCP 390 414 238 410 21Pentachlorophenol 880 1090 2760 3740 200Picric acid* 560 336 1040 1110 639Tetraphenylboron* 7280 4350 4650 5000

Equal volumes of aqueous and organic phases (1 ml each) werevortexed for 0.5 min and then centrifuged to separate the twophases completely. The organic phase was a mixture of 55% byvolume of toluene and 45% by volume of n-butanol. The concen-tration of uncoupler added to the organic phase was 10 mM. Theaqueous phase was 10 mM in Tris adjusted to pH 8.3 with HCl andthe concentration of cation added as the chloride was 100 mM.4SCa2+, 22Na+, SSMn2+, and SSRb+ were determined by measure-ment of radioactivity in an aliquot of the organic phase (usually0.50 ml). Mg2+ was determined by atomic absorption in a Perkin-Elmer flame spectrophotometer. All values in the table have beencorrected for the small blank without uncoupler.* Picric acid and tetraphenylboron are components of uncouplercombinations and not uncouplers in their own right.

t The partition studies with Mn2+ were carried out at pH 7.0 withTris-chloride. At pH 8.3, precipitation of Mn2+ complicated themeasurement of partition.

comparable ionophoric capability. The classical uncouplersexamined by us for ionophoric activity include nitrophenols(2,4-dinitrophenol and 2,5-dinitrophenol), pentachlorophenol,derivatives of carbonyl cyanide phenylhydrazone (FCCP andm-ClGCP), a hydroxybenzylidinemalononitrile derivative (SF6847), a benzimidazole derivative (TTFB), a nitrosalicylanilidederivative (S13) a fluoroketone derivative (1799), and desapidin(a reagent that contains a substituted quinone ring linked bya methine bridge to a substituted quinol ring, and whichils usedto uncouple photosynthetic phosphorylation). In each of theseeight types of classical uncouplers, there are known variants

2,4-Dnp

200_

C

E 10O _C"

20 40 60

% n-Butanol in toluene

FIG. 1. Effect of polarity on Ca2+ binding: uncoupler-mediatedpartition of Ca2+ into the organic phase as a function of the ratio ofn-butanol to toluene. Details are as in the legend for Table 1. The pHof the aqueous phase was 8.3.

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C

300/

E

100_

5 6 7 8

pH

FIG. 2. Effect ofpH on Ca2+ binding by uncouplers: uncoupler-mediated partition of Ca2+ into the organic phase as a function ofthepH of the aqueous phase. Details are as in the legend for Table 1. Theorganic phase was a 70:30 mixture by volume of n-butanol and tolu-ene. The aqueous phase was buffered with 10mM buffer: pH 5 (ca-codylate) and pH 7-8 (Tris-chloride).

with more or less uncoupler activity than the particular variantwe have tested. Based on our results thus far with variants inthe nitrophenol and carbonylcyanide hydrazone classes, weexpect that all variants will be indistinguishable in respect toionophoric capability.

2,4,6-Trinitrophenol (picric acid) and tetraphenylboron are

Table 2. Uncoupler-mediated partition of Ca2+ between an

aqueous phase (pH 10.0) and an organic phase

% Theory forCa2+ bound,* a 2:1 molarng-atoms/ ratio of,umol uncoupler

Uncoupler uncoupler to Ca2+

Pentachlorophenol 363 73SF 6847 254 51FCCP 366 73TTFB 395 79Desapidin 369 73S13 389 78Dicoumarolt 124 252,5-Dinitrophenol 114 232,4-Dinitrophenol 105 211799 296 59CCPt 198 40

* Net 45Ca2+ extracted into the organic phase after subtractingout a blank in which uncoupler was omitted from the assay.45Ca2+ concentration in the organic phase of the blank was 23uM. On an uncoupler basis, this represents a background level of2.3 ng-atoms of Ca2+/Mmol of uncoupler. Radioactivity was mea-sured in the presence of acetic acid (0.20 ml/10 ml of Aquasol) toeliminate quenching by chromophoric uncouplers.

t Insoluble material was found at the interface after centrifugation.At 10 mM, dicoumarol is not totally soluble in either phase. Theconcentration of uncoupler in the organic phase was 10 mM.Details are as in the legend for Table 1. The aqueous phase con-tained 50 mM ethylenediamine (pH 10); the organic phase was a30:70 mixture by volume of toluene and n-butanol.

t Carbonyl cyanide phenylhydrazone.

200-

C 1000- / Dnp

FCCP

1 2 3Time(hours)

FIG. 3. Uncoupler-mediated transport of Rb+ in a Pressman cell.The design of the cell was as described previously by Tyson et al. (24).The organic phase (7 ml) was a 70:30 mixture by volume of chloroformand n-butanol. The concentration of the uncoupler added to the or-ganic phase was 10 mM. The donor aqueous phase (2 ml) contained0.1 M rubidium chloride and 25 mM Tris-chloride (pH 8.3); the re-ceiver aqueous phase (2 ml) contained 0.1 M tetramethylammoniumcitrate (pH 5.0). The rates of transport have been corrected for thesmall blank without added uncoupler. PCP is pentachlorophenol; Dnprefers to the 2,4 isomer.

reagents which, in submitochondrial particles, can uncoupleonly in combination with nigericin and K+ or in equivalentcombinations (16). These reagents should be ionophores, sincethey should be acting in the same fashion as valinomycin. Asshown at the bottom of Table 1, both picric acid and tetra-phenylboron are potent ionophores for both monovalent anddivalent cations.

There is good correlation between the partition studies andthe direct measurement of net transport in a Pressman cell (seeFig. 3 for a representative experiment documenting suchtransport). Tyson et al. (24) have already established that thephospholipid-mediated partition of cations into the organicphase is a measure of the ionophoric capability of phospholipidsas determined by net transport in a Pressman cell.Release of membrane-bound cations by uncouplersMitochondria contain a complement of cations which are notleached out by extensive washing in 0.25 M sucrose (15, 22, 25).For example, beef heart mitochondria in a state appropriatefor oxidative phosphorylation usually contain 30 nmol of Ca2+and 3040 nmol of Mg2+. Whether these cations are encapsu-lated within the membrane phase [presumably bound to acidicphospholipids (24)] or are in solution in the matrix space, theirrelease from the mitochondrion has to be mediated by iono-phores. Divalent metal ionophores such as X537A and A23187can readily induce this release of "bound" Ca2+ and Mg2+ (22,23).

Table 3 shows that there is a close relationship between theconcentration of classical uncoupler required for maximaluncoupling and the concentration required for maximal releaseof bound Ca2+. This correlation was established for 2,4-dini-trophenol, m-CICCP, and SF6847. It is of interest that picrate(100 AM), which is not a classical uncoupler, had no effect onthe release of bound Ca2+. Not only Ca2+, but also other cations

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Table 3. Uncoupler-mediated release of membrane-bound Ca2+ versus uncoupler-mediated release ofrespiration in beef heart mitochondria

Respirationrate, ng-atoms Ca2+ release,

Concentration 02/mg of ng-atoms/mgUncoupler of uncoupler (MM) protein of protein

None 18 0.7m-ClCCP 0.1 61 1.6

2.0 120 12.62,4-Dinitrophenol 5.0 33 1.0

20.0 115 13.6None 24 0SF 6847 0.0005 77 0.8

0.001 106 1.60.002 161 6.60.005 189 10.20.01 212 13.80.05 234 14.3

The mitochondrial suspension was 0.25 M in sucrose, 10 mM in Tris-chloride, 2 mM in pyruvate and malate (K+ salts), and uncoupler wasused at the concentration indicated in the table. The suspension was incubated for 2 min at 300 before sedimentation of the mitochondria in a*high-speed Eppendorf centrifuge (time 8 sec). Released Ca2+ was determined in the supernatant fluid. The uncoupler-induced release ofrespiration was measured in an oxygen electrode during the incubation phase.

(K+, Mg2+), are released by uncouplers, depending upon theconditions.The uncoupler-mediated release of Ca2+ from the mito-

chondrial membrane can be greatly potentiated by monovalentcations such as K+ and Na+ (doubling or trebling of the release).The potentiation of release by cations represents an additionaldimension of the ionophoric capability of uncouplers (cation-cation exchange via successive cation-proton and proton-cationexchange).The above-documented uncoupler-mediated release of Ca2+

from isolated mitochondria under physiological conditions ofpH represents a third demonstration of the ionophoric capa-bility of uncouplers. What is significant about this particularmethod is that the uncoupler is being tested over the concen-tration range in which uncoupling takes place and at the pHwhere effective uncoupling is demonstrable.

Since the cationic requirement for the action of uncouplercan be satisfied by the usual level of bound cations in the mi-tochondrion, it is obvious that this requirement cannot bedemonstrated until the complement of bound cations in themitochondrion is reduced below a critical concentration. Therequirement of cations for the action of uncouplers is a facetwhich will be considered in a separate publication.

DISCUSSIONThe complete correspondence between the uncoupling actionof classical uncouplers and the uncoupling action of the com-bination of valinomycin and nigericin in the presence of K+ isthe foundation stone for the thesis that uncoupling is the sub-stitution of coupled cyclical transport of cations for any coupledprocess occurring in mitochondria. This cyclical transport hasbeen conclusively demonstrated for the combination of vali-nomycin and nigericin (16). With the evidence presented abovethat classical uncouplers are potent cation ionophores, and theevidence in the literature that classical ionophores can bind ordissociate a proton in the pH range where uncoupling takesplace (6, 26), the necessary requirements are met to justify thethesis that classical uncouplers act in the same way as thecombination of valinomycin and nigericin. It is significant thatthe uncoupler-mediated release of cations from the mito-

chondrial inner membrane depends upon the combination ofthese two properties (ionophoric and protonophoric) and, asshown in Table 3, there is an exact parallelism between theconcentration of uncoupler required for graded release of cationand the concentration required for graded uncoupling.What distinguishes classical uncouplers from acidic iono-

phores like X537A and A23187 is still not entirely clear. Whyare these two ionophores not classical uncouplers (22, 23)? Itwould appear that they cannot act electrogenically, i.e., electronflow cannot be coupled to the cation flow mediated by thesetwo ionophores. To act electrogenically, an ionophore must becapable of binding a cation when in a neutral form. This ca-pability appears to be intrinsic to classical uncouplers, butmissing in carboxylic ionophores. Picric acid is an ionophorethat can act electrogenically (16), but that lacks the capacityto carry protons in the range of pH appropriate for uncoupling(27). This ionophore must pair with a nigericin-type ionophoreor with amines in order to form an uncoupler combination. § Ifuncoupling could take place at pH 1, picric acid might possiblyshow the properties of a classical ionophore.

There are still two critical experimental pieces that have tobe put in place before the thesis of uncoupling via the imposi-tion of coupled cyclical transport can be considered to be rig-orously established. First and foremost is the essentiality ofcations for uncoupling via classical ionophores. Second is theneed for complete documentation that the valinomycin-nig-ericin combination can duplicate all the effects of classicaluncouplers (this duplication can be inferred from many ob-servations in the literature, but has never been directly tested).When these two experimental tasks are completed, it wouldthen be important to specify the detailed molecular mechanismby which classical uncouplers can mediate the coupling ofelectron flow to cyclical transport of cations.A recent study of SF6847 has led to the conclusion that un-

couplers can act catalytically, since the molar concentration ofelectron transfer chains can be shown to be 10 to 50 times higherthan the molar concentration of uncoupler estimated forcomplete uncoupling (28). The thesis we are proposing for themechanism of uncoupling would require 1:1 stoichiometry

§ Unpublished observations of H. Komai.

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between a given electron transfer complex and an uncouplerduring the catalytic cycle. Allowance has to be made for the factthat this stoichiometry can be evaluated only when all sites forelectron transfer are operative (see Table 3), and for the possi-bility that the intervals between electron flow in a given com-plex may greatly exceed in time the intervals committed toelectron flow (between committed intervals the uncoupler isfree to interact at another site). Margolis et al (29), who madesuch allowance in analyzing their data, concluded that thestoichiometry is indeed 1:1.The mechanism of uncoupling cannot be separated from the

mechanism of coupling. These are two sides of the same coin.Until now, there have been only speculations about the mech-anism of uncoupling-no hard evidence. But if, as it now ap-pears, the mechanism of uncoupling can be defined with pre-cision, the moment of truth will have arrived for all currenttheories of energy coupling. How do these theories rationalizeand accommodate the experimentally verifiable mechanismof uncoupling?Note Added in Proof. The full set of known uncouplers has been ex-amined for ionophoric activity and 24 in all have been found to beactive without exception. Uncouplers form 1:1 complexes in presenceof cations with electrogenic ionophores such as valinomycin; thesemixed complexes are 10-20 times more active as ionophores at pH 7.0than either of the component ionophores. It thus appears that thesynergistic action of uncouplers and intrinsic ionophores underliesuncoupling via cyclical transport of cations. At the point of maximalrelease of respiration, there is a 1:1:1 molar relation of uncoupler(SF6847), intrinsic electrogenic ionophore, and electron transfercomplex.

This investigation was supported in part by Program Project GrantGM-12847 of the National Institute of General Medical Sciences. Theexpert technical assistance of Ms. Karen Senzig is gratefully ac-knowledged. Our thanks is due to Dr. Peter G. Heytler of the duPontdeNemours Company for samples of carbonyl cyanide phenylhydra-zone and 1799; to Dr. D. Amnon for a sample of desapidin; and to theSumitomo Chemical Co. in Osaka, Japan for a sample of SF6847.

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