Eur. J. Biochem. 77, 349-356 (1977)
The Effective Proton Conductance of the Inner Membrane of Mitochondria from Brown Adipose Tissue Dependency on Proton Electrochemical Potential Gradient David G. NlCHOLLS Department of Psychiatry, Ninewells Medical School, Dundee University (Received January 4, 1977)
The nucleotide-sensitive H+(OH-) conducting pathway of mitochondria from the brown-adipose tissue of cold-adapted guinea-pigs passes an effective proton current which is directly proportional to the proton electrochemical gradient. At 23 "C and pH 7.0 this conductance is 16 nmol H + . min-' . mg-' . mV-'. Addition of 0.2 mM GDP results in a conductance which is linear and low (0.7 nmol H + . min-' . mg-' . mV-') until d,iiH+ exceeds 220 mV. At higher values of d,iiFI+, which can be attained by glycerol 3-phosphate oxidation but not palmitoyl-L-carnitine plus malate oxidation, the membrane conductance greatly increases, effectively limiting the maximal d,iiH+ to 240 mV. High glycerol 3-phosphate concentrations which have the thermodynamic potential to exceed this value of d,iiH+ instead create a greatly increased rate of controlled respiration. The generality and significance of this device to limit A D H + , and its relation to the nucleotide-sensitive conductance, are discussed.
An essential requirement of the chemiosmotic theory of oxidative phosphorylation [I - 31 is that the effective proton conductance of the mitochondrial inner membrane (C,,,+) is sufficiently low to avoid excessive shunting of the notional proton circuit linking the respiratory chain and the proton-translocating ATP synthetase. Brown-adipose-tissue mitochondria, however, are distinctive in possessing an ion-conducting pathway which may be inhibited by the binding of certain purine nucleotides to a site on the outer face of the inner membrane [4,5]. The operation of this pathway increases C,,H+ by an order of magnitude, while at the same time increasing the conductivity of the membrane to anions such as CIor Br- [4,6,7]. The modulation of the ion conductances by exogenous purine nucleotides appears to represent a physiological event, namely the control of energy dissipation during non-shivering thermogenesis [4], and this conductance pathway can therefore act as a chemically-gated translocator. As the existence of a specific binding site for the inhibitory purine nucleotides on the outer face of the inner membrane [5] raises the possibility of the identification of the comSymbols. A G,(out), adenine nucleotide phosphorylation potential in the extra-matrix space; A pH+,proton electrochemical gradient (proton-motiLc force); A E, membrane potential; A pH, transmembrane pH gradient; Cm."+,effective proton conductance of the inner membrane.
ponents responsible for the conductance pathway, this system represents one which is potentially fruitful for the elucidation of the mode of action of such translocators. The purpose of the present communication is to extend the information on this translocator by considering the way in which the proton current leaking back across the inner membrane varies with the magnitude of the applied proton electrochemical gradient (LIP"+). Proton conductance is defined as the effective proton current crossing the membrane per unit of proton electrochemical gradient, thus it is necessary to determine both these parameters simultaneously. A direct approach, which has been employed both for chromatophores [8] and mitochondria [9] is to study the decay of a transient proton gradient under conditions where A E is eliminated by the inclusion of valinomycin and high external potassium concentrations. However, while chromatophores can maintain a high ApH+ under these conditions, their applicability to mitochondrial systems is limited to sub-optimal levels of A,&+, as in state 4 conditions the major component of ApH+appears of necessity to be A E [3,12], with the result that A pH alone would greatly under-estimate the driving force for proton re-entry. Additionally, studies of the rate of decay of A ~ H would + not reveal change in proton current occurring at constant A i&+, of the type described in this paper. Therefore the approach taken here is to
350
determine proton current and A pH+under a series of steady-state conditions. In order to determine the dependency of the effective proton current across the membrane on A ,El,+, it is necessary to modulate the latter parameter by means which do not a pviori affect the effective proton conductance of the membrane. In this paper, this is accomplished by progressively limiting the substrate dehydrogenase activity by decreasing substrate concentrations. The results indicate that the nucleotide-sensitive translocator conducts protons (or OH-) at a rate proportional to d p H + , whereas when this pathway is inhibited by the addition of exogenous purine nucleotide, then flavoprotein-linked substrates can raise A p H + to levels at which there is a marked increase in C,,,,H+,the generality and significance of which is discussed. MATERIALS AND METHODS Mitochondria were prepared as previously described [lo, l l ] from the brown adipose tissue of hamsters and guinea-pigs which had been acclimatised at 4-6°C for at least one week before sacrifice. Protein was determined by the biuret method. The components of A,&+ were determined as previously described [12]. The volumes for the matrix compartments of mitochondria from both animals were taken from previous studies [11,131, allowance being made where appropriate for the increased osmolarity created by the addition of high concentrations of DL-glycerol 3-phosphate. Respiration was monitored by a Clark-type oxygen electrode in a thermostated chamber of 1.2-ml capacity. For the calculation of the trans-membrane effective proton current, an integral stoichiometry of 3+H+/site/2eP was assumed [ l l ,14- 161. The phosphorylation state of extra-mitochondria1 adenine nucleotides was determined by polyethylimine thinlayer chromatography, as previously described [5,12]. The fluorescence of mitochondrial NAD(P)H was determined in an Eppendorf photometer with fluorescence attachment, the exciting wavelength being 366 nm, and with a secondary filter transmitting above 430 nm. Oxidised and reduced nicotinamide nucleotides were extracted from mitochondria and assayed by the methods of Klingenberg and Slenczka [17], employing an Eppendorf photometer with fluorescence attachment. Radioactive isotopes were obtained from the Radiochemical Centre (Amersham, U.K.). Enzymes for the assay of nicotinamide nucleotides, G D P and carbonylcyanide p-trifluoromethoxyphenylhydrazone were supplied by the Boehringer Corp. (London). Palmitoyl-L-carnitine was supplied by PL Biochemicals
Pathways of Mitochondria1 Proton Conductance
(Milwaukee, Wisconsin, U.S.A.). All other reagents were of reagent grade or better. RESULTS In the absence of exogenous purine nucleotides, brown-adipose-tissue mitochondria from both coldadapted hamsters [18] and guinea-pigs [ l l ] are capable of oxidising substrates at rates approaching 200 nmol 0 . min-' . mg-' at 23 "C without developing a proton electrochemical gradient in excess of 80 mV. As the mitochondria continue to extrude protons under these conditions [18] this indicates that a high conductance pathway for H + (or OH-) must exist across the inner membrane. In Fig. 1A, respiration and A&+ are plotted for the guinea-pig mitochondria under these conditions. In the absence of exogenous CaZ the mitochondrial glycerol-3-phosphate dehydrogenase, located on the outer face of the mitochondrial inner membrane, possesses a low affinity for its substrate [19], so that it is possible to vary the respiratory rate by limiting the concentration of glycerol 3-phosphate. In the absence of substrate a A&+ of - 3 mV is obtained, indicating that under these conditions protons are essentially at electrochemical equilibrium across the membrane. As the respiratory rate increases, the steady-state A pH+across membrane increase proportionately, until a A p t , + of 70 mV is attained when respiration reaches 200 nmol 0 . min-' . mg-'. In Fig. 1B, these results are replotted to show the dependency of A ,GH+ on the respiratory rate. In order to confirm that these results do not depend upon the substrate oxidised, results are also depicted which were obtained from a parallel experiment in which palmitoyl-L-carnitine (which requires the presence of malate for rapid oxidation by the guinea-pig mitochondria [20]) was the substrate, varying malate concentrations being employed to vary the rate of respiration. The slightly lower slope in this latter case (Fig.1B) is most likely a consequence of a predicted + H + / O stoichiometry of 6 for glycerol-3phosphate oxidation, but 8 for the oxidation of palmitoyl-L-carnitine plus malate to citrate [I I ] . With both substrates it is clear that the effective proton conductance is constant over the range examined, i.e. that the proton current is directly proportional to d,&,+. From Fig. 1 B, the value of this conductance can be calculated to be 16nmol H + . min-' . mg-' . mV-' in the presence of glycerol3-phosphate, and 19 nmol H + . min-' . mg-' . mV-l when palmitoyl-L-carnitine plus malate are substrates. With brown-adipose-tissue mitochondria from hamsters, a similar ohmic relationship was obtained in the presence of varying glycerol 3-phosphate. From these results it is apparent that the conductance pathway which is unique to these mitochondria +
D. G. Nicholls
351 mitochondria
I
mitochondria
mitochondria
II
Iu
I
80
60
->
5
F .
40
0
-
I I d
0
d
c
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0
8 0
I
5 min 274
Fig.2. Enhanced state 4 respiration in the presence qf high glycerol 3-phosphate concentrurions. Guinea-pig mitochondria (0.5 mg protein/ml incubation) were incubated at 23 "C and pH 7.0 on a medium containing 100 mM sucrose, 10 mM Na/N-tris(hydroxymethyl)methyl-2-aminoethane sulphonate, 32 pM albumin, 0.5 mM KCI, 0.5 mM EDTA and 0.5 mM sodium phosphate. In trace I 60 pM palmitoyl-L-carnitine and 1 mM malate were present initially. In trace I1 2 mM DL-glycerol3-phosphate and 2 pM rotenone were present, while in trace 111 2 pM rotenone and 20 mM DLglycerol 3-phosphate were present. Further additions where indicated were of 0.5 mM GDP, 1 mM MgC12 or 1 pM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). Rates are in nmol 0 .min-' . mg-'
/4 0
20
40
60
\
80
db,+(mv)
Fig. 1. The variation of respiratory Fate with proton electrochemical gradient ,for guinea-pig mitochondria respiring in the absence of exogenous purine nucleotide. (A) Mitochondria (0.5 mg proteinjinl incubation) were incubated at 23 'C and pH 7.0 in a medium containing 100 m M sucrose, 10 mM NajN-tris(hpdroxymethy1)methyl-2-aminoethane sulphonate, 0.5 mM KC1, 0.5 mM NaEDTA, 32 pM albumin, 0.5 pM valinomycin, 2 pM rotenone, 50 pM "RbCI (0.05 pCijmI), 50 pM ['4C]methylamine (0.1 pCi/ ml), 0.2 mM [3H]acetate (1 pCi/ml), with the further additions of glycerol 3-phosphate at final concentrations from 0.2 to 10 mM. Respiration (0) and A&+ (0) were determined as described in Methods following a 2-min incubation. (B) The data from A is replotted to show the variation of A&+ with respiratory rate (A). Also shown are points from a parallel experiment where the substrate was 60 pM palmitoyl-L-carnitine, respiration being varied by the addition of malate at concentrations from 0 to 2 mM (A); conditions were otherwise as described for glycerol 3-phosphate, except that rotenone was absent
[4,18] is operative even when A,&+ is vanishingly small. This is in contrast to a pathway which is formed as a response to a membrane potential or electrochemical gradient, as has been shown for the voltage-gated channel forming antibiotic alamethicin [21]. The operation of the H' (or OH-) pathway
under conditions of very low AD"+ is consistent with experiments involving osmotic swelling of non-respiring mitochondria under conditions where proton translocation is required for charge neutralisation [61. The chemiosmotic theory proposes that the rate of controlled state 4 respiration is limited by the effective proton conductance of the inner membrane [l - 31, and should therefore be independent of the activity of the respiratory chain or substrate dehydrogenase, as long as these are in excess. Thus, Fig.2, the rate of state 4 respiration following GDP addition with palmitoyl-L-carnitine plus malate as substrates, or with 2 mM DL-glycerol 3-phosphate imply similar rates of proton cycling across the inner membrane. In the presence of high concentrations of glycerol 3-phosphate, however (Fig. 2) state 4 respiration can increase to a rate double that in the presence of low glycerol 3-phosphate concentrations. That the increase in rate is not due to a chelation of divalent cations by the high glycerol 3-phosphate concentrations is indicated both by the failure of 1 mM MgClz to reduce significantly the state 4 respiration, and by the low state 4 rates in the other traces of Fig. 2 even though the incubation medium included 0.5 mM EDTA. Oligomycin, 2 pg/ mg mitochondria1 protein was without effect on the increased respiration in the presence of high glycerol 3-phosphate concentrations.
352
Pathways of Mitochondria1 Proton Conductance
8o
1
WH+( m V ) Fig. 3. The variation of respiratory rate with proton electrochemical gradient for guinea-pig mitochondria respiring in the presence of exogenous GDP. The experimental conditions were precisely as described in the legend to Fig. 1, except that 0.2 mM GDP was present in all incubations, and the concentration o f glycerol 3-phosphate where present was varied from 0 to 20 mM. Varied DL-glycerol 3-phosphate (A), varied malate in the presence of palmitoyl-L-carnitine (A)
ao
--
60
m
E
-
-
L
E
-
0
-
40
c c
0
+ m
L a rn
20
C
0
160
260
360
400
500
600
700
A Gp - (out) (mV)
Fig. 4. The variation of re.spir.atosy rate with the phosphorylation potential of external adenine nucleotides ,for guiiiea-pig mitochondria in the presence of GDP. Mitochondria (0.5 mg protein/ml incubation) were incubated at 23 "C and pH 7.0 under the conditions of Fig. 1, but without valinomycin or the isotopic indicators of A,?"+ and with the further additions of 0.5 mM sodium phosphate and 0.1 mM [3H]ADP (10 pCi/ml). After 6 min of incubation, 100-pl aliquots were taken and quenched in 5 M perchloric acid as previously described [I 21. The phosphorylation potentials were calculated employing the values of Rosing and Slater [27] for the standard free energy of ATP hydrolysis. The experimental points were obtained by varying glycerol 3-phosphate concentrations (A) in the presence of rotenone, or by varying malate concentrations in the presence of 60 pM palmitoyl-L-carnithe (A)
The increased state 4 respiration could result either from an increase in C,,,, H + , or from a decreased stoichiometry of proton extrusion under the conditions produced by the high glycerol 3-phosphate concentrations. The latter would imply that under the near thermodynamic equilibrium conditions pertaining in state 4 [ l l , 22- 241, the oxido-reduction potential available from the respiratory chain would be capable of maintaining an increased A,&+ at the expense of the decreased stoichiometry. However, experiments performed under the conditions of Fig. 3 showed that increasing m-glycerol 3-phosphate from 2 mM to 20 mM changed A,&+ only marginally, from 237mV to 239mV. An unchanged stoichiometry is also implied by the same reasoning from an unchanged AG, (out) when the same transition is made (Fig. 4). The conclusion is thus reached that C m , ~ in+ creases in the presence of high glycerol 3-phosphate concentrations under the conditions of Fig. 2. With the assumption of an invariant stoichiometry of proton extrusion this conductance may be derived from the slope of a respiration/A pH+ trace obtained under identical conditions to those of Fig. 1, but with the inclusion of 0.2 mM GDP. Results are presented in Fig.3, which shows that in the presence of GDP to inhibit the conductance pathway [I81 there is a greatly decreased conductance (cJ. Fig. l), and that furthermore there is a clear discontinuity when ApH+ rises above about 220 mV, resulting in a disproportionate increase in respiration and hence proton current across the membrane. This increase in the effective proton conductance of the membrane could have a variety of causes. A direct uncoupling effect of glycerol 3-phosphate per se is however unlikely, because as will be discussed below, a similar non-linear relation between respiration and A ,CH+ has been previously observed with liver mitochondria [12], when the rate of succinate oxidation was increased by decreasing the concentration of malonate in the incubation ;clearly a direct uncoupling effect cannot account for the increased conductance observed under those conditions. Additionally, a direct uncoupling effect would manifest itself in a decreased A ~ H +[ l l , l S ] , whereas as shown in F i g 3 A P H t is maintained as respiration increases. The non-linear response could also be an artifact inherent in the techniques employed to determine A,&+ [ll]. Thus any technical limit on the observable magnitudes of the ion gradients used in the estimation of A E or A pH, could result in an artifactual plateau of the measured A pH+.However this is not probable, as the values of A p H + calculated by the isotope distribution technique employed here for liver mitochondria in state 4 are in excellent agreement with those obtained by continuous monitoring with ionspecific electrodes, as originally employed by Mitchell
D. G. Nicholls
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Table 1. The components ofthe proton electrochemical gradient across the inner membrane of hamster brown-adipose-tissue mitochondria oxidising glycerol 3-phosphate in the presence of varying KCl Mitochondria (1 mg protein/ml) were incubated at 23 "C and pH 7.2 in a medium containing 100 mM sucrose, 10 mM choline chloride, 10 mM glycyl-glycine, 6 mM m-glycerol 3-phosphate, 0.5 pM valinomycin, 140 pM albumin, 1 mM GDP, SO pM "RbCl (0.05 pCi/ml), 50 pM [14C]methylammoniumchloride (0.1 pCi/ml) 100 pM [3H]acetate (1 pCi/ml) together with KCI as indicated. After incubating for 4 min, aliquots were filtered for the determination of the components of A ptIt
mM
mV
0.25 0.5 1.o
154 133 125
61 80 84
21 5 21 3 209
and Moyle [25]. In addition Table 1 demonstrates that it is possible to vary the magnitude of the individual components of A pH+under state 4 conditions without significantly influencing the observed A ,&+, indicating that neither the Rb' nor acetate gradients have attained their experimental limits under state 4 conditions. A third possible artifact could result from the necessity to include valinomycin in the incubation media in order to estimate LIE from the Nernst distribution of "Rb+ across the membrane [12,25]. As brown-adipose-tissue mitochondria possess an electroneutral exchange activity [6] of the type first described by Mitchell and Moyle [26] for the exchange of H' for Na', or less effectively K + , this results in the possibility of an energy-dissipating cycling of K' or Rb' across the membrane in concert with valinomycin, the cycle being driven by A&+. While control experiments [18] have established that the activity of the antiporter with K + or R b + is low, the possibility remains that as in the presence of excess valinomycin the electroneutral exchange is driven by the full A,iiH+ (Eqn 1) then the increase in C m , ~could + be due to greatly increased activity of the antiporter itself.
In order to eliminate both these technical factors as possible causes of the non-ohmic response, the extra-mitochondria1 phosphorylation potential of adenine nucleotides [ AG, (out)] was determined in place of A ptI+.The ability to omit valinomycin under these conditions lessens the possibility of a dissipative cycling across the membrane of K' or Rb'. Under conditions where the proton electrochemical gradient is dissipated directly by an increase in Cm,H+ there is little net synthesis of ATP once a steady-state is
attained [ I l l , and it has been previously shown for guinea-pig brown-adipose-tissue mitochondria [l1, 161 that under these conditions A p H + and AG,(out) remain close to thermodynamic equilibrium when ApH+ is varied over the range from 230 to 190 mV, although differing results have been reported for liver mitochondria [15]. As shown in Fig.4, a non-linear relationship between respiration and A G,(out) is obtained which is virtually indistinguishable from that obtained when A pH+ was determined (Fig. 3). Thus both dissipative cation cycling and technical errors in the determination of A fiH+ can be eliminated as factors contributing to a possibly artifactual electrochemical-potential-dependent conductance of the membrane. While a non-linear relation between proton current and A pH+is invariably attained when brown-adiposetissue mitochondria from both guinea-pigs and hamsters (experiments not shown) oxidise glycerol 3-phosphate, when palmitoyl-L-carnitine plus malate are present as substrate, no significantly disproportionate increase in C,,H+is observed (Fig. 3) even when sufficient malate is present for uncontrolled rates of respiration of 160 nmol 0 . min-' . mg-' (Fig.2). In a similar way, this substrate pair does not show a non-linear increase in respiration when this is plotted as a function of AG,(out), Fig. 4. It is however significant that the maximum values of A,&+ or AG,(out) obtained with palmitoyl-L-carnitine plus malate are somewhat lower than those reached with glycerol 3-phosphate as substrate. These results are consistent with a hypothesis whereby brown-adipose-tissue mitochondria in state 4 are operating with a transmembrane A&+ which is close to the maximum which a component in the membrane can withstand without a substantial nonohmic increase in conductance. This would imply that at least under the conditions employed in the present experiments a substrate such as glycerol 3-phosphate is thermodynamically capable of generating a ApH+in excess of this limiting potential, while palmitoyl-carnitine plus malate are not. To decide whether the increase in C,,,H+is dependent on the magnitude of AbH+rather than either of its components A E or ApH, the experiment shown in Table 1 was performed. As the increase in Cm,Habove the limiting potential essentially prevents any further increase in d pH+ from occurring (Fig. 3), an increase in the relative contribution of A pH to A i i H + by increasing [K'] [12,25] in the medium would be predicted to lower ApH+ in state 4 if the magnitude of A pH determined the breakdown potential, to increase A &+ if A E was the determinant of the breakdown, and to leave the state 4 Aj&+ unchanged if A,&,+ itself was relevant parameter. As can be seen, the evidence is in favour of the last possibility, A , l H + being the least variable parameter,
Pathways of Mitochondria1 Proton Conductance
354
fl
1 'L :aEj;yl-~-
carnitine
Rotenone
-1
GDP
I
7Nicotinamide nucleotide fluorescence
Glycerol 3-phosphate
GDP
GDP
I
7
7
IT
m
5rnin
Fig. 5. Fluorescence o/ endogetiou.Y reduced nicotinamide nucleotides of guinea-pig brol.1.n-adipose-tissue mitochondria. Mitochondria (0.5 mg proteinjml) were incubated at 23 "C and pH 7.0 in a medium containing 100 mM sucrose, 20 mM Na/N-tris(hydroxymethyl)methyl-2-aminoethane sulphonate, 32 pM albumin, 0.5 niM KCI, 0.5 m M EDTA and 0.5 mM sodium phosphate. In trace I. 60 pM palmitoyl-L-carnitine and 1 mM malate were initially present, and further additions were made where indicated of 0.5 mM GDP, 10 m M DL-glycerol 3-phosphate and 2 pM rotenone. In trace 11, 0.4 mM DL-glycerol 3-phosphate was initially present, and 0.5 mM GDP was added where indicated. In trace 111, 10 mM m-glycerol 3-phosphate was initially present, and additions were made of 0.5 mM GDP, 60 pM palniitoyl-L-carnitine plus 2 mM malate, and 2 pM rotenone
of producing a partial reduction. Assays performed on nicotinamide nucleotides extracted from hamster brown-adipose-tissue mitochondria oxidising 3 0 m M glycerol 3-phosphate in the presence of 160 pM albumin and 1 mM G D P at pH 7.2 revealed a total pool of NAD(H) of 6.3 nmol . mg-' and 0.5 nmol .mg-' of total NADP(H). The N A D + pool was 83 % reduced, confirming an extensive reduction by glycerol 3-phosphate in Fig. 5. The results reported above show that the first proton-translocating region of the respiratory chain is only in thermodynamic equilibrium with the A pH+generated by the oxidation of high glycerol 3-phosphate concentrations when the NAD' pool is 83% reduced. Clearly, palmitoylL-carnitine plus malate oxidation is incapable of maintaining the even more extensive reduction of NAD' which would be required for there to be net electron transport through this first region under these conditions (Fig. 5), and the conclusion is that electron transport from NAD-linked substrates to quinone becomes inhibited by the approach to thermodynamic equilibrium at values of ApH+ which are below those which the second and third regions can sustain. DISCUSSION
while substantial variations in the magnitude of A E and - 59 ApH occur. An increase in Cm,H+appears not to be restricted to brown-adipose-tissue mitochondria, as rat liver mitochondria oxidising succinate in the presence of varying concentrations of malonate have been reported to decrease AplI+[12] or AG,(out) [23] disproportionately as respiration is progressively inhibited. The ability of two flavoprotein-linked substrates to invoke a non-linear response, and the ability of the flavoprotein-linked glycerol 3-phosphate to maintain a greater A j i ~ +than palmitoyl-L-carnitine plus malate, raises the possibility that at least under the conditions employed here, the second and third regions of proton translocation in the respiratory chain have the ability, for a given rate of electron transfer, to maintain a il,iiilgreater + than that produced by the first site between NADH and ubiquinone. This can be experimentally verified by comparing the extent of reduction of endogenous nicotinamide nucleotides when palmitoyl-L-carnitine is oxidised in the controlled state with that maintained by glycerol 3-phosphate oxidation, as a consequence of the thermodynamic reversibility of the first site [28]. The experiments reproduced in Fig. 5 were performed under conditions parallel to those of Fig. 2 - 4. It is clear that high concentrations of glycerol 3-phosphate cause extensive reduction of nicotinamide nucleotides while low glycerol 3-phosphate levels or palmitoyl-L-carnitine plus malate are each only capable
The Conductance of the Inner Membrane in the Absence of Exogenous Purine Nucleotides From Fig. 1 B the conductance of the nucleotide sensitive pathway is about 16 nmol H + . min-' . mg-'. Analysis of the binding of inhibitory purine nucleotides to the outer face of the inner mitochondria1 membrane reveal 0.5-0.8 nmol . mg protein-' of binding sites for both hamster and guinea-pig mitochondria [5,30]. The good correlation obtained between binding of nucleotides and the inhibition of C m , ~[31,32] + strongly suggests that all these binding sites are active in inhibiting the conducting pathway. On the assumption that there is about one binding site per ion conducting pathway, this implies a conductance of about 20 H . min-' . mV-' per pathway, or when d,iiH+ is 60 mV, 1200 H + . min-'. This conductance is many orders of magnitude lower than that catalysed by pore-forming antibiotics such as gramicidin [29]. On the other hand the existence of specific nucleotide-binding sites on the outer face only of the membrane [5,30] argues against a lowmolecular-weight mobile carrier. The evidence is therefore in favour of a component which spans the inner membrane. The conductance of CI- across the membrane of brown-adipose-tissue mitochondria is abnormally high and is inhibited to levels characteristic of liver or heart mitochondria by the same purine nucleotides which inhibit Cm,H+[6,7,31]. In addition there is an ohmic dependency of CIK transport on the C1- electro-
355
D. G. Nicholls
chemical gradient in the absence of inhibitory purine nucleotides, and a non-ohmic relation in their presence [7]. All this, together with evidence of competition for transport between the ions [4] suggests that a common pathway is involved, and that, as previously proposed [4], this is most easily reconciled with a pathway which conducts not protons into the matrix, but OH- out of the matrix, the two being bioenergetically indistinguishable. In the course of catalysing anion uniport across the membrane, uncompensated charge is transferred. The ohmic nature of the conductance suggests that one way in which this might be accomplished is by the diffusion under the influence of an electrochemical gradient of anions through a conducting channel lined with positively charged groups.
The Conductance of the Inner Membrane in the Presence of Exogenous Purine Nucleotides The ohmic conduction region of Fig. 3 (i.e. up to 200 mV) indicates a C,,,H+ of 0.7 nmol H + . min-' . mg-' . mV-', which is only 4 % of the level in the absence of purine nucleotide. The nucleotide inhibition is thus extremely effective. The non-ohmic increase in ion conductance at high electrochemical gradients appears not only for the effective proton conductance of brown-adiposetissue mitochondria and liver mitochondria [12,23], and for the chloride conductance of the former mitochondria [7], but may also be inferred from the 'energydependent' chloride permeability which has been reported for heart mitochondria [33]. Such conductances demand a pathway for ion conduction, even though this pathway is non-ohmic and is only significant at high electrochemical gradients. The components for this pathway could either be distinct from the nucleotide-sensitive conductance of brown-adipose-tissue mitochondria, which would help to explain the similar behaviour of mitochondria from a variety of organs, or, in view of the ability of brown-adiposetissue mitochondria to conduct the same ions, namely C1- and OH-, under both ohmic and non-ohmic conditions, it is also possible that a single conductance pathway is present in all mitochondria, but that only in brown-adipose-tissue mitochondria is it modified to produce a high, ohmic conducting state, together with a specific binding site for purine nucleotides, occupancy of which would cause the pathway to revert to the normal state of low and non-ohmic conductance. The rate of controlled respiration of both hamster [IS] and guinea-pig [l 11 brown-adipose-tissue mitochondria has been shown to be proportional to the extent of the disequilibrium between ApH+ and the oxido-reduction potential span available from the substrate couple. The increased respiration obtained
500
I '
j
t
Fig. 6 . Analysis (if the non-ohmic relution between effective proton current and proton electrochemical grudient produced by mitochondria oxidising varying concentrations of glycerol 3-phosphate in the presence qf exogenous GDP. The empirical relation between effective proton current and A & + obtained in Fig.3 is replotted ( 0 )and compared with that generated (solid line) on the assumptions that the membrane possesses both a conductance of 0.66 nmol H min- ' . mg- ' . mV- ' (given by I) and an infinite conductance operative only at or above a A,&+ of 240 mV (given by IT). On the assumption that the empirically observed drop of 1 mV in A ~ I I +which is required to drive an increase in respiration of 2 nmol 0 . min-' . mg-' 1111 holds for varying glycerol 3-phosphate concentrations, the discontinuous lines 111, IV and V represent the extrapolation back to static head conditions of A&+ observed at respectively 0.6 mM, 2 mM and 20 mM glycerol 3-phosphate +
at high concentrations of glycerol 3-phosphate therefore implies that, as ADH+ does not alter (Fig.3), the oxido-reduction potential span must increase. Under conditions closely similar to those of Fig. 3 , it has previously been shown that guinea-pig brownadipose-tissue mitochondria increase their rate of controlled respiration by 2 nmol 0 . min-' . mg-' for each millivolt that ApH+ decreases, under conditions where substrate concentration does not vary [ l l ] . Extrapolating this relationship back to zero respiration, it is possible to calculate the value of A p H + which is maintained by the oxidation of glycerol 3-phosphate under static head equilibrium conditions. As the rate of controlled respiration is therefore proportional to the disequilibrium between the static head A,&,+ and the observed A&+, this would imply that the driving force for the increased respiration observed during the oxidation of high glycerol 3-phosphate concentrations is an increased disequilibrium between the static head and observed values of Aj&+. AS the observed values of A ~ H +are virtually constant at the limiting value of ADH+ (Fig.3), this requires that the static head A&+
356
should increase as the concentration of glycerol 3-phosphate increases. In Fig. 6, the experimental points obtained in Fig. 3 are replotted, and compared with the relation between A,&+ and proton current which would be obtained if the mitochondria possessed both a constant conductance of 0.66 nmol H + . min-' . mg-' . mV-l, and an infinite conductance operative only at or above a A b H + of 240 mV, given respectively by the solid lines I and TI in the figure. The dashed lines represent the extrapolation back to static head conditions of the empirical values of A,&+ obtained during the oxidation of respectively 0.6 mM, 2 mM and 20 mM glycerol 3-phosphate. From Fig.6 the corresponding static head ApH+ which is in equilibrium at these concentrations is respectively 230 mV, 258 mV and 273 mV. In conclusion, it appears that mitochondria possess a component in the inner membrane which effectively limits the attainable A,iiH+ across the membrane. With liver and brown-adipose-tissue mitochondria, this limiting potential appears to be set at a level just above that usually attained by NAD+-linked respiration, but which can be reached by active flavoprotein-linked dehydrogenases. Such a limiting device could clearly act to ensure that while A&+ is maintained at very high levels, it cannot rise to levels which cause possibly catastrophic dialectric breakdown of the membrane. This work is financed by the Science Research Council, grant number B,'RG,'84050.0. The expert technical assistance of Aileen Kiddie is gratefully acknowledged.
REFERENCES 1. Mitchell, P. (1961) .Nature (Lond.) 191, 144-148. 2. Mitchell, P. (1968) Chenzio.smotic Coupling and Energy Transduction, Glynn Research, Bodmin, U.K. 3. Mitchell, P. (1976) Biochem. Soc. Trans. 4, 399-430. 4. Nicholls, D. G. (1976) FEBS Lett. 61, 103-110. 5. Nicholls, D. G. (1976) Eur. J . Biochem. 42, 223-228. 6. Nicholls, D. G. & Lindberg, 0. (1973) Eur. J . Biorhem. 37, 523 - 530.
D G. Nicholls : Pathways of Mitochondria1 Proton Conductance
7. Nicholls, D. G. (1974) Eur. J . Biochem. 49, 585-593. 8. Jackson, J. B., Saphon, S. & Witt, H. T. (1975) Riochim. Biophys. Acta, 408, 83 92. 9. Mitchell, P. & Moyle, J. (1967) Biochem. J . 104, 588-600. 10. Hittelman, K. J., Lindberg, 0. & Cannon, B. (1969) Eur. J . Biochem. 11, 183-192. 11. Nicholls, D. G. & Bernson, V. S. M. (1977) Eur. J . Biochem. 75,601 -612. 12. Nicholls, D. G. (1974) Eur. J . BiochiJm. 50, 305-315. 13. Nicholls, D. G., Grav, H. .I. & Lindberg, 0. (1972) Eur. J. Biochem. 31, 526-533. 14. Brand, M. D., Reynafarje, R. & Lehninger, A. L. (1976) Proc. Nut1 Acad. Sci. U.S.A. 73, 437--441. 15. Weichmann, A. H. C. A , , Beem, E. P. & van Dam, K. (1975) in Electron Transfer Chains and Oxidative Phosphorylation (Quagliariello, F., Papa, S., Palmieri, F., Slater, E. C. & Siliprandi, N., eds) pp. 335-342, North Holland, Amsterdam. 16 Nicholls, D. G. (1977) Biochrm. Soc. Trans. 5,200-203. 17 Klingenberg, M. & Slenczka, W. (1959) Biochem. Z . 331, 486 - 517. 18. Nicholls, D. G. (1974) Eur. J . Biochem. 49, 573-583. 19. Bukowiecki, L. J. & Lindberg, 0. (1974) Biochim. Bioph.vs. Acts, 348, 115 125. 20. Bernson, V. S. M. & Nicholls, D. G. (1974) Eur. J . Biochem. 47,517-525. 21. Baumann. G. & Mueller, P. (1974) J. Supramol. Struc.t. 2, 538 - 557. 22. Klingenberg, M. & Schollmeyer, P . (1961) Biochem. Z. 335, 243 262. 23. Slater, E. C . , Rosing, J. & Mol, A. (1973) Biochim. Biophyx. Acta, 292, 534-553. 24. Wilson, D. F. & Erecinska, M. (1973) Mitochondria: Biogenesis and Bioenergetics (van den Bergh, S. G., Borst, P. & Slater, E. C., eds) pp. 119-132, North Holland, Amsterdam. 25. Mitchell, P. & Moyle, J. (1969) Eur. J . Biochem. 7, 471 -484. 26. Mitchell, P. & Moyle, J. (1969) Eur. J . Biochem. Y, 149-155. 27. Rosing, J. & Slater, E. C. (1972) Biochim. Biophys. Actn, 267, 275 - 290. 28. Chance, B. & Hollunger, G. (1960) Nature (Lond.) 185. 666672. 29. Mueller, P. & Rudin, D. 0. (1969) Curr. Top. Bioenergetic.s, 3, 159 -249. 30. Rafael, J. & Heldt, H. W. (1976) FEBS Lett. 43, 304-308. 31. Cannon, B., Nicholls, D. G. & Lindberg, 0. (1973) in Mechanisms in Bioenergetics, pp. 357 - 363, Academic Press, New York. 32. Heaton, G. M. & Nicholls, D. G. (1977) Biochem. Soc. Trans. 5,210-212. 33. Brierley, G. P. (1970) Biochemistry, Y, 697-707. -
-
-
D G Nicholls, Department of psychiatry, University of Dundee Medical School, Ninewells Hospital, Dundee, Great Britain, DD2 1UD
The Effective Proton Conductance of the Inner Membrane of Mitochondria from Brown Adipose Tissue Dependency on Proton Electrochemical Potential Gradient David G. NlCHOLLS Department of Psychiatry, Ninewells Medical School, Dundee University (Received January 4, 1977)
The nucleotide-sensitive H+(OH-) conducting pathway of mitochondria from the brown-adipose tissue of cold-adapted guinea-pigs passes an effective proton current which is directly proportional to the proton electrochemical gradient. At 23 "C and pH 7.0 this conductance is 16 nmol H + . min-' . mg-' . mV-'. Addition of 0.2 mM GDP results in a conductance which is linear and low (0.7 nmol H + . min-' . mg-' . mV-') until d,iiH+ exceeds 220 mV. At higher values of d,iiFI+, which can be attained by glycerol 3-phosphate oxidation but not palmitoyl-L-carnitine plus malate oxidation, the membrane conductance greatly increases, effectively limiting the maximal d,iiH+ to 240 mV. High glycerol 3-phosphate concentrations which have the thermodynamic potential to exceed this value of d,iiH+ instead create a greatly increased rate of controlled respiration. The generality and significance of this device to limit A D H + , and its relation to the nucleotide-sensitive conductance, are discussed.
An essential requirement of the chemiosmotic theory of oxidative phosphorylation [I - 31 is that the effective proton conductance of the mitochondrial inner membrane (C,,,+) is sufficiently low to avoid excessive shunting of the notional proton circuit linking the respiratory chain and the proton-translocating ATP synthetase. Brown-adipose-tissue mitochondria, however, are distinctive in possessing an ion-conducting pathway which may be inhibited by the binding of certain purine nucleotides to a site on the outer face of the inner membrane [4,5]. The operation of this pathway increases C,,H+ by an order of magnitude, while at the same time increasing the conductivity of the membrane to anions such as CIor Br- [4,6,7]. The modulation of the ion conductances by exogenous purine nucleotides appears to represent a physiological event, namely the control of energy dissipation during non-shivering thermogenesis [4], and this conductance pathway can therefore act as a chemically-gated translocator. As the existence of a specific binding site for the inhibitory purine nucleotides on the outer face of the inner membrane [5] raises the possibility of the identification of the comSymbols. A G,(out), adenine nucleotide phosphorylation potential in the extra-matrix space; A pH+,proton electrochemical gradient (proton-motiLc force); A E, membrane potential; A pH, transmembrane pH gradient; Cm."+,effective proton conductance of the inner membrane.
ponents responsible for the conductance pathway, this system represents one which is potentially fruitful for the elucidation of the mode of action of such translocators. The purpose of the present communication is to extend the information on this translocator by considering the way in which the proton current leaking back across the inner membrane varies with the magnitude of the applied proton electrochemical gradient (LIP"+). Proton conductance is defined as the effective proton current crossing the membrane per unit of proton electrochemical gradient, thus it is necessary to determine both these parameters simultaneously. A direct approach, which has been employed both for chromatophores [8] and mitochondria [9] is to study the decay of a transient proton gradient under conditions where A E is eliminated by the inclusion of valinomycin and high external potassium concentrations. However, while chromatophores can maintain a high ApH+ under these conditions, their applicability to mitochondrial systems is limited to sub-optimal levels of A,&+, as in state 4 conditions the major component of ApH+appears of necessity to be A E [3,12], with the result that A pH alone would greatly under-estimate the driving force for proton re-entry. Additionally, studies of the rate of decay of A ~ H would + not reveal change in proton current occurring at constant A i&+, of the type described in this paper. Therefore the approach taken here is to
350
determine proton current and A pH+under a series of steady-state conditions. In order to determine the dependency of the effective proton current across the membrane on A ,El,+, it is necessary to modulate the latter parameter by means which do not a pviori affect the effective proton conductance of the membrane. In this paper, this is accomplished by progressively limiting the substrate dehydrogenase activity by decreasing substrate concentrations. The results indicate that the nucleotide-sensitive translocator conducts protons (or OH-) at a rate proportional to d p H + , whereas when this pathway is inhibited by the addition of exogenous purine nucleotide, then flavoprotein-linked substrates can raise A p H + to levels at which there is a marked increase in C,,,,H+,the generality and significance of which is discussed. MATERIALS AND METHODS Mitochondria were prepared as previously described [lo, l l ] from the brown adipose tissue of hamsters and guinea-pigs which had been acclimatised at 4-6°C for at least one week before sacrifice. Protein was determined by the biuret method. The components of A,&+ were determined as previously described [12]. The volumes for the matrix compartments of mitochondria from both animals were taken from previous studies [11,131, allowance being made where appropriate for the increased osmolarity created by the addition of high concentrations of DL-glycerol 3-phosphate. Respiration was monitored by a Clark-type oxygen electrode in a thermostated chamber of 1.2-ml capacity. For the calculation of the trans-membrane effective proton current, an integral stoichiometry of 3+H+/site/2eP was assumed [ l l ,14- 161. The phosphorylation state of extra-mitochondria1 adenine nucleotides was determined by polyethylimine thinlayer chromatography, as previously described [5,12]. The fluorescence of mitochondrial NAD(P)H was determined in an Eppendorf photometer with fluorescence attachment, the exciting wavelength being 366 nm, and with a secondary filter transmitting above 430 nm. Oxidised and reduced nicotinamide nucleotides were extracted from mitochondria and assayed by the methods of Klingenberg and Slenczka [17], employing an Eppendorf photometer with fluorescence attachment. Radioactive isotopes were obtained from the Radiochemical Centre (Amersham, U.K.). Enzymes for the assay of nicotinamide nucleotides, G D P and carbonylcyanide p-trifluoromethoxyphenylhydrazone were supplied by the Boehringer Corp. (London). Palmitoyl-L-carnitine was supplied by PL Biochemicals
Pathways of Mitochondria1 Proton Conductance
(Milwaukee, Wisconsin, U.S.A.). All other reagents were of reagent grade or better. RESULTS In the absence of exogenous purine nucleotides, brown-adipose-tissue mitochondria from both coldadapted hamsters [18] and guinea-pigs [ l l ] are capable of oxidising substrates at rates approaching 200 nmol 0 . min-' . mg-' at 23 "C without developing a proton electrochemical gradient in excess of 80 mV. As the mitochondria continue to extrude protons under these conditions [18] this indicates that a high conductance pathway for H + (or OH-) must exist across the inner membrane. In Fig. 1A, respiration and A&+ are plotted for the guinea-pig mitochondria under these conditions. In the absence of exogenous CaZ the mitochondrial glycerol-3-phosphate dehydrogenase, located on the outer face of the mitochondrial inner membrane, possesses a low affinity for its substrate [19], so that it is possible to vary the respiratory rate by limiting the concentration of glycerol 3-phosphate. In the absence of substrate a A&+ of - 3 mV is obtained, indicating that under these conditions protons are essentially at electrochemical equilibrium across the membrane. As the respiratory rate increases, the steady-state A pH+across membrane increase proportionately, until a A p t , + of 70 mV is attained when respiration reaches 200 nmol 0 . min-' . mg-'. In Fig. 1B, these results are replotted to show the dependency of A ,GH+ on the respiratory rate. In order to confirm that these results do not depend upon the substrate oxidised, results are also depicted which were obtained from a parallel experiment in which palmitoyl-L-carnitine (which requires the presence of malate for rapid oxidation by the guinea-pig mitochondria [20]) was the substrate, varying malate concentrations being employed to vary the rate of respiration. The slightly lower slope in this latter case (Fig.1B) is most likely a consequence of a predicted + H + / O stoichiometry of 6 for glycerol-3phosphate oxidation, but 8 for the oxidation of palmitoyl-L-carnitine plus malate to citrate [I I ] . With both substrates it is clear that the effective proton conductance is constant over the range examined, i.e. that the proton current is directly proportional to d,&,+. From Fig. 1 B, the value of this conductance can be calculated to be 16nmol H + . min-' . mg-' . mV-' in the presence of glycerol3-phosphate, and 19 nmol H + . min-' . mg-' . mV-l when palmitoyl-L-carnitine plus malate are substrates. With brown-adipose-tissue mitochondria from hamsters, a similar ohmic relationship was obtained in the presence of varying glycerol 3-phosphate. From these results it is apparent that the conductance pathway which is unique to these mitochondria +
D. G. Nicholls
351 mitochondria
I
mitochondria
mitochondria
II
Iu
I
80
60
->
5
F .
40
0
-
I I d
0
d
c
20
0
8 0
I
5 min 274
Fig.2. Enhanced state 4 respiration in the presence qf high glycerol 3-phosphate concentrurions. Guinea-pig mitochondria (0.5 mg protein/ml incubation) were incubated at 23 "C and pH 7.0 on a medium containing 100 mM sucrose, 10 mM Na/N-tris(hydroxymethyl)methyl-2-aminoethane sulphonate, 32 pM albumin, 0.5 mM KCI, 0.5 mM EDTA and 0.5 mM sodium phosphate. In trace I 60 pM palmitoyl-L-carnitine and 1 mM malate were present initially. In trace I1 2 mM DL-glycerol3-phosphate and 2 pM rotenone were present, while in trace 111 2 pM rotenone and 20 mM DLglycerol 3-phosphate were present. Further additions where indicated were of 0.5 mM GDP, 1 mM MgC12 or 1 pM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). Rates are in nmol 0 .min-' . mg-'
/4 0
20
40
60
\
80
db,+(mv)
Fig. 1. The variation of respiratory Fate with proton electrochemical gradient ,for guinea-pig mitochondria respiring in the absence of exogenous purine nucleotide. (A) Mitochondria (0.5 mg proteinjinl incubation) were incubated at 23 'C and pH 7.0 in a medium containing 100 m M sucrose, 10 mM NajN-tris(hpdroxymethy1)methyl-2-aminoethane sulphonate, 0.5 mM KC1, 0.5 mM NaEDTA, 32 pM albumin, 0.5 pM valinomycin, 2 pM rotenone, 50 pM "RbCI (0.05 pCijmI), 50 pM ['4C]methylamine (0.1 pCi/ ml), 0.2 mM [3H]acetate (1 pCi/ml), with the further additions of glycerol 3-phosphate at final concentrations from 0.2 to 10 mM. Respiration (0) and A&+ (0) were determined as described in Methods following a 2-min incubation. (B) The data from A is replotted to show the variation of A&+ with respiratory rate (A). Also shown are points from a parallel experiment where the substrate was 60 pM palmitoyl-L-carnitine, respiration being varied by the addition of malate at concentrations from 0 to 2 mM (A); conditions were otherwise as described for glycerol 3-phosphate, except that rotenone was absent
[4,18] is operative even when A,&+ is vanishingly small. This is in contrast to a pathway which is formed as a response to a membrane potential or electrochemical gradient, as has been shown for the voltage-gated channel forming antibiotic alamethicin [21]. The operation of the H' (or OH-) pathway
under conditions of very low AD"+ is consistent with experiments involving osmotic swelling of non-respiring mitochondria under conditions where proton translocation is required for charge neutralisation [61. The chemiosmotic theory proposes that the rate of controlled state 4 respiration is limited by the effective proton conductance of the inner membrane [l - 31, and should therefore be independent of the activity of the respiratory chain or substrate dehydrogenase, as long as these are in excess. Thus, Fig.2, the rate of state 4 respiration following GDP addition with palmitoyl-L-carnitine plus malate as substrates, or with 2 mM DL-glycerol 3-phosphate imply similar rates of proton cycling across the inner membrane. In the presence of high concentrations of glycerol 3-phosphate, however (Fig. 2) state 4 respiration can increase to a rate double that in the presence of low glycerol 3-phosphate concentrations. That the increase in rate is not due to a chelation of divalent cations by the high glycerol 3-phosphate concentrations is indicated both by the failure of 1 mM MgClz to reduce significantly the state 4 respiration, and by the low state 4 rates in the other traces of Fig. 2 even though the incubation medium included 0.5 mM EDTA. Oligomycin, 2 pg/ mg mitochondria1 protein was without effect on the increased respiration in the presence of high glycerol 3-phosphate concentrations.
352
Pathways of Mitochondria1 Proton Conductance
8o
1
WH+( m V ) Fig. 3. The variation of respiratory rate with proton electrochemical gradient for guinea-pig mitochondria respiring in the presence of exogenous GDP. The experimental conditions were precisely as described in the legend to Fig. 1, except that 0.2 mM GDP was present in all incubations, and the concentration o f glycerol 3-phosphate where present was varied from 0 to 20 mM. Varied DL-glycerol 3-phosphate (A), varied malate in the presence of palmitoyl-L-carnitine (A)
ao
--
60
m
E
-
-
L
E
-
0
-
40
c c
0
+ m
L a rn
20
C
0
160
260
360
400
500
600
700
A Gp - (out) (mV)
Fig. 4. The variation of re.spir.atosy rate with the phosphorylation potential of external adenine nucleotides ,for guiiiea-pig mitochondria in the presence of GDP. Mitochondria (0.5 mg protein/ml incubation) were incubated at 23 "C and pH 7.0 under the conditions of Fig. 1, but without valinomycin or the isotopic indicators of A,?"+ and with the further additions of 0.5 mM sodium phosphate and 0.1 mM [3H]ADP (10 pCi/ml). After 6 min of incubation, 100-pl aliquots were taken and quenched in 5 M perchloric acid as previously described [I 21. The phosphorylation potentials were calculated employing the values of Rosing and Slater [27] for the standard free energy of ATP hydrolysis. The experimental points were obtained by varying glycerol 3-phosphate concentrations (A) in the presence of rotenone, or by varying malate concentrations in the presence of 60 pM palmitoyl-L-carnithe (A)
The increased state 4 respiration could result either from an increase in C,,,, H + , or from a decreased stoichiometry of proton extrusion under the conditions produced by the high glycerol 3-phosphate concentrations. The latter would imply that under the near thermodynamic equilibrium conditions pertaining in state 4 [ l l , 22- 241, the oxido-reduction potential available from the respiratory chain would be capable of maintaining an increased A,&+ at the expense of the decreased stoichiometry. However, experiments performed under the conditions of Fig. 3 showed that increasing m-glycerol 3-phosphate from 2 mM to 20 mM changed A,&+ only marginally, from 237mV to 239mV. An unchanged stoichiometry is also implied by the same reasoning from an unchanged AG, (out) when the same transition is made (Fig. 4). The conclusion is thus reached that C m , ~ in+ creases in the presence of high glycerol 3-phosphate concentrations under the conditions of Fig. 2. With the assumption of an invariant stoichiometry of proton extrusion this conductance may be derived from the slope of a respiration/A pH+ trace obtained under identical conditions to those of Fig. 1, but with the inclusion of 0.2 mM GDP. Results are presented in Fig.3, which shows that in the presence of GDP to inhibit the conductance pathway [I81 there is a greatly decreased conductance (cJ. Fig. l), and that furthermore there is a clear discontinuity when ApH+ rises above about 220 mV, resulting in a disproportionate increase in respiration and hence proton current across the membrane. This increase in the effective proton conductance of the membrane could have a variety of causes. A direct uncoupling effect of glycerol 3-phosphate per se is however unlikely, because as will be discussed below, a similar non-linear relation between respiration and A ,CH+ has been previously observed with liver mitochondria [12], when the rate of succinate oxidation was increased by decreasing the concentration of malonate in the incubation ;clearly a direct uncoupling effect cannot account for the increased conductance observed under those conditions. Additionally, a direct uncoupling effect would manifest itself in a decreased A ~ H +[ l l , l S ] , whereas as shown in F i g 3 A P H t is maintained as respiration increases. The non-linear response could also be an artifact inherent in the techniques employed to determine A,&+ [ll]. Thus any technical limit on the observable magnitudes of the ion gradients used in the estimation of A E or A pH, could result in an artifactual plateau of the measured A pH+.However this is not probable, as the values of A p H + calculated by the isotope distribution technique employed here for liver mitochondria in state 4 are in excellent agreement with those obtained by continuous monitoring with ionspecific electrodes, as originally employed by Mitchell
D. G. Nicholls
353
Table 1. The components ofthe proton electrochemical gradient across the inner membrane of hamster brown-adipose-tissue mitochondria oxidising glycerol 3-phosphate in the presence of varying KCl Mitochondria (1 mg protein/ml) were incubated at 23 "C and pH 7.2 in a medium containing 100 mM sucrose, 10 mM choline chloride, 10 mM glycyl-glycine, 6 mM m-glycerol 3-phosphate, 0.5 pM valinomycin, 140 pM albumin, 1 mM GDP, SO pM "RbCl (0.05 pCi/ml), 50 pM [14C]methylammoniumchloride (0.1 pCi/ml) 100 pM [3H]acetate (1 pCi/ml) together with KCI as indicated. After incubating for 4 min, aliquots were filtered for the determination of the components of A ptIt
mM
mV
0.25 0.5 1.o
154 133 125
61 80 84
21 5 21 3 209
and Moyle [25]. In addition Table 1 demonstrates that it is possible to vary the magnitude of the individual components of A pH+under state 4 conditions without significantly influencing the observed A ,&+, indicating that neither the Rb' nor acetate gradients have attained their experimental limits under state 4 conditions. A third possible artifact could result from the necessity to include valinomycin in the incubation media in order to estimate LIE from the Nernst distribution of "Rb+ across the membrane [12,25]. As brown-adipose-tissue mitochondria possess an electroneutral exchange activity [6] of the type first described by Mitchell and Moyle [26] for the exchange of H' for Na', or less effectively K + , this results in the possibility of an energy-dissipating cycling of K' or Rb' across the membrane in concert with valinomycin, the cycle being driven by A&+. While control experiments [18] have established that the activity of the antiporter with K + or R b + is low, the possibility remains that as in the presence of excess valinomycin the electroneutral exchange is driven by the full A,iiH+ (Eqn 1) then the increase in C m , ~could + be due to greatly increased activity of the antiporter itself.
In order to eliminate both these technical factors as possible causes of the non-ohmic response, the extra-mitochondria1 phosphorylation potential of adenine nucleotides [ AG, (out)] was determined in place of A ptI+.The ability to omit valinomycin under these conditions lessens the possibility of a dissipative cycling across the membrane of K' or Rb'. Under conditions where the proton electrochemical gradient is dissipated directly by an increase in Cm,H+ there is little net synthesis of ATP once a steady-state is
attained [ I l l , and it has been previously shown for guinea-pig brown-adipose-tissue mitochondria [l1, 161 that under these conditions A p H + and AG,(out) remain close to thermodynamic equilibrium when ApH+ is varied over the range from 230 to 190 mV, although differing results have been reported for liver mitochondria [15]. As shown in Fig.4, a non-linear relationship between respiration and A G,(out) is obtained which is virtually indistinguishable from that obtained when A pH+ was determined (Fig. 3). Thus both dissipative cation cycling and technical errors in the determination of A fiH+ can be eliminated as factors contributing to a possibly artifactual electrochemical-potential-dependent conductance of the membrane. While a non-linear relation between proton current and A pH+is invariably attained when brown-adiposetissue mitochondria from both guinea-pigs and hamsters (experiments not shown) oxidise glycerol 3-phosphate, when palmitoyl-L-carnitine plus malate are present as substrate, no significantly disproportionate increase in C,,H+is observed (Fig. 3) even when sufficient malate is present for uncontrolled rates of respiration of 160 nmol 0 . min-' . mg-' (Fig.2). In a similar way, this substrate pair does not show a non-linear increase in respiration when this is plotted as a function of AG,(out), Fig. 4. It is however significant that the maximum values of A,&+ or AG,(out) obtained with palmitoyl-L-carnitine plus malate are somewhat lower than those reached with glycerol 3-phosphate as substrate. These results are consistent with a hypothesis whereby brown-adipose-tissue mitochondria in state 4 are operating with a transmembrane A&+ which is close to the maximum which a component in the membrane can withstand without a substantial nonohmic increase in conductance. This would imply that at least under the conditions employed in the present experiments a substrate such as glycerol 3-phosphate is thermodynamically capable of generating a ApH+in excess of this limiting potential, while palmitoyl-carnitine plus malate are not. To decide whether the increase in C,,,H+is dependent on the magnitude of AbH+rather than either of its components A E or ApH, the experiment shown in Table 1 was performed. As the increase in Cm,Habove the limiting potential essentially prevents any further increase in d pH+ from occurring (Fig. 3), an increase in the relative contribution of A pH to A i i H + by increasing [K'] [12,25] in the medium would be predicted to lower ApH+ in state 4 if the magnitude of A pH determined the breakdown potential, to increase A &+ if A E was the determinant of the breakdown, and to leave the state 4 Aj&+ unchanged if A,&,+ itself was relevant parameter. As can be seen, the evidence is in favour of the last possibility, A , l H + being the least variable parameter,
Pathways of Mitochondria1 Proton Conductance
354
fl
1 'L :aEj;yl-~-
carnitine
Rotenone
-1
GDP
I
7Nicotinamide nucleotide fluorescence
Glycerol 3-phosphate
GDP
GDP
I
7
7
IT
m
5rnin
Fig. 5. Fluorescence o/ endogetiou.Y reduced nicotinamide nucleotides of guinea-pig brol.1.n-adipose-tissue mitochondria. Mitochondria (0.5 mg proteinjml) were incubated at 23 "C and pH 7.0 in a medium containing 100 mM sucrose, 20 mM Na/N-tris(hydroxymethyl)methyl-2-aminoethane sulphonate, 32 pM albumin, 0.5 niM KCI, 0.5 m M EDTA and 0.5 mM sodium phosphate. In trace I. 60 pM palmitoyl-L-carnitine and 1 mM malate were initially present, and further additions were made where indicated of 0.5 mM GDP, 10 m M DL-glycerol 3-phosphate and 2 pM rotenone. In trace 11, 0.4 mM DL-glycerol 3-phosphate was initially present, and 0.5 mM GDP was added where indicated. In trace 111, 10 mM m-glycerol 3-phosphate was initially present, and additions were made of 0.5 mM GDP, 60 pM palniitoyl-L-carnitine plus 2 mM malate, and 2 pM rotenone
of producing a partial reduction. Assays performed on nicotinamide nucleotides extracted from hamster brown-adipose-tissue mitochondria oxidising 3 0 m M glycerol 3-phosphate in the presence of 160 pM albumin and 1 mM G D P at pH 7.2 revealed a total pool of NAD(H) of 6.3 nmol . mg-' and 0.5 nmol .mg-' of total NADP(H). The N A D + pool was 83 % reduced, confirming an extensive reduction by glycerol 3-phosphate in Fig. 5. The results reported above show that the first proton-translocating region of the respiratory chain is only in thermodynamic equilibrium with the A pH+generated by the oxidation of high glycerol 3-phosphate concentrations when the NAD' pool is 83% reduced. Clearly, palmitoylL-carnitine plus malate oxidation is incapable of maintaining the even more extensive reduction of NAD' which would be required for there to be net electron transport through this first region under these conditions (Fig. 5), and the conclusion is that electron transport from NAD-linked substrates to quinone becomes inhibited by the approach to thermodynamic equilibrium at values of ApH+ which are below those which the second and third regions can sustain. DISCUSSION
while substantial variations in the magnitude of A E and - 59 ApH occur. An increase in Cm,H+appears not to be restricted to brown-adipose-tissue mitochondria, as rat liver mitochondria oxidising succinate in the presence of varying concentrations of malonate have been reported to decrease AplI+[12] or AG,(out) [23] disproportionately as respiration is progressively inhibited. The ability of two flavoprotein-linked substrates to invoke a non-linear response, and the ability of the flavoprotein-linked glycerol 3-phosphate to maintain a greater A j i ~ +than palmitoyl-L-carnitine plus malate, raises the possibility that at least under the conditions employed here, the second and third regions of proton translocation in the respiratory chain have the ability, for a given rate of electron transfer, to maintain a il,iiilgreater + than that produced by the first site between NADH and ubiquinone. This can be experimentally verified by comparing the extent of reduction of endogenous nicotinamide nucleotides when palmitoyl-L-carnitine is oxidised in the controlled state with that maintained by glycerol 3-phosphate oxidation, as a consequence of the thermodynamic reversibility of the first site [28]. The experiments reproduced in Fig. 5 were performed under conditions parallel to those of Fig. 2 - 4. It is clear that high concentrations of glycerol 3-phosphate cause extensive reduction of nicotinamide nucleotides while low glycerol 3-phosphate levels or palmitoyl-L-carnitine plus malate are each only capable
The Conductance of the Inner Membrane in the Absence of Exogenous Purine Nucleotides From Fig. 1 B the conductance of the nucleotide sensitive pathway is about 16 nmol H + . min-' . mg-'. Analysis of the binding of inhibitory purine nucleotides to the outer face of the inner mitochondria1 membrane reveal 0.5-0.8 nmol . mg protein-' of binding sites for both hamster and guinea-pig mitochondria [5,30]. The good correlation obtained between binding of nucleotides and the inhibition of C m , ~[31,32] + strongly suggests that all these binding sites are active in inhibiting the conducting pathway. On the assumption that there is about one binding site per ion conducting pathway, this implies a conductance of about 20 H . min-' . mV-' per pathway, or when d,iiH+ is 60 mV, 1200 H + . min-'. This conductance is many orders of magnitude lower than that catalysed by pore-forming antibiotics such as gramicidin [29]. On the other hand the existence of specific nucleotide-binding sites on the outer face only of the membrane [5,30] argues against a lowmolecular-weight mobile carrier. The evidence is therefore in favour of a component which spans the inner membrane. The conductance of CI- across the membrane of brown-adipose-tissue mitochondria is abnormally high and is inhibited to levels characteristic of liver or heart mitochondria by the same purine nucleotides which inhibit Cm,H+[6,7,31]. In addition there is an ohmic dependency of CIK transport on the C1- electro-
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D. G. Nicholls
chemical gradient in the absence of inhibitory purine nucleotides, and a non-ohmic relation in their presence [7]. All this, together with evidence of competition for transport between the ions [4] suggests that a common pathway is involved, and that, as previously proposed [4], this is most easily reconciled with a pathway which conducts not protons into the matrix, but OH- out of the matrix, the two being bioenergetically indistinguishable. In the course of catalysing anion uniport across the membrane, uncompensated charge is transferred. The ohmic nature of the conductance suggests that one way in which this might be accomplished is by the diffusion under the influence of an electrochemical gradient of anions through a conducting channel lined with positively charged groups.
The Conductance of the Inner Membrane in the Presence of Exogenous Purine Nucleotides The ohmic conduction region of Fig. 3 (i.e. up to 200 mV) indicates a C,,,H+ of 0.7 nmol H + . min-' . mg-' . mV-', which is only 4 % of the level in the absence of purine nucleotide. The nucleotide inhibition is thus extremely effective. The non-ohmic increase in ion conductance at high electrochemical gradients appears not only for the effective proton conductance of brown-adiposetissue mitochondria and liver mitochondria [12,23], and for the chloride conductance of the former mitochondria [7], but may also be inferred from the 'energydependent' chloride permeability which has been reported for heart mitochondria [33]. Such conductances demand a pathway for ion conduction, even though this pathway is non-ohmic and is only significant at high electrochemical gradients. The components for this pathway could either be distinct from the nucleotide-sensitive conductance of brown-adipose-tissue mitochondria, which would help to explain the similar behaviour of mitochondria from a variety of organs, or, in view of the ability of brown-adiposetissue mitochondria to conduct the same ions, namely C1- and OH-, under both ohmic and non-ohmic conditions, it is also possible that a single conductance pathway is present in all mitochondria, but that only in brown-adipose-tissue mitochondria is it modified to produce a high, ohmic conducting state, together with a specific binding site for purine nucleotides, occupancy of which would cause the pathway to revert to the normal state of low and non-ohmic conductance. The rate of controlled respiration of both hamster [IS] and guinea-pig [l 11 brown-adipose-tissue mitochondria has been shown to be proportional to the extent of the disequilibrium between ApH+ and the oxido-reduction potential span available from the substrate couple. The increased respiration obtained
500
I '
j
t
Fig. 6 . Analysis (if the non-ohmic relution between effective proton current and proton electrochemical grudient produced by mitochondria oxidising varying concentrations of glycerol 3-phosphate in the presence qf exogenous GDP. The empirical relation between effective proton current and A & + obtained in Fig.3 is replotted ( 0 )and compared with that generated (solid line) on the assumptions that the membrane possesses both a conductance of 0.66 nmol H min- ' . mg- ' . mV- ' (given by I) and an infinite conductance operative only at or above a A,&+ of 240 mV (given by IT). On the assumption that the empirically observed drop of 1 mV in A ~ I I +which is required to drive an increase in respiration of 2 nmol 0 . min-' . mg-' 1111 holds for varying glycerol 3-phosphate concentrations, the discontinuous lines 111, IV and V represent the extrapolation back to static head conditions of A&+ observed at respectively 0.6 mM, 2 mM and 20 mM glycerol 3-phosphate +
at high concentrations of glycerol 3-phosphate therefore implies that, as ADH+ does not alter (Fig.3), the oxido-reduction potential span must increase. Under conditions closely similar to those of Fig. 3 , it has previously been shown that guinea-pig brownadipose-tissue mitochondria increase their rate of controlled respiration by 2 nmol 0 . min-' . mg-' for each millivolt that ApH+ decreases, under conditions where substrate concentration does not vary [ l l ] . Extrapolating this relationship back to zero respiration, it is possible to calculate the value of A p H + which is maintained by the oxidation of glycerol 3-phosphate under static head equilibrium conditions. As the rate of controlled respiration is therefore proportional to the disequilibrium between the static head A,&,+ and the observed A&+, this would imply that the driving force for the increased respiration observed during the oxidation of high glycerol 3-phosphate concentrations is an increased disequilibrium between the static head and observed values of Aj&+. AS the observed values of A ~ H +are virtually constant at the limiting value of ADH+ (Fig.3), this requires that the static head A&+
356
should increase as the concentration of glycerol 3-phosphate increases. In Fig. 6, the experimental points obtained in Fig. 3 are replotted, and compared with the relation between A,&+ and proton current which would be obtained if the mitochondria possessed both a constant conductance of 0.66 nmol H + . min-' . mg-' . mV-l, and an infinite conductance operative only at or above a A b H + of 240 mV, given respectively by the solid lines I and TI in the figure. The dashed lines represent the extrapolation back to static head conditions of the empirical values of A,&+ obtained during the oxidation of respectively 0.6 mM, 2 mM and 20 mM glycerol 3-phosphate. From Fig.6 the corresponding static head ApH+ which is in equilibrium at these concentrations is respectively 230 mV, 258 mV and 273 mV. In conclusion, it appears that mitochondria possess a component in the inner membrane which effectively limits the attainable A,iiH+ across the membrane. With liver and brown-adipose-tissue mitochondria, this limiting potential appears to be set at a level just above that usually attained by NAD+-linked respiration, but which can be reached by active flavoprotein-linked dehydrogenases. Such a limiting device could clearly act to ensure that while A&+ is maintained at very high levels, it cannot rise to levels which cause possibly catastrophic dialectric breakdown of the membrane. This work is financed by the Science Research Council, grant number B,'RG,'84050.0. The expert technical assistance of Aileen Kiddie is gratefully acknowledged.
REFERENCES 1. Mitchell, P. (1961) .Nature (Lond.) 191, 144-148. 2. Mitchell, P. (1968) Chenzio.smotic Coupling and Energy Transduction, Glynn Research, Bodmin, U.K. 3. Mitchell, P. (1976) Biochem. Soc. Trans. 4, 399-430. 4. Nicholls, D. G. (1976) FEBS Lett. 61, 103-110. 5. Nicholls, D. G. (1976) Eur. J . Biochem. 42, 223-228. 6. Nicholls, D. G. & Lindberg, 0. (1973) Eur. J . Biorhem. 37, 523 - 530.
D G. Nicholls : Pathways of Mitochondria1 Proton Conductance
7. Nicholls, D. G. (1974) Eur. J . Biochem. 49, 585-593. 8. Jackson, J. B., Saphon, S. & Witt, H. T. (1975) Riochim. Biophys. Acta, 408, 83 92. 9. Mitchell, P. & Moyle, J. (1967) Biochem. J . 104, 588-600. 10. Hittelman, K. J., Lindberg, 0. & Cannon, B. (1969) Eur. J . Biochem. 11, 183-192. 11. Nicholls, D. G. & Bernson, V. S. M. (1977) Eur. J . Biochem. 75,601 -612. 12. Nicholls, D. G. (1974) Eur. J . BiochiJm. 50, 305-315. 13. Nicholls, D. G., Grav, H. .I. & Lindberg, 0. (1972) Eur. J. Biochem. 31, 526-533. 14. Brand, M. D., Reynafarje, R. & Lehninger, A. L. (1976) Proc. Nut1 Acad. Sci. U.S.A. 73, 437--441. 15. Weichmann, A. H. C. A , , Beem, E. P. & van Dam, K. (1975) in Electron Transfer Chains and Oxidative Phosphorylation (Quagliariello, F., Papa, S., Palmieri, F., Slater, E. C. & Siliprandi, N., eds) pp. 335-342, North Holland, Amsterdam. 16 Nicholls, D. G. (1977) Biochrm. Soc. Trans. 5,200-203. 17 Klingenberg, M. & Slenczka, W. (1959) Biochem. Z . 331, 486 - 517. 18. Nicholls, D. G. (1974) Eur. J . Biochem. 49, 573-583. 19. Bukowiecki, L. J. & Lindberg, 0. (1974) Biochim. Bioph.vs. Acts, 348, 115 125. 20. Bernson, V. S. M. & Nicholls, D. G. (1974) Eur. J . Biochem. 47,517-525. 21. Baumann. G. & Mueller, P. (1974) J. Supramol. Struc.t. 2, 538 - 557. 22. Klingenberg, M. & Schollmeyer, P . (1961) Biochem. Z. 335, 243 262. 23. Slater, E. C . , Rosing, J. & Mol, A. (1973) Biochim. Biophyx. Acta, 292, 534-553. 24. Wilson, D. F. & Erecinska, M. (1973) Mitochondria: Biogenesis and Bioenergetics (van den Bergh, S. G., Borst, P. & Slater, E. C., eds) pp. 119-132, North Holland, Amsterdam. 25. Mitchell, P. & Moyle, J. (1969) Eur. J . Biochem. 7, 471 -484. 26. Mitchell, P. & Moyle, J. (1969) Eur. J . Biochem. Y, 149-155. 27. Rosing, J. & Slater, E. C. (1972) Biochim. Biophys. Actn, 267, 275 - 290. 28. Chance, B. & Hollunger, G. (1960) Nature (Lond.) 185. 666672. 29. Mueller, P. & Rudin, D. 0. (1969) Curr. Top. Bioenergetic.s, 3, 159 -249. 30. Rafael, J. & Heldt, H. W. (1976) FEBS Lett. 43, 304-308. 31. Cannon, B., Nicholls, D. G. & Lindberg, 0. (1973) in Mechanisms in Bioenergetics, pp. 357 - 363, Academic Press, New York. 32. Heaton, G. M. & Nicholls, D. G. (1977) Biochem. Soc. Trans. 5,210-212. 33. Brierley, G. P. (1970) Biochemistry, Y, 697-707. -
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D G Nicholls, Department of psychiatry, University of Dundee Medical School, Ninewells Hospital, Dundee, Great Britain, DD2 1UD
