Conformational Model of Active Transport John H

Proceedings of the National Academy of Sciences Vol. 67, No. 2, pp. 550-559, October 1970

Conformational Model of Active Transport John H. Young*, George A. Blondin, G. Vanderkooi, and D. E. Green

INSTITUTE FOR ENZYME RESEARCH AND THEORETICAL CHEMISTRY INSTITUTE UNIVERSITY OF WISCONSIN, MADISON 53706 Read as an Invited Paper on the Conformational Basis of an Energy Transduction, April 29, 1970 Abstract. A model of active transport of monovalent cations in mitochondria is developed. The model is based on the coupling of electron transfer to the generation of a metastable protein conformation which in turn leads to the generation of an asymmetric surface charge, a membrane potential, and a redistribution of diffusible ions across the inner mitochondrial membrane. The ions at all times move spontaneously down an electrochemical potential gradient in this model so that there is no need to invoke the concept of an ion pump. It is shown that a wide variety of experimental facts can be rationalized in terms of the present model. In seeking an explanation for active transport, two mutually exclusive alterna- tives present themselves-either the ions move spontaneously down an electrochemical potential gradient or they do not. Whereas many investigators in the field of active transport believe that ions are pumped against an electrochemical potential gradient, the model we will present involves no ion pumps but only the spontaneous movement of ions down an electrochemical potential gradient. We will consider only one active transport system, the mitochondrion, and we will consider only monovalent cation translocation in the mitochondrion. Even for this special case we cannot yet claim to have a complete explanation of active transport. Yet certain fundamental points have emerged with very great clarity, and it is these points that we would like to present to you today. Fig. 1 is a schematic representation of the mitochondrion. It consists of two membranes, an inner and an outer membrane, each of which forms a closed surface. Active transport in the mitochondrion consists of coupling either electron transfer or ATP hydrolysis to the flow of ions from the outside of the inner membrane to the inside or matrix space. The increased concentration of ions in the inside space is accompanied by a flow of water across the membrane and an increase in volume or swelling of the inside space in order to prevent a signif- icant increase in internal osmotic pressure. In Fig. 2 is shown the trace of a potassium electrode in a solution containing mitochondria and 6 m1l\ potassium acetate. Upon initiation of electron transfer by the addition of succinate, the potassium concentration of the external solution decreases as the potassium is being actively concentrated by the mitochondrion. When steady state is attained, the ratio of the potassium concentrations, inside to out, is approximately 80:1. Upon the addition of a respiratory inhibitor, 550 Downloaded by guest on September 25, 2021 VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 551

such as antimycin, or an uncoupler of oxidative phosphorylation, there is an efflux of potassium out of the mitochon- CM drion back into solution, confirming that the concentration of potassium by the IBM mitochondrion is indeed an energy-requiring process. This experiment illustrates a very in- -C M teresting effect. Far more potassium is concentrated by the mitochondrion when a trace amount of the antibiotic valinomycin is included in the reaction mixture than when it is not. In Table 1 are given the amounts of potassium concentrated with and without val-

inomycin. With only 2 X 10-1" mol ______of valinomycin per mg protein present in the medium, the mitochondrion can FIG. 1. Schematic representation of the 6-7 mitochondrion. OM, outer membrane; concentrate times more potassium M, matrix space; CM, cristal membrane; than without valinomycin. IBM, inner boundary membrane; IC, intra- What is valinomycin, and how does it cristal space. work? In Fig. 3 is shown a model of the valinomycin molecule. It is a cyclic polypeptide with six oxygen atoms on the inside of the ring, arranged in octahedral symmetry. This cyclic molecule can form a complex with an alkali metal ion. The size of the ring is such that valinomycin exhibits specificity for potassium, rubidium, and cesium as opposed to sodium and lithium. It has two conformations, a closed one shown here, and an open one such that it can accept or release an ion. The exterior is mostly hydrophobic so that the ion-valinomycin complex tends to be soluble in an

7 EXvalinomn crn without succinate Et.-ris succinate

E~~~~A Us 51 \0i B ithout

Fig. 2. Valinomycin-induced swellinghinmyci sucrose-containing media. This figure is re-i produced from the article of Blondin and Green, Arch. B3iochem. Biophys., 132, 509 (1969). FIG. 3. Molecular model of valinomycin. Downloaded by guest on September 25, 2021 552 N. A. S. SYMPOSIUM: YOUNG ET AL. PROC. N. A. S.

organic solvent. Because of this property valinomycin can facilitate the partitioning of potassium, rubidium, and cesium from an aqueous into an organic phase. Because of exactly the same property valinomycin can increase the passive permeability of synthetic lipid bilayers and biological membranes to these ions. For example, Table 2 shows the half-time of equilibration (T12) of 8Rb across TABLE 2. Effect of valinomycin on the rubidium ion flux of nonrespiring TABLE 1. Effect of valinomycin on active mitochondria after swelling in concentration of potassium ion 0.15 Ml rubidium acetate. in 0.006 11 potassium acetate Valinomycin T1/, of equilibration [K+Iout K +/mg protein (M) (min) Valinomycin (mM) (nmol) 0 36.5 - 6.00 200 1.3 X 10-9 10 + 3.75 1325 2.7 X 10-9 7.7 1.3 X 10-8 4.5 2.7 X 10-8 3.0

the inner mitochondrial membrane as a function of valinomycin. We see that in going from no valinomycin to 2.7 X 10-8 M, T1/, decreases by a factor of 12, indicating a dramatic increase in the permeability of the inner membrane. There are now a number of small molecular weight polypeptides like valinomycin, collectively designated ionophores, that can facilitate the partitioning of alkali metal ions from an aqueous into an organic phase, increase the passive permeability of synthetic lipid bilayers and biological membranes to these ions and induce active transport in the mitochondrion. However, explaining the mechanism by which these ionophores facilitate the movement of ions across biological membranes does not explain how they induce active transport. Some have proposed that the ionophores participate in the operation of a cation pump. This would presumably involve the inversion of a binding site from one side of the membrane to the other. Some have proposed that the ionophores participate in some manner in the operation of an ion pump. However, there are a large number of factors that argue against this hypothesis. In Table 3 are listed a set of facts on ionophores that bear upon this question. TABLE 3. Pertinent facts on ionophores. 1. Partition cations from aqueous into organic phase. 2. Increase permeability of synthetic lipid bilayers. 3. Increase permeability of inner mitochondrial membrane. 4. Different ionophores can perform same function. 5. Ionophores of microbiological origin not endogenous to mitochondrion. 6. Need be present only in catalytic amounts. 7. Ionophore-induced transport not stoichiometric. 8. Ionophore unnecessary in high salt media. The first three points relate to the properties already discussed i.e., the partitioning and facilitated transport. The established capacity of valinomycin to increase the permeability of the inner mitochondrial membrane opposes its hy- pothesized capacity to participate in the operation of an ion pump, for the ions Downloaded by guest on September 25, 2021 VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 553

will tend to leak back out as they are pumped in against an electrochemical potential gradient. The more valinomycin that is added, the more readily would they tend to leak out. However, the fact is that the more valinomycin that is added, the more potassium that is concentrated. The fourth point is that a number of ionophores can induce active transport in the mitochondrion. One would expect that if an ion pump required for its operation the presence of an ionophore there would be a stereospecific requirement such that only one and not several different ionophores could fulfill this function. The fifth point is that the ionophores of microbiological origin are not endogenous to the mitochondrion. Thus under normal physiological conditions the mitochondrion never sees these ionophores. There is therefore, no physiological rationale why the mitochondrion should have an ion pump which requires for its operation an ionophore of microbiological origin. The sixth point is that the ionophores need to be present only in catalytic amounts to have their effect, for instance less than one valinomycin molecule per 104 tripartite repeating units. The tripartite repeating unit is the functional unit of the inner mitochondrial membrane and there would supposedly be at least one ion pump per tripartite repeating unit. The seventh point is that there is variable stoichiometry between the numler of ions moved per oxygen atom absorbed or per molecule of ATP hydrolyzed. For example, as one varies the valinomycin from zero to 80 ng/mg protein, the initial number of potassium ions moved per oxygen atom varies from 0.6 to 9.5. Note that more potassiums are moved per oxygen atom as the membrane be- comes more permeable. Finally, the mitochondrion can concentrate several hundred nanomoles of potassium per mg protein even without any added ionophore if the external salt concentration is increased from a few millimolar to 0.1 M. Thus, the ionophores cannot be essential for active transport in the mitochondrion. None of these arguments is in itself rigorous or conclusive, yet they all point to the same conclusion-that whatever the mechanism by which the ionophores induce active transport in the mitochondrion, there are no cation pumps in the mitochondrion. What are the consequences of rejecting the hypothesis of ion pumps? First, if there are no ion pumps, the ions must be spontaneously moving down an electrochemical potential gradient under -conditions of active transport. Note that this is completely in accord with the experimentally established capacity of the ionophores to facilitate the movement of ions across the inner mitochondrial membrane. Second, since the ions are moving against a large concentration gradient, there must be a compensating electrical potential gradient, negative inside, generated under active transport conditions. We are, therefore, forced to the corollary that a membrane potential is generated under active transport conditions simply as a consequence of our conclusion that there are no ion pumps in the mitochondrion. This argument based on the rejection of the hypothesis of ion pumps is our first argument for a membrane potential. Downloaded by guest on September 25, 2021 554 N. A. S. SYMPOSIUM: YOUNG ET AL. PROC. N. A. S.

Now let us consider a second aspect of this problem which confirms this conclusion. We mentioned that in 0.1 M potassium acetate, the mitochondrion can concentrate several hundred nanomoles of potassium per mg protein without any ionophore. In Table 4 is shown Tl1, of '"Rb in 0.1 M rubidium acetate for respiring and nonrespiring mitochondria. This is a measurement of the time of iso- topic exchange under steady state conditions, i.e., no net movement of ions and water across the membrane. We see that the permeability is essentially the same under both respiring and nonrespiring conditions. I Now let us consider a different situation involving a net flux of ions and water across the membrane. In Table 5 is shown the initial flux of potassium across TABLE 4. Rubidium ion flux under respir- TABLE 5. Potassium ion influx under re- ing versus nonrespiring condi- spiring versus nonrespiring con- tions in 0.15 M rubidium acetate. ditions in 0.116 AI potassium ace- '1/2 of Equilibration* tate. (min) Potassium influx Nonrespiring 25.2 (nmol/mg protein/min) Respiring 29.2 Nonrespiring* t 14.2 * Equivalent to exchange across mitochond- Respiring* 517 rial membrane of 1.0 Amol/mg protein. * In presence of glutamate and malate. t In presence of antimycin and rotenone. the inner mitochondrial membrane under these same two conditions. We should note that the transport properties of potassium and rubidium are essentially identical in mitochondria. We see there is a 35-fold increase in the ion influx under respiring as opposed to nonrespiring conditions. In Table 4 it was shown that the permeability is essentially constant so that only a large difference in the driving force could account for the large difference in the flux, i.e., there must be generated a difference in the electrochemical potential on the two sides of the membrane under respiring con- 4co --- ditions. Thus, again we are forced to the conclusion, by a second, completely indepen- 120 dent argument, that a membrane potential 00 is generated under the energizing conditions kK+ TOTAL required for active transport. E 800 Let us now consider a third aspect of this 'A \problem, the anion requirement of cation + 600 translocation. In Fig. 4 is shown the effect upon the amount of potassium concentrated 400- Nas we vary the anion from 100% acetate on c \*the left to 100% chloride on the right. We 200- An see that six times more potassium is concen- l80 06 0 02 N trated with acetate as the anion than with MOLE PARTS ACETATE ANIO 0 chloride. The inner mitochondrial membrane is freely permeable to the chlorides ion so that FIG. 4. Magnitude of K+ trans- impermeability cannot be the explanation for location as a function of the ratio of this difference mole fraction of acetate versus chloride. ThisThisdifference.difference between weak- and strong- Downloaded by guest on September 25, 2021 VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 533

acid anions is fairly general.3 In Fig. 5 is shown the absorbance vs. time as a function of various anions. Decreased absorbance is a measure of increased volume because of the increased concentration of osmotically active ions in the mitochondrion. Thus qualitatively, decreased absorbance is equivalent to decreased potassium ion concentration in the external solution. We see that as a general rule, cacodylate being the exception, the weak acid anions generally lead to much greater swelling than that induced by the strong acid anions. We should note here that the phosphate ion also belongs in the category of weak-acid anions as one that produces large amplitude swelling.

0.8 _

chloride and bromide>

E 0.6- cacodylate C FIG. 5. Effect of different anions on po- 0 tassium-induced swelling in a sucrose-con- a > taining medium. The medium was 0.2 M in sucrose, containing 1 mg of mitochondrial \ protein and t0.1 nmol of valinomycirl per ml. W 0.4 - .-monochloroacetate This figure is reproduced from the article of z Blondin and Green, Arch. Biochem. Bio- 4co phys., 132, 509 (1969). O aet\ 0 )formatey 100.2 _ 4oF ~~~~~~acetate

0 2 ~~4 6 8 TIME (minutes)

What is the origin of this specificity for weak acid anions in cation translocation? Specificity implies binding. This means that weak-acid anions could be bound under energizing conditions, whereas strong-acid anions could not be bound. Can we pursue this further, and say where the weak-acid anions are bound? The fact that cation translocation leads to swelling implies that the osmotically active ions are on the inside of the inner mitochondrial membrane. Since the cation concentration is anion-dependent, and since electroneutrality requires an equal number of positive and negative charges on the inside, we conclude that the weak-acid anions must also be on the inside and must, therefore, be bound on the inner surface of the inner membrane. What are the implications of binding anions on the inner surface of the inner mitochondrial membrane? In Fig. 6 is presented a schematic representation of the mitochondrion as two aqueous solutions separated by a semipermeable membrane. Side 1 denotes the inside of the inner membrane, side 2 the outside, and the semipermeable membrane is the inner mitochondrial membrane. Here R- designates the concentration of impermeant negative charge (which for simplicity we assume stands only for the concentration of bound anions), y + z Downloaded by guest on September 25, 2021 556 N. A. S. SYMPOSIUM: YOUNG ET AL. Puoc. N. A. S.

DONNAN EQUILIBRIUM AND MEMBRANE POTENTIAL

1 2 (R-) Z (M)1 Z + Y (M)2 X FIG. 6. Schematic representation of (A-) y (A-)2 X the inner mitochondrial membrane as a semipermeable barrier between two r s tM+)1 X (A )2 aqueous solutions. Symbols explained (M )2 (A-)1 ill text.

r XY X

E(mv) X RT In r - 59.1lograt250 F

stands for the concentration of cation on the inside, y the concentration of diffusible anion on the inside (note the distinction between diffusible and bound anions), and x the concentration of salt on the outside. Assuming, for simplicity, unit activity coefficients for the diffusible species, we obtain the familiar equa- tions of the Donnan equilibrium for the ratio of the concentrations of cations in to cations out or anions out to diffusible anions in. Because of the fixed charge, a membrane potential is also generated whose magnitude is given by the bottom equation in Fig. 6. We can now see the implications of the anion requirement for cation translocation. The specificity for weak-acid anions implies that they are bound site- specifically. The increased osmotic activity of the inner space and the requirement of electroneutrality implies that the weak-acid anions are bound on the inner surface of the inner membrane. Finally, the asymmetric generation of bound surface charge produces a membrane potential and an asymmetric distribution of diffusible ions across the membrane. The key ideas here are the asymmetric binding of weak-acid anions and the consequent asymmetric distribution of diffusible ions. Let us explore further the implications of this model. We note from the equation for the ion ratio in Fig. 6 that for r >> 1, as in the present case, both the concentration of cations and the total concentration of anions in the inside space are approximately equal to the concentration of bound anions. Binding all of the anions concentrated during active transport would appear to greatly exceed any reasonable estimate of the number of binding sites available in the mitochondrion. This fact alone indicates that the quasi-equilibrium model being developed here can at best provide only a partial rationalization of what is in- trinsically a nonequilibrium phenomenon. Nonetheless, it is instructive to pursue this line of reasoning further. If predominantly one, singly ionized anionic species is bound, electroneutrality requires that the ratio of cation concentration to total anion concentration be approximately unity. This assumes of course, that the anions, as well as the cations, are monovalent. We see from Table 6 that under energizing conditions the ratio of potassium to phosphate is indeed essentially unity in the inside space. This result may appear somewhat surprising at first since the external pH is 6.5, and under these conditions the second pK of phosphoric acid is in the neigh- Downloaded by guest on September 25, 2021 VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 557

TABLE 6. Valinomycin-induced uptake of potaesiurn and inorganic phosphate by mitochondria. Nonanergized Energized mg H20per mg protein 3.1 7.3 nmol Pi per mg protein 122 677 nmol K+ per mg protein 652 borhood of 7.0, so that the second dissociable hydrogen of phosphoric acid would be partially ionized. However, if the proposed mechanism is correct, the concentration ratio of all permeant cations must be approximately the same as that of potassium, which is of the order of 100:1. Assuming a mechanism for the equilibration of protons and/or hydroxyl ions across the membrane under these conditions, this would imply a drop of approximately two pH units across the membrane. Thus the phosphate ion in the inside space would be only singly ionized, and the potassium: phosphate ratio should be unity, as is observed. These results in a potassium phosphate medium lend further support to the model proposed here. We have noted from the equation for the ion ratio in Fig. 6 that for r >> 1 the concentration of diffusible anions in the inside space must be much less than either the concentration of cations or the concentration of bound anions. The present model would, therefore, predict that if a nonbonding anion, such as chloride, were present in a medium containing an alkali metal-weak-acid salt, then an efflux of the nonbonding anion should accompany the influx of the cation and weak acid anion during active transport. The results of such an experiment are shown in Fig. 7. We see that the efflux of chloride almost valimomycin M-CI-CCP

FIG. 7. Chloride and potassium con- C centrations in the external medium dur-1 P 4 / P ing active transport in the mitochon- -\107g 1 drion. uptakes I 1mi exactly parallels the influx of potassium. The net amount of either ion trans- ported across the membrane equals the difference between the product of the concentration and the volume before and after induction of active transport. The initial concentrations of both ions on the inside are assumed to be similar, but whereas the concentration of potassium is greatly increased, the concentration of chloride is greatly decreased upon induction of active transport. Thus the net movement of chloride should be very small relative to that of the potassium, as is observed. It appears that the only viable explanation for the movement of the chloride is the generation of a membrane potential, negative inside, under active transport conditions. Downloaded by guest on September 25, 2021 558 N. A. S. SYM1POSIUM: YOUNG ET AL. PROC. N. A. S.

We therefore conclude from a wide variety of completely independent arguments that a membrane potential is generated under active transport conditions and that it is the driving force for cation transport. The mechanism of generat- ing the membrane potential is the asymmetric binding of weak-acid anions that occurs under energizing conditions. Having established this conclusion, we must now ask the central question: How can the free energy released by electron transport be coupled to the generation of a membrane potential, or more specifically to increased binding of weak- acid anions? It is well known that the conformation of a protein greatly affects its affinity for small ions and molecules. The cooperative binding of oxygen by hemoglobin is one well-known example. We now see how the conformational model can very simply rationalize the increased binding of weak-acid anions under energizing conditions. Electron transfer is coupled to the generation of a metastable protein conformation, and the metastable protein conformation has a greatly increased affinity for weak-acid anions. We can now tie everything together. In Table 7 is a summary of the confor-

TABLE 7. Conformational model of active transport. 1. Redox Reactions A Conformation 2. A Conformation - A Surface Charge 3. A Surface Charge A Membrane Potential 4. A Membrane Potential - Ion Redistribution 51. Ion Redistribution A Osmotic Pressure 6. A Osmotic Pressure - Water Movement (Swelling)

mational model of active transport. The first two points are those just mentioned: the coupling of the redox reactions to a conformational transition and the asymmetric generation of a surface charge. The next two points refer to the generation of the membrane potential and the ion redistribution according to the Donnan equilibrium. The last two points concern the increase in osmotic pressure due to the concentrated cations and the subsequent water movements and swelling. We might note here that by coupling electron transfer to a conformational transition with the consequent asymmetric weak-acid anion binding, a vectorial character is imparted to the electron-transfer process. It is just this vectorial character that is required by Curie's principle for coupling electron transfer, a chemical reaction, with ion transport, a vectorial process.4 There are additional points that we have not been able to discuss adequately for lack of time, such as the role of an impermeant solute such as sucrose in com- pelling large ion gradients and the correlation between the configuration of the inner membrane and antion binding and cation transport. These experimental facts can also be rationalized in terms of the conformational model of active transport. Thus, even though we do not yet have a complete explanation for active transport in the mitochondrion, there is already a very large body of experimental facts that can be successfully correlated and rationalized in terms of a few physically well-defined concepts. We should emphasize that the schematic summary in Table 7 is not meant to Downloaded by guest on September 25, 2021 VOL. 67, 1970 CONFORMATION AND ENERGY TRANSDUCTION 559

indicate the chronological sequence of events-it is not a kinetic scheme. All of these events take place simultaneously until steady state is attained, at which point the net flux of ions and water across the membrane is zero. Even at steady state, however, the metastable protein conformations are continually relaxing and requiring regeneration. Thus there is a constant need for an energy input even at steady state. Finally, we wish to mention that the model presented here is basically an at- tempt to focus attention on certain key experimental facts that appear to us to point the way to the final solution. We have used the terms Donnan equilibrium and fixed-charge in describing this system, but this is really an oversimplification for there is really a kinetic dimension to this model not present in a simple fixed- charge, Donnan-type system. The implications of this kinetic aspect of the model have yet to be fully developed. In particular, we do not yet understand the consequences of coupling the absorption and desorption of the anions to the generation and relaxation of the metastable conformation. In conclusion, although we do not yet have a complete explanation for monovalent cation transport in the mitochondrion, two fundamental points have emerged with great clarity: (1) There are no cation pumps in the mitochondrion, but rather the cations diffuse spontaneously across the membrane. (2) The driving force for monovalent cation transport arises from the conformationally de- termined binding of weak acid anions. We feel that these two points must form an integral part of any successful theory of active transport in the mitochondrion. *This work was supported in part by training grant GM-88 and Program Project Grant GM- 12847 from the National Institute of General Medical Sciences (USPHS) and also NGL5O-002- 001 of the National Aeronautics and Space Administration. ' We might note here that this result further confirms our conclusion that there are no ion pumps in the mitochondrion, for if there were, Ti,, would be much less for respiring than for nonrespiring mitochondria. 2 Blondin, G. A., and D. E. Green, J. Bioenergetie2, 1, 193 (1970). 8 We use the term "weak-acid anion" here to denote any species that has a dissociable hydrogen in the neutral pH range. 4Katchalsky, A., and P. F. Curran, Nonequilibrium Thermodynamics in Biophysics (Cam- bridge, Massachusetts: Harvard University Press, 1965), pp. 88-89. Downloaded by guest on September 25, 2021