PARTIX. PERMEATION MECHANISM ACROSS BIOLOGICAL MEMBRANES:
111
DRIVING FORCES OF AMINO ACID TRANSPORT IN ANIMAL CELLS" E. Heinz, P. Geck, and C. Pietrzyk Abteilung fur Physikalische Biochemie Gustav-Embden-Zentrum der Biologischen Chemie Johann Wolfgang Goethe Universitiit Frankfurt am Main, Federal Republic of Germany
Amino acids, neutral ones as well as charged ones, are accumulated in most animal cells, especially in tumor cells. There is now little doubt that this accumulation is by active transport by the usual definition of this term. There is, on the other hand, still much controversy as to the immediate driving force for this transport: Is it primary active, i.e., immediately coupled to a metabolic chemical reaction such as ATP hydrolysis; or is it secondary active, i.e., coupled immediately to the thermodynamically downhill movement of electrolyte ions, by co- or countertransport? In the first case the immediate driving force would derive directly from the affinity of the chemical reaction, and in the second case, from the electrochemical potential gradient of one or more electrolyte ions. Since the driving ion gradient is continuously restored at the expense of metabolic energy, also secondary amino acid transport is ultimately but indirectly coupled to a chemical reaction of metabolism. In the last years, considerable evidence has been accumulated showing that the transport of amino acid is indeed coupled to the inward movement of Na' ions and that energy of the Na' gradient does drive amino acid uphill. Also, the (inverse) gradient of potassium ions appears to contribute some energy for this transport, but since under certain conditions valinomycin stimulates this effect, the potassium efflux is probably not directly linked to the amino acid influx via countertransport, so that the energy from the K gradient is contributed via the electrical potential difference (PD) across the cell membrane, thereby enhancing the electrochemical potential gradient of Na' ions.' In view of all evidence, the question can no longer be whether the ion gradient is a driving force for amino acid transport or not. Rather, the question presently whether the ion gradient is the only driving force for amino acid transport, or whether in addition a major part of this force stems directly from a metabolic reaction. There are significant observations that indeed appear to support the latter alternative and that have been interpreted to indicate that part of the amino acid transport is primary, i.e., directly linked to metabolism. Even though the ion gradient may be effective in the absence of metabolic activity, the converse has not unequivocally been shown. Hence the two hypothetical pathways of energy transfer could not likely be separate and parallel, but would have to be geared to each other in a more or less intricate way. The major arguments that have been raised against the gradient hypothesis are the following: *This work has been supported by Grant No. He 102112 from the Deutsche Forschungsgemeinschaft. 428
Heinz et al.:
Driving Forces
429
1. The total force available from the electrochemical potential gradient (in the following simply called "gradient") of Na+, the stoichiometric coupling ratio taken to be unity, appears to be grossly inadequate to account for the amino acid accumulation observed.' Even the sum of both the Na' and the (inverted) K gradients would barely be fully adequate. As mentioned before, however, the K gradient is probably not fully available as the driving force for amino acid transport, neither via direct linkage nor indirectly via the electrical PD, so that the true driving force seems nontheless far from adequate. 2. Respiring cells accumulate glycine about three times more effectively than do metabolically inhibited cells at the same ion di~tribution.~ So it seems that the ion gradient contributes only a minor, perhaps merely accidental portion of the total transport energy. 3. Active uptake of amino acids, although at a reduced rate, may still take place in respiring cells, if the Na' and K gradients are abolished or even inverted.' This transport appears to be against an opposing driving force, at least as long as the latter does not exceed a limiting value of 4000 to 5000 J/mol. We shall now scrutinize these arguments and see that each of them is based on certain assumptions which can be refuted or at least weakened. 1. As to the energetic inadequacy of the ion gradients, previous calculations were based on two assumptions: (1) that the activity coefficient of Na' and K is about the same inside and outside the cell; and (2) that the ions are evenly distributed over the whole intracellular space. Whereas assumption (1) may not be too far off, assumption (2) is wrong. By fractionating the cells, we found that under normal conditions Na' and C1- ions are strongly sequestered in the cell nucleus, which covers about one-third of the cellular volume, whereas amino acid and K ions appear to be evenly distributed inside the cell.' It turned out that the effective electrochemical potential gradients of Na+, i.e., after correction for nuclear sequestration and in consideration of the fact that the electrical component, as determined by various methods, is at least 25 mV (Reference 6), should under these conditions account energetically for the highest accumulation ratios of amino acid.',' From this fact it does of course not necessarily follow that this driving force is actually used in full by the transport mechanism. We could meanwhile show, however, applying the principles of irreversible thermodynamics, that the direct coupling between amino acid transport and Na+ influx is tight enough to warrant about adequate energy transfer from the ion gradient to amino acid transport.* Analogous investigations on the direct coupling between amino acid transport and ATP hydrolysis did not demonstrate any coupling at all, so that on these grounds ATP is very unlikely to contribute directly to the transport energy' (TABLE 1). The apparent stimulation of active transport by cellular metabolism can at least in part also be explained by the nuclear sequestration of both Na' and Cl-. Though no direct experimental evidence is available, we may safely presume that during metabolic inhibition, owing to a paralyzed Na' pump, the cytoplasmic Na' and C1- will rise without much change of the nuclear portion of these ions. It can easily be shown that under these circumstances the overall, i.e., uncorrected, distribution of Na' and C1- between cell and medium will be affected by metabolic inhibition much less than the effective distribution of these ions between cytoplasm and medium. This difference may not account for the full discrepancy between metabolizing and nonmetabo-
Annals New York Academy of Sciences
430
TABLE1 COUPLING OF AIB TRANSPORT T o NA INFLUX
AND
ATP HYDROLYSIS Ji
Degree of coupling Efficacy of accumulation
9
--XA
i = Na+ (Na influx)
i = ATP (ATP hydrolysis)
0.5 - 0.6
Not detectable
~0.6
Xi
Efficiency of energy transfer
I)
w
*
vNa 0.6
0 0
lizing cells. There is likely to be an additional effect, however, which may concern the electrical membrane PD, as will be discussed later. 3. The last of the above arguments rests on cases of apparent incongruity in direction between amino acid transport and ion gradients; that is, active amino acid transport and the driving gradients appear to oppose each other. This is the hardest argument to disprove. Nuclear sequestration of Na' was found to vanish if the extracellular Na' was reduced to the low value required to invert the gradient.5 As far as the concentration gradients of Na' and K' are concerned, their inversion thus appears to be real under these circumstances, and certainly cannot be explained away by nuclear sequestration. With inverted gradients active transport of aminoisobutyrate ( AIB) is still possible, ever. after correction for nuclear sequestration, provided that the opposing driving 1) . The gradient hypothesis force does not exceed about 5000 J/mol (FIGURE would in the present case still hold if the electrical potential across the cellular membrane were high enough to outweigh the opposed chemical potential gradients, so that the total driving force would still be in the direction congruent to the amino acid transport. Since active transport is observed as long as the opposing driving force is less than 4000 to 5000 J/mol, the electrical potential difference, in order to overcome this force, has to be higher than 40 to 50 mV, inside negative. In other words, with inverted gradients this PD should be substantially higher than that with normal gradients. Before looking into the experimental evidence on this point, let us first consider its theoretical basis and ask whether, and under what conditions, we could reasonably expect a PD of the postulated magnitude here from what we know about the origin of the PD in these cells. Clearly any reasonable prediction as to this p.d. depends on the alleged mechanism of electrical PD generation. There is little known about this mechanism, but there are several hypothetical possibilities, which we shall briefly discuss: Most arguments on the electrical PD in these cells appear to have been more or less explicitly based on the assumption that the electrical PD in these cells represents a membrane-diffusion potential resulting from differential passive permeability through the cell membrane of Na' and K ions, which are maintained at a disequilibrium distribution by an electroneutral exchange pump (FIGURE2 ) . The C1- ions, which are not subject to any pumping, should instead attain equilibrium distribution, from which the electrical PD can be derived directly.', lo In view of these assumptions the electrical PD
Heinz et al.:
15000
10000
43 1
Driving Forces
5000
-5000
-10000
S X ~joule/mole , ~
FIGURE 1. The net influx of 2-aminoisobutyrate as a function of the ionic driving forces. The final results of eight typical experiments with varying Na' and K' gradients are summarized. Abscissa: XNa+- XI
111
DRIVING FORCES OF AMINO ACID TRANSPORT IN ANIMAL CELLS" E. Heinz, P. Geck, and C. Pietrzyk Abteilung fur Physikalische Biochemie Gustav-Embden-Zentrum der Biologischen Chemie Johann Wolfgang Goethe Universitiit Frankfurt am Main, Federal Republic of Germany
Amino acids, neutral ones as well as charged ones, are accumulated in most animal cells, especially in tumor cells. There is now little doubt that this accumulation is by active transport by the usual definition of this term. There is, on the other hand, still much controversy as to the immediate driving force for this transport: Is it primary active, i.e., immediately coupled to a metabolic chemical reaction such as ATP hydrolysis; or is it secondary active, i.e., coupled immediately to the thermodynamically downhill movement of electrolyte ions, by co- or countertransport? In the first case the immediate driving force would derive directly from the affinity of the chemical reaction, and in the second case, from the electrochemical potential gradient of one or more electrolyte ions. Since the driving ion gradient is continuously restored at the expense of metabolic energy, also secondary amino acid transport is ultimately but indirectly coupled to a chemical reaction of metabolism. In the last years, considerable evidence has been accumulated showing that the transport of amino acid is indeed coupled to the inward movement of Na' ions and that energy of the Na' gradient does drive amino acid uphill. Also, the (inverse) gradient of potassium ions appears to contribute some energy for this transport, but since under certain conditions valinomycin stimulates this effect, the potassium efflux is probably not directly linked to the amino acid influx via countertransport, so that the energy from the K gradient is contributed via the electrical potential difference (PD) across the cell membrane, thereby enhancing the electrochemical potential gradient of Na' ions.' In view of all evidence, the question can no longer be whether the ion gradient is a driving force for amino acid transport or not. Rather, the question presently whether the ion gradient is the only driving force for amino acid transport, or whether in addition a major part of this force stems directly from a metabolic reaction. There are significant observations that indeed appear to support the latter alternative and that have been interpreted to indicate that part of the amino acid transport is primary, i.e., directly linked to metabolism. Even though the ion gradient may be effective in the absence of metabolic activity, the converse has not unequivocally been shown. Hence the two hypothetical pathways of energy transfer could not likely be separate and parallel, but would have to be geared to each other in a more or less intricate way. The major arguments that have been raised against the gradient hypothesis are the following: *This work has been supported by Grant No. He 102112 from the Deutsche Forschungsgemeinschaft. 428
Heinz et al.:
Driving Forces
429
1. The total force available from the electrochemical potential gradient (in the following simply called "gradient") of Na+, the stoichiometric coupling ratio taken to be unity, appears to be grossly inadequate to account for the amino acid accumulation observed.' Even the sum of both the Na' and the (inverted) K gradients would barely be fully adequate. As mentioned before, however, the K gradient is probably not fully available as the driving force for amino acid transport, neither via direct linkage nor indirectly via the electrical PD, so that the true driving force seems nontheless far from adequate. 2. Respiring cells accumulate glycine about three times more effectively than do metabolically inhibited cells at the same ion di~tribution.~ So it seems that the ion gradient contributes only a minor, perhaps merely accidental portion of the total transport energy. 3. Active uptake of amino acids, although at a reduced rate, may still take place in respiring cells, if the Na' and K gradients are abolished or even inverted.' This transport appears to be against an opposing driving force, at least as long as the latter does not exceed a limiting value of 4000 to 5000 J/mol. We shall now scrutinize these arguments and see that each of them is based on certain assumptions which can be refuted or at least weakened. 1. As to the energetic inadequacy of the ion gradients, previous calculations were based on two assumptions: (1) that the activity coefficient of Na' and K is about the same inside and outside the cell; and (2) that the ions are evenly distributed over the whole intracellular space. Whereas assumption (1) may not be too far off, assumption (2) is wrong. By fractionating the cells, we found that under normal conditions Na' and C1- ions are strongly sequestered in the cell nucleus, which covers about one-third of the cellular volume, whereas amino acid and K ions appear to be evenly distributed inside the cell.' It turned out that the effective electrochemical potential gradients of Na+, i.e., after correction for nuclear sequestration and in consideration of the fact that the electrical component, as determined by various methods, is at least 25 mV (Reference 6), should under these conditions account energetically for the highest accumulation ratios of amino acid.',' From this fact it does of course not necessarily follow that this driving force is actually used in full by the transport mechanism. We could meanwhile show, however, applying the principles of irreversible thermodynamics, that the direct coupling between amino acid transport and Na+ influx is tight enough to warrant about adequate energy transfer from the ion gradient to amino acid transport.* Analogous investigations on the direct coupling between amino acid transport and ATP hydrolysis did not demonstrate any coupling at all, so that on these grounds ATP is very unlikely to contribute directly to the transport energy' (TABLE 1). The apparent stimulation of active transport by cellular metabolism can at least in part also be explained by the nuclear sequestration of both Na' and Cl-. Though no direct experimental evidence is available, we may safely presume that during metabolic inhibition, owing to a paralyzed Na' pump, the cytoplasmic Na' and C1- will rise without much change of the nuclear portion of these ions. It can easily be shown that under these circumstances the overall, i.e., uncorrected, distribution of Na' and C1- between cell and medium will be affected by metabolic inhibition much less than the effective distribution of these ions between cytoplasm and medium. This difference may not account for the full discrepancy between metabolizing and nonmetabo-
Annals New York Academy of Sciences
430
TABLE1 COUPLING OF AIB TRANSPORT T o NA INFLUX
AND
ATP HYDROLYSIS Ji
Degree of coupling Efficacy of accumulation
9
--XA
i = Na+ (Na influx)
i = ATP (ATP hydrolysis)
0.5 - 0.6
Not detectable
~0.6
Xi
Efficiency of energy transfer
I)
w
*
vNa 0.6
0 0
lizing cells. There is likely to be an additional effect, however, which may concern the electrical membrane PD, as will be discussed later. 3. The last of the above arguments rests on cases of apparent incongruity in direction between amino acid transport and ion gradients; that is, active amino acid transport and the driving gradients appear to oppose each other. This is the hardest argument to disprove. Nuclear sequestration of Na' was found to vanish if the extracellular Na' was reduced to the low value required to invert the gradient.5 As far as the concentration gradients of Na' and K' are concerned, their inversion thus appears to be real under these circumstances, and certainly cannot be explained away by nuclear sequestration. With inverted gradients active transport of aminoisobutyrate ( AIB) is still possible, ever. after correction for nuclear sequestration, provided that the opposing driving 1) . The gradient hypothesis force does not exceed about 5000 J/mol (FIGURE would in the present case still hold if the electrical potential across the cellular membrane were high enough to outweigh the opposed chemical potential gradients, so that the total driving force would still be in the direction congruent to the amino acid transport. Since active transport is observed as long as the opposing driving force is less than 4000 to 5000 J/mol, the electrical potential difference, in order to overcome this force, has to be higher than 40 to 50 mV, inside negative. In other words, with inverted gradients this PD should be substantially higher than that with normal gradients. Before looking into the experimental evidence on this point, let us first consider its theoretical basis and ask whether, and under what conditions, we could reasonably expect a PD of the postulated magnitude here from what we know about the origin of the PD in these cells. Clearly any reasonable prediction as to this p.d. depends on the alleged mechanism of electrical PD generation. There is little known about this mechanism, but there are several hypothetical possibilities, which we shall briefly discuss: Most arguments on the electrical PD in these cells appear to have been more or less explicitly based on the assumption that the electrical PD in these cells represents a membrane-diffusion potential resulting from differential passive permeability through the cell membrane of Na' and K ions, which are maintained at a disequilibrium distribution by an electroneutral exchange pump (FIGURE2 ) . The C1- ions, which are not subject to any pumping, should instead attain equilibrium distribution, from which the electrical PD can be derived directly.', lo In view of these assumptions the electrical PD
Heinz et al.:
15000
10000
43 1
Driving Forces
5000
-5000
-10000
S X ~joule/mole , ~
FIGURE 1. The net influx of 2-aminoisobutyrate as a function of the ionic driving forces. The final results of eight typical experiments with varying Na' and K' gradients are summarized. Abscissa: XNa+- XI
