Mitochondrial transport systems


Normal operation of the respiratory chain creates both a pH differential and a voltage gradient of 30,000,000 volts/metre across the inner mitochondrial membrane. This demands highly specific transport proteins to control the movement of small ions across the membrane, and to prevent the dissipation of the various gradients.

The majority of inner membrane carriers are antiporters which exchange one molecule for another. If the electrical charges on the two molecules exactly balance the exchange will be an electroneutral process and the huge membrane potential will have no effect on the result. However, the pH gradient may affect the equilibrium position for an electroneutral exchange if one or more participants are acidic or basic.

Symports transport two molecules in the same direction. If the molecules have opposite charges this may still be an electroneutral process. It is experimentally difficult to distinguish between a proton symport and a hydroxyl antiport.

If the charges differ on the transported molecules for either symports or antiports, then there will be an electrical (sometimes called electrogenic) transport process. Such carriers can "feel" the membrane potential, which will exert a major influence on the result. In general the gradients affected by the pH component of the proton motive force are fairly modest, but those influenced by the membrane potential are substantial.

In a very few cases (e.g. for calcium uptake) the carrier simply allows the charged ion to traverse the membrane. This constitutes an electrical (or electrogenic) uniporter. The membrane potential exerts its full effect, leading to substantial concentration gradients if the reaction were able to reach equilibrium.

The principal transport systems are listed in the table below, which also shows some of the main inhibitors.

porter

stoichiometry

inhibitors

comments

phosphate electroneutral exchange of
H2PO4- for OH-
NEM
mersalyl
all mitochondria
dicarboxylate random electroneutral exchange of
malate2- for succinate2- or HPO42-
mersalyl
malonate
all mitochondria
tricarboxylate electroneutral exchange of
(citrate3- + H+ ) for malate2-
benzene 1,2,3
tricarboxylate
mammalian adipose tissue
and liver: needed for fatty
acid biosynthesis
oxoglutarate electroneutral exchange of
malate2- for oxoglutarate2-

_

most mammalian tissues
needed for the malate
aspartate cycle
adenine
nucleotides
electrical ATP4-/ADP3- exchange atractyloside
bongkrekic
acid
all mitochondria
glutamate electroneutral exchange of
glutamate- for OH-

_

mammalian liver: needed
for the urea cycle
aspartate electrical exchange of
(glutamate- + H+ ) for aspartate-

_

most mammalian tissues
needed for the malate
aspartate cycle
calcium electrical uniport for Ca++

_

calcium uptake system
calcium electroneutral exchange of
Ca++ for 2H+

_

mitochondrial calcium
export system in liver
calcium /
sodium
electroneutral exchange of
Ca++ for 2Na+

_

mitochondrial calcium
export system in heart
sodium /
proton
electroneutral exchange of
Na+ for H+

_

widely distributed

The charge imbalance associated with the adenine nucleotide carrier leads to a large difference in the ATP/ADP ratio between matrix space and cytosol. ATP is effectively "worth more" in the cytosol, because the ATP:ADP couple is maintained further away from equilibrium:

Cells do not, however, get something for nothing. The transport of ATP, ADP and phosphate across the inner mitochondrial membrane costs 33% additional energy, over the minimum required for the synthesis of ATP within the mitochondrial matrix compartment. This extra energy must be supplied by the respiratory chain. One additional proton is used to drive both of these transport systems: the positive charge on this single proton drives adenine nucleotide exchanger, while its acidity drives the phosphate uptake. The electrical and pH components of the proton motive force are exploited separately.

There has recently been great interest in the ATP:ADP carrier protein as a possible auto-antigen in dilated cardiomyopathy, a common and debilitating cardiac disease. This same enzyme is involved in the mitochondrial permeability transition and in apoptosis.

The high cytosolic ATP/ADP implies a very low cytosolic AMP concentration, as a result of the myokinase equilibrium. This enables 5' AMP to serve as an emergency signal, which indicates a threat to the ATP supply. Several key enzymes (notably glycogen phosphorylase and phospho- fructokinase) are strongly activated by low concentrations of 5'AMP. This nucleotide also gives rise to adenosine which stimulates blood flow to active tissues. Note that 5'AMP differs from 3'5' cyclic AMP produced by adenyl cyclase.

Muscle contraction apparently requires a very high ATP/ADP ratio, and it is difficult to maintain a low myofibrillar ADP because it will not diffuse quickly enough back to the mitochondria at low ADP levels. (Diffusion rate is directly proportional to metabolite concentration.) Active muscles contain creatine phosphokinase, (CPK) and large amounts of creatine and creatine phosphate. These compounds equilibrate with the adenine nucleotide pools. It is thought that the high concentrations of these highly diffusible energy carriers increase the maximum energy transport rate.

Gene knockout experiments have shown that mice lacking both the mitochondrial and myofibrillar CPK iso-enzymes exhibit abnormal development, however mutants deficient in only one iso-enzyme are superficially healthy. It is difficult to see why both CPK genes should be widely retained if they don't do anything. It remains to be established whether the single mutants can maintain the sustained work outputs achieved by the wild-type controls.

One of the most important asymmetric transporters is the aspartate carrier, which in mammals plays a key role in the re-oxidation of glycolytic NADH by the malate-aspartate cycle. This cycle is necessary because the inner membrane is not permeable to either NAD or NADH. The component reactions must revolve twice for each molecule of glucose oxidised by the cell. The two enzymes involved, malate dehydrogenase (MDH) and glutamate oxaloacetate transaminase (GOT) are among the most active in the body. Both enzymes exist in mitochondrial and cytosolic variants, but all four proteins are coded by nuclear genes.

Aspartate- swops for glutamate plus a proton, so the full proton motive force is applied to the aspartate porter. The overall effect is to bias this otherwise symmetrical cycle, and maintain the cytosolic compartment in a relatively "oxidising" state (with a low NADH/NAD ratio) while the mitochondrial compartment is kept correspondingly reduced. This arrangement suppresses lactate formation during aerobic glycolysis. Invertebrate species employ alternative shuttles to achieve the same effect, and it is doubtful if eukaryotic cells could work at all without some such arrangement.

It is important to realise that no porter means no transport. Oxaloacetate and fumarate, for example, bind very badly to the dicarboxylate carrier, and are not transported at any significant rate. If both malate and oxaloacetate were transported it would be impossible to maintain the redox differential for NADH / NAD which exists between the mitochondrial matrix space and the cytosol.

Metabolite porter genes are located in the nucleus and are only expressed in those tissues which require them. For example, liver mitochondria have porters for ornithine and citrulline, which are required for the urea cycle, but these are not found in other tissues. The citrate carrier is found only in liver cells and adipocytes, where it is needed for lipogenesis

The outcome of many substrate oxidation experiments in vitro is determined by the porter specificity. For example: most animal mitochondria oxidise succinate almost quantitatively to malate, because neither fumarate nor oxaloacetate can be transported out of the matrix space and there is no source of acetyl-CoA to produce citrate.

Even in liver mitochondria, which possess a glutamate / hydroxyl antiporter, glutamate added alone mainly yields aspartate via glutamate oxaloacetate transaminase (GOT), oxoglutarate dehydrogenase (OGDH), succinate thiokinase (STK), succinate dehydrogenase (SDH), fumarase (FUM), malate dehydrogenase (MDH) and the aspartate porter.

However, if glutamate and malate are added together, mammalian mitochondria initially produce equal amounts of aspartate and oxoglutarate using the mitochondrial half of the malate - aspartate cycle. The aspartate and oxoglutarate porters, GOT, MDH and the respiratory chain are the only enzymes involved under these conditions.

Self assessment question: If mitochondria were incubated with oxoglutarate alone, which pathways would be operative and which compound(s) would be formed?

In some cases, e.g. for calcium ions, an electrical carrier is used for uptake and an electroneutral system is used for export. The two components of the proton motive force are used to drive the ion in opposite directions. This may be exploited by cells to regulate intra-mitochondrial calcium to a different value to the cytosolic concentration.

Many important metabolites show an asymmetric distribution across the mitochondrial inner membrane. The operation of the electroneutral anion carriers leads to a modest accumulation of polybasic acids (especially citrate) within the matrix space. In calculating the equilibrium distribution, it is only the "bottom line" which counts - the net number of protons which crossed the membrane by whatever process in order to accumulate the anion. The detailed mechanism is irrelevant, and any intermediate "swops" are discounted. If the overall reaction for electroneutral metabolite accumulation is:

anionn-(out) + nH+(out) <=> anionn-(in) + nH+(in)

then simple mass action considerations lead to the conclusion that:

[anionn-in ] / [anionn-out ] = ([H+out] / [H+in])n

This is helpful for the operation of the citric acid cycle, and also has implications for the regulation of glycolysis and lipogenesis by phosphofructokinase and acetyl CoA carboxylase, where citrate is an allosteric effector.

Mitochondrial swelling experiments: If it is desired to study membrane transport in isolation it is often necessary to block further metabolism with cyanide or rotenone. The most accurate studies of transport kinetics involve the rapid separation of mitochondria from their incubation medium using ultrafiltration or centrifugation through silicone oil. These complex and time consuming experiments require radioactive tracers to correct for "carry over" of the incubation medium into the mitochondrial fraction. Large scale metabolite movements will, however, produce alterations in matrix volume through osmotic mechanisms. A qualitative estimate of transport rates can thus be obtained by measuring the changes in light scattering from a turbid mitochondrial suspension.

These results were obtained from 0.5 mg liver mitochondria suspended at pH 7 in a spectrophotometer cuvette with 2.5 ml of 150 mM potassium acetate. Acetate ions can penetrate rapidly by an electroneutral process as free acetic acid, but no swelling is observed initially because the potassium cannot enter. Addition of valinomycin has no effect because this ionophore catalyses electrical movement. Nigericin allows an electroneutral exchange of potassium ions for protons and rapid swelling takes place.

Passive swelling in isotonic ammonium salt solutions can be used to study the major anion transport systems in cyanide-blocked mitochondria. The solutes move passively down their electrochemical gradients. Ammonium ions penetrate rapidly as free ammonia without movement of charge, and swelling is observed if the anion is also transported by an electroneutral process. It can be shown that phosphate, malate, succinate, oxoglutarate, citrate and sodium are all taken up by electroneutral mechanisms, and the phosphate / hydroxyl, phosphate / dicarboxylate, dicarboxylate / tricarboxylate and malate / oxoglutarate exchangers can be demonstrated.

It is also possible to observe "energised swelling" using intact mitochondria where solutes are accumulated against a concentration gradient using energy from respiration or from external ATP. There are substantial differences between the energised and passive processes, but in every case (except for ATP-driven pumps) net ion movements are always down the electro-chemical gradient.

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