Andrew D. Beavis

Properties of the inner membrane anion channel in intact mitochondria

The mitochondrial inner membrane possesses an anion channel (IMAC) which mediates the electrophoretic transport of a wide variety of anions and is believed to be an important component of the volume homeostatic mechanism. IMAC is regulated by matrix Mg2+ (IC50=38 µM at pH 7.4) and by matrix H+ (pIC50=7.7). Moreover, inhibition by Mg2+ is pH-dependent. IMAC is also reversibly inhibited by many cationic amphiphilic drugs, including propranolol, and irreversibly inhibited byN,N′-dicyclohexylcarbodiimide. Mercurials have two effects on its activity: (1) they increase the IC50 values for Mg2+, H+, and propranolol, and (2) they inhibit transport. The most potent inhibitor of IMAC is tributyltin, which blocks anion uniport in liver mitochondria at about 1 nmol/mg. The inhibitory dose is increased by mercurials; however, this effect appears to be unrelated to the other mercurial effects. IMAC also appears to be present in plant mitochondria; however, it is insensitive to inhibition by Mg2+, mercurials, andN,N′-dicyclohexylcarbodiimide. Some inhibitors of the adenine nucleotide translocase also inhibit IMAC, including Cibacron Blue, agaric acid, and palmitoyl CoA; however, atractyloside has no effect.

Evidence for the existence of an inner membrane anion channel in mitochondria

Mitochondria normally exhibit very low electrophoretic permeabilities to physiologically important anions such as chloride, bicarbonate, phosphate, succinate, citrate, etc. Nevertheless, considerable evidence has accumulated which suggests that heart and liver mitochondria contain a specific anion-conducting channel. In this review, a postulated inner membrane anion channel is discussed in the context of other known pathways for anion transport in mitochondria. This anion channel exhibits the following properties. It is anion-selective and inhibited physiologically by protons and magnesium ions. It is inhibited reversibly by quinine and irreversibly by dicyclohexylcarbodiimide. We propose that the inner membrane anion channel is formed by inner membrane proteins and that this pathway is normally latent due to regulation by matrix Mg2+. The physiological role of the anion channel is unknown; however, this pathway is well designed to enable mitochondria to restore their normal volume following pathological swelling. In addition, the inner membrane anion channel provides a potential futile cycle for regulated non-shivering thermogenesis and may be important in controlled energy dissipation.

On the nature of ion leaks in energy-transducing membranes

Diffusion is the implicit null hypothesis for ion transport across biological membranes. A proper model of ionic diffusion across the permeability barrier is needed to distinguish among leaks, channels and carriers and to determine whether changes in flux reflect changes in permeability (regulation) or merely changes in the driving force. These issues arise in all biomembranes, but they are particularly confounding in energy-transducing membranes on account of their characteristically high electrical gradients. This paper examines the nature of the barrier to ion leaks, using the classical Eyring rate theory. We introduce new practical procedures for estimating permeability coefficients from ion flux data. We also reach some general conclusions regarding ion leaks across energy-transducing membranes. (1) The dependence of ion flux on the electrical membrane potential is invariably non-linear (non-ohmic). (2) Non-ohmic behavior does not imply variable permeability. (3) Ohmic behavior is exceptional and its occurrence should alert us to the possibility of an underlying carrier or channel. (4) Leak pathways are very likely localized to protein-lipid interfaces and will exhibit quasi-specific properties such as saturation and competition. (5) The inherent non-ohmicity of leaks and the requirement for efficient energy transduction impose constraints upon the magnitude of allowable Gibbs free-energy changes in biological systems. (6) Nature adapts to these constraints by devising mechanisms for step-wise splitting of the partial reactions of energy transduction.

The mitochondrial inner membrane anion channel is inhibited by DIDS

The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg2+ and H+. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2′-disulfonates. Inhibition of malonate transport approaches 100% with IC50 values of 26, 44, and 88 ΜM for DIDS, H2-DIDS, and SITS respectively and Hill coefficients ≤1. In contrast, inhibition of Cl− transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC50 which is severalfold higher than that for malonate. The IC50 for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC50 by about 50%, but under conditions where only the matrix pH is made alkaline the IC50 is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other “Cl−” channels than previously thought, but also suggest that other Cl− channels may be sensitive to some of the many regulators of IMAC which have been identified.

MUTATIONS IN LIS1 (ERG6) GENE CONFER INCREASED SODIUM AND LITHIUM UPTAKE IN SACCHAROMYCES CEREVISIAE

A Saccharomyces cerevisiae mutant, lis1-1, hypersensitive to Li+ and Na+ was isolated from a wild-type strain after ethylmethane sulfonate mutagenesis. The rates of Li+ and Na+ uptake of the mutant are about 3-4-times higher than that of the wild-type; while the rates of cation efflux from the mutant and wild-type strains are indistinguishable. The LIS1 was isolated from a yeast genomic library by complementation of the cation hypersensitivity of the lis1-1 strain. LIS1 is a single copy, nonessential gene. However, the deletion of LIS1 from the wild-type results in a growth defect in addition to the cation hypersensitive phenotype. The order of increasing cation uptake rates of the wild-type and mutant strains, LIS1 < lis1-1 < lis1-delta 1::LEU2, correlates perfectly with the degree of cation hypersensitivity, suggesting that the cation hypersensitivity is primarily due to increased rates of cation influx. LIS1 encodes a membrane associated protein 384 amino acids long. Data base searches indicate that LIS1 is identical to ERG6 in S. cerevisiae which encodes a putative S-adenosylmethionine-dependent methyltransferase in the ergosterol biosynthetic pathway. Cell membranes of lis1 (erg6) mutants are known to be devoid of ergosterol and have altered sterol composition. Since membrane sterols can influence the activity of cation transporters, the increased cation uptake of the lis1 mutants may stem from an altered function of one or many different membrane transporters.

MECHANISMS FOR THE TRANSPORT OF ALPHA ,OMEGA -DICARBOXYLATES THROUGH THE MITOCHONDRIAL INNER MEMBRANE

α,ω-Dicarboxylates have antibacterial properties, have been used in the treatment of hyperpigmentary disorders, are active against various melanoma cell lines, and can also undergo β-oxidation. Little, however, is known about their transport. In this paper, we examine the mitochondrial transport of α,ω-dicarboxylates ranging from oxalate (DC2) to sebacate (DC10). DC2-DC10 are transported by the inner membrane anion channel (IMAC). DC6-DC10 are also transported by an electroneutral mechanism that appears to reflect transport of the acid through the lipid bilayer. At 37°C and pH 7.0, DC10 is transported very rapidly at 3 μmol/min·mg, and respiring mitochondria swell in the K+ salts of these acids. This transport mechanism is probably the major pathway by which the longer dicarboxylates enter cells, bacteria, and mitochondria. We also demonstrate that DC5-DC10 can also be transported by an electroneutral mechanism mediated by tributyltin, a potent inhibitor of IMAC. The mechanism appears to involve electroneutral exchange of a TBT-dicarboxylate-H complex for TBT-OH. Finally, we present evidence that of all the dicarboxylates tested only DC2-DC4 can be transported by the classical dicarboxylate carrier.

On the regulation of Na+/H+ and K+/H+ antiport in yeast mitochondria: Evidence for the absence of an Na+-selective Na+/H+ antiporter

Unlike mammalian mitochondria, yeast mitochondria swell spontaneously in both NaOAc and KOAc. This swelling reflects the activity of an electroneutral cation/H+ antiport pathway. Transport of neither salt is stimulated by depletion of endogenous divalent cations; however, it can be inhibited by addition of exogenous divalent cations (Mg2+ IC50 = 2.08 mM, Ca2+ IC50 = 0.82 mM). Transport of both Na+ and K+ can be completely inhibited by the amphiphilic amines propranolol (IC50 = 71 microM) and quinine (IC50 = 199 microM) with indistinguishable IC50 values. Dicyclohexylcarbodiimide inhibits with a second-order rate constant of 1.6 x 10(-4) (nmol DCCD/mg)-1 min-1 at 0 degrees C; however, with both Na+ and K+ inhibition reaches a maximum of about 46%. The remaining transport can still be inhibited by propranolol. Transport of both cations is sensitive to pH; yielding linear Hill plots and Dixon plots with a pIC50 value of 7.7 for both Na+ and K+. These properties are qualitatively the same as those of the non-selective K+/H+ antiporter of mammalian mitochondria. However, the remarkable similarity between the data obtained in Na+ and K+ media suggests that an antiporter akin to the Na(+)-selective Na+/H+ antiporter of mammalian mitochondria, which is inhibited by none of these agents, is absent in yeast. In an attempt to reveal the activity of a propranolol-insensitive Na(+)-selective antiporter, we compared the rates of Na+/H+ and K+/H+ antiport in the presence of sufficient propranolol to block the K+/H+ antiporter. Between pH 4.6 and 8.8 no difference could be detected. Consequently, we conclude that yeast mitochondria lack the typical Na(+)-selective Na+/H+ antiporter of mammalian mitochondria.

A Channel Model to Explain Regulation of the Mitochondrial Inner Membrane Anion Channel (IMAC)

The mitochondrial inner membrane anion channel (IMAC) is a transport pathway in the inner mitochondrial membrane which is able to transport a wide variety of anions ranging from Cl- to ATP4- (see Beavis (1992) and references therein for a review). In addition to its very broad selectivity, it differs from all the other anion transporters of the inner membrane in that it mediates rapid electrophoretic uniport. On the basis of these properties, we call it a channel. In all other respects, however, its: properties are indistinguishable from those of a uniport carrier. Its turnover number is not known, since it has not yet been identified or purified and therefore its abundance is not known.

Upper and Lower Limits of Charge, Proton and ATP Stoichiometries of Oxidative Phosphorylation

It is now generally accepted that the flow of electrons down the mitochondrial respiratory chain is coupled to phosphorylation of ADP via a chemiosmotic mechanism. Thus, electron flow drives the electrogenic ejection of protons from the mitochondrial matrix and the electrophoretic influx of protons drives the synthesis of ATP. For this model the mechanistic ATP/O ratio for a given substrate is only dependent on the mechanistic H+/O ratio for the substrate and the mechanistic H+/ATP ratio. Thus, there is no need for the mechanistic ATP/O ratio to be integral. Since Mitchell1 first suggested that the H+/O ratio was 6 for NADH oxidation and 4 for succinate oxidation and that the H+/ATP ratio was 2, many different groups have measured these quantities with different results. In recent years values proposed for the H+/O ratios have included 82,3, 104,5, 126–9 and 1310,11 for NADH oxidation and 62,3,12 and 86–11 for succinate oxidation. Furthermore, values of 313,14 and 48,9,15–18 have been proposed for the net H+/ATP ratio. Now, if one assumes that the true H+/O ratio for NADH lies somewhere between 8 and 13 and the H+/ATP ratio is either 3 or 4, the mechanistic ATP/O ratio for NADH oxidation could equal any one of 11 values between 2.0 and 4.33! To determine which of these is correct from ATP/O measurements alone would require one to determine the ATP/O ratio to within 0.16 units or 5%. This would be difficult if the fluxes were completely coupled; however, since the fluxes are incompletely coupled and the uncoupled flux could account for up to 20% of the electron flow, it is possible that the measured flux ratio could underestimate the mechanistic stoichiometry by up to 20%. Thus, to establish the mechanistic ATP/O ratio with any certainty it is necessary to measure the ATP/O ratio, the H+/O ratio and/or the H+/ATP ratio. In order to eliminate some of the previously proposed stoichiometries, I have designed experiments to determine upper and lower limits to the mechanistic ATP/O and H+/O stoichiometries19. The results of these studies, presented in this paper, are inconsistent with all ATP/O ratios previously proposed by others and suggest that the true mechanistic ATP/O ratios are 2.75 for NADH oxidation and 1.75 for succinate oxidation19–22.