Effect of leader peptides on the permeability of mitochondria.

Peptides with sequences based on the leader sequence of yeast cytochrome c oxidase subunit IV (pCOX IV-(1-25)) activate the electrophoretic uptake of K+ and other cations such as tetraethylammonium and lysine by rat liver mitochondria with EC50 = 11-15 microM. Uptake of these cations is dependent on respiration and is prevented by uncoupling agents, and the Vmax for K+ is 1.2-1.5 micromol/min/mg. Albeit more slowly, the non-electrolytes mannitol and sucrose are also transported by this pathway. Treatment of the peptides with proteinase K eliminates the stimulatory effect. Since the stimulated rate is not inhibited by ATP or by cyclosporin, we conclude that this pathway is not related to the mitochondrial KATP channel or the Ca2+-dependent permeability transition pore. Transport is stimulated by pCOX IV-(1-23), pCOX IV-(1-22), and pCOX IV-(1-12)Y, but not by a 13-amino acid peptide representing the nuclear location sequence of the SV40 large T antigen, which is responsible for directing that protein to the nucleus. Spermine, which has four positive charges, also has no stimulatory effect, and an amphiphilic 22-residue peptide derived from antithrombin III with seven net charges is only one-twentieth as effective as pCOX IV-(1-22). Thus, these data indicate that the sequence/structure is important for activation of transport. We also demonstrate that mitochondrial uncoupling, previously reported to be induced by these peptides, actually reflects coupled accumulation of salt. In view of our findings, it is also likely that the lytic effects attributed to these peptides are secondary to swelling and are not due to membrane damage per se. Finally, we show that, in non-ionic media, the peptide is an inhibitor of cytochrome c oxidase.

Mechanisms for the transport of alpha,omega-dicarboxylates through the mitochondrial inner membrane.

alpha,omega-Dicarboxylates have antibacterial properties, have been used in the treatment of hyperpigmentary disorders, are active against various melanoma cell lines, and can also undergo beta-oxidation. Little, however, is known about their transport. In this paper, we examine the mitochondrial transport of alpha, omega-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 degrees C and pH 7.0, DC10 is transported very rapidly at 3 micromol/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.

Temperature dependence of the mitochondrial inner membrane anion channel. The relationship between temperature and inhibition by protons.

In this paper, we investigate the temperature and pH dependence of the mitochondrial inner membrane anion channel (IMAC) that is believed to be involved in mitochondrial volume homeostasis. At pH 7. 4, the flux of malonate is highly temperature-dependent with rates increasing from 1 nmol/min mg at 5 degrees C to 1900 nmol/min mg at 45 degrees C. The Arrhenius plot is nonlinear with the activation energy increasing from 21 kJ/mol (Q10 = 1.3) to 193 kJ/mol (Q10 = 13) as the temperature is decreased. This temperature dependence is unusual and not seen with solutes that are transported through the bilayer such as NH4OAc, malonamide, and KSCN (plus valinomycin) or even for cytochrome c oxidase-dependent uptake of potassium (plus valinomycin). The temperature dependence of IMAC is closely related to the inhibition of IMAC by protons. Thus, we find that the pIC50 for protons decreases from 9.3 (Hill coefficient = 1.0) at 5 degrees C to 7.1 (Hill coefficient = 2.5) at 45 degrees C. This behavior is explained on the basis of a new kinetic model for IMAC in which the net open probability is not only modulated by the binding of three protons but also by temperature via effects on the open probability of the unprotonated channel and the pK of one of the inhibitory protonation sites.

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 mu M for DIDS, H2-DIDS, and SITS respectively and Hill coefficients < or = 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 with N-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.

On the relationship between the mitochondrial inner membrane anion channel and the adenine nucleotide translocase.

The mitochondrial inner membrane anion channel (IMAC) is a transport pathway which is believed to be involved in mitochondrial volume homeostasis. The protein, however, has not been identified. In this paper, we examine the relationship between IMAC and the adenine nucleotide translocator. Many inhibitors of the adenine nucleotide translocase are shown to block IMAC, including Cibacron blue 3GA, bromcresol green, alizarin red S, agaric acid, palmitoyl-CoA, and the fluorescein derivatives erythrosin B, erythrosin isothiocyanate, rose bengal, and eosin Y. The following evidence suggests that Cibacron blue, agaric acid, and palmitoyl-CoA inhibit by binding to a common site. 1) They all only partially block the transport of small anions such as Cl-, NO3-, and HCO3-, but completely block the transport of larger anions such as malonate. 2) They decrease the IC50 values of each other in a manner consistent with competitive binding. 3) N-Ethylmaleimide decreases their IC50 values by a similar extent. 4) Inhibition by all shows no dependence on matrix pH and only a small dependence on medium pH. It is suggested that these agents may selectively bind to an open state of IMAC and inhibit by decreasing its conductance. The physiological nucleotides CoA, NAD+, NADH, NADP+, NADH, and ATP do not inhibit; in fact, IMAC is shown to transport ATP. Despite these similarities between IMAC and the adenine nucleotide translocase, IMAC appears to be a separate entity, since some of the IC50 values differ by up to 8-fold, and carboxyatracyloside, the most selective inhibitor of the adenine nucleotide translocase, has no effect on IMAC. In addition, IMAC is also able to transport AMP, while the adenine nucleotide translocase does not.

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.

On the regulation of K+ uniport in intact mitochondria by adenine nucleotides and nucleotide analogs.

Respiring mitochondria drive the electrophoretic uptake of K+ and other cations. In the presence of permeant acids this transport leads to mitochondrial swelling if it is not compensated by electroneutral K+/H+ exchange mediated by the K+/H+ antiporter. The mechanism of influx has yet to be established; however, evidence is accumulating that in addition to leak pathways a specific K+ channel or uniporter may be involved. We examine some of the properties of K+ uniport which are consistent with the existence of a specific ATP-regulated K+ channel. In contrast to the K+/H+ antiporter, K+ uniport shows little dependence on pH. K+ uniport is, however, very sensitive to inhibition by adenine nucleotides. The maximum percent inhibition is increased from 40 to 60% by treatment of mitochondria with N-ethylmaleimide (30 nmol/mg) which stimulates K+ uniport 3.6-fold. N-Ethylmaleimide, however, has no effect on the IC50 values which are 0.5, 2.3, and 8 microM for ADP, ATP, and AMP, respectively. GDP has no effect, while carboxyatractyloside is found to inhibit. The nucleotide analogs Cibacron blue 3GA and erythrosin B exhibit three effects on K+ uniport. Low doses partially inhibit K uniport (I50 = 0.13 microM Cibacron Blue), while higher doses stimulate (EC50 = 13 microM Cibacron Blue). Stimulation is especially apparent in N-ethylmaleimide-treated mitochondria. These analogs also antagonize inhibition by ATP. Since the EC50 values for this antagonism for these two drugs are similar, while the IC50 values for inhibition of ATP transport differ by a factor of five, we suggest that inhibition of K+ uniport by ATP is not mediated via the adenine nucleotide translocase. These data are consistent with the existence of an ATP-regulated K+ channel in the inner mitochondrial membrane.

Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria.

The transport properties of mitochondria are such that net potassium flux across the inner membrane determines mitochondrial volume. It has been known that K+ uptake is mediated by diffusive leak driven by the high electrical membrane potential maintained by redox-driven, electrogenic proton ejection and that regulated K+ efflux is mediated by an 82-kDa inner membrane K+/H+ antiporter. There is also long-standing suggestive evidence for the existence of an inner membrane protein designed to catalyze electrophoretic K+ uptake into mitochondria. We report reconstitution of a highly purified inner membrane protein fraction from rat liver and beef heart mitochondria that catalyzes electrophoretic K+ flux in liposomes and channel activity in planar lipid bilayers. The unit conductance of the channel at saturating [K+] is about 30 pS. Reconstituted K+ flux is inhibited with high affinity by ATP and ADP in the presence of divalent cations and by glibenclamide in the absence of divalent cations. The mitochondrial ATP-dependent K+ channel is selective for K+, with a Km of 32 mM, and does not transport Na+. K+ transport depends on voltage in a manner consistent with a channel activity that is not voltage-regulated. Thus, the mitochondrial ATP-dependent K+ channel exhibits properties that are remarkably similar to those of the ATP-dependent K+ channels of plasma membranes.

Anion uniport in plant mitochondria is mediated by a Mg(2+)-insensitive inner membrane anion channel.

It has long been established that the inner membrane of plant mitochondria is permeable to Cl-. Evidence has also accumulated which suggests that a number of other anions such as Pi and dicarboxylates can also be transported electrophoretically. In this paper, we present evidence that anion uniport in plant mitochondria is mediated via a pH-regulated channel related to the so-called inner membrane anion channel (IMAC) of animal mitochondria. Like IMAC, the channel in potato mitochondria transports a wide variety of anions including NO3-, Cl-, ferrocyanide, 1,2,3-benzene-tricarboxylate, malonate, Pi, alpha-ketoglutarate, malate, adipate, and glucuronate. In the presence of nigericin, anion uniport is sensitive to the medium pH (pIC50 = 7.60, Hill coefficient = 2). In the absence of nigericin, transport rates are much lower and much less sensitive to pH, suggesting that matrix H+ inhibit anion uniport. This conclusion is supported by measurements of H+ flux which reveal that "activation" of anion transport at high pH by nigericin and at low pH by respiration is associated with an efflux of matrix H+. Other inhibitors of IMAC which are found to block anion uniport in potato mitochondria include propranolol (IC50 = 14 microM, Hill coefficient = 1.28), tributyltin (IC50 = 4 nmol/mg, Hill coefficient = 2.0), and the nucleotide analogs Erythrosin B and Cibacron Blue 3GA. The channel in plant mitochondria differs from IMAC in that it is not inhibited by matrix Mg2+, mercurials, or N,N'-dicyclohexylcarbodiimide. The lack of inhibition by Mg2+ suggests that the physiological regulation of the plant channel may differ from IMAC and that the plant IMAC may have functions such as a role in the malate/oxaloacetate shuttle in addition to its proposed role in volume homeostasis.

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 microM 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 by N,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, and N,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.

Triorganotins inhibit the mitochondrial inner membrane anion channel.

The inner membrane of liver and heart mitochondria possesses an anion uniport pathway, known as the inner membrane anion channel (IMAC). IMAC is inhibited by matrix Mg2+, matrix H+, N,N'-dicyclohexycarbodiimide, mercurials and amphiphilic amines such as propranolol. Most of these agents react with a number of different mitochondrial proteins and, therefore, more selective inhibitors have been sought. In this paper, we report the discovery of a new class of inhibitors, triorganotin compounds, which block IMAC completely. One of the most potent, tributyltin (TBT) inhibits malonate uniport via IMAC 95% at 0.9 nmol/mg. The only other mitochondrial protein reported to react with triorganotins, the F1F0ATPase, is inhibited by about 0.75 nmol/mg. The potency of inhibition of IMAC increases with hydrophobicity in the sequence trimethyltin much less than triethyltin much less than tripropyltin less than triphenyltin less than tributyltin; which suggests that the binding site is accessible from the lipid bilayer. It has long been established that triorganotins are anionophores able to catalyze Cl-/OH- exchange; however, TBT is able to inhibit Cl- and NO3- transport via IMAC at doses below those required to catalyze rapid rates of Cl-/OH- exchange. Consistent with previous reports, the data indicate that about 0.8 nmol of TBT per mg of mitochondrial protein is tightly bound and not available to mediate Cl-/OH- exchange. We have also shown that the mercurials, p-chloromercuribenzene sulfonate and mersalyl, which only partially inhibit Cl- and NO3- transport can increase the IC50 for TBT 10-fold. This effect appears to result from a reaction at a previously unidentified mercurial reactive site. The inhibitory dose is also increased by raising the pH and inhibition by TBT can be reversed by S2- and dithiols but not by monothiols.

N-ethylmaleimide and mercurials modulate inhibition of the mitochondrial inner membrane anion channel by H+, Mg2+ and cationic amphiphiles.

Previously it has been shown that the mitochondrial inner membrane anion channel is reversibly inhibited by matrix Mg2+, matrix H+ and cationic amphiphiles such as propranolol. Furthermore, the IC50 values for both Mg2+ and cationic amphiphiles are dependent on matrix pH. It is now shown that pretreatment of mitochondria with N-ethylmaleimide, mersalyl and p-chloromercuribenzenesulfonate increases the IC50 values of these inhibitors. The effect of the mercurials is most evident when cysteine or thioglycolate is added to the assay medium to reverse their previously reported inhibitory effect (Beavis, A.D. (1989) Eur. J. Biochem. 185, 511-519). Although the IC50 values for Mg2+ and propranolol are shifted they remain pH dependent. Mersalyl is shown to inhibit transport even in N-ethylmaleimide-treated mitochondria indicating that N-ethylmaleimide does not react at the inhibitory mercurial site. However, the effects of N-ethylmaleimide and mersalyl on the IC50 for H+ are not additive which suggests that mercurials and N-ethylmaleimide react at the same 'regulatory' site. It is suggested that modification of this latter site exerts an effect on the binding of Mg2+, H+ and propranolol by inducing a conformational change. It is also suggested that a physiological regulator may exist which has a similar effect in vivo.

Evidence for the allosteric regulation of the mitochondrial K+/H+ antiporter by matrix protons.

It is well accepted that the mitochondrial K+/H+ antiporter is regulated by matrix Mg2+; however, this is not the only factor controlling its activity. The precise conditions used to deplete divalent cations have profound effects on the subsequent activity of the antiporter in a KOAc assay medium. Examination of the proton fluxes during both pretreatment and subsequent assay of K+/H+ antiport reveals that differences in K+/H+ antiport activity correlate very well with differences in matrix pH. Thus, inhibition of the K+/H+ antiporter following depletion of Mg2+ appears to result from inhibition by matrix protons. To test this hypothesis, we have examined the effect of modulating matrix pH in three different ways on the activity of the K+/H+ antiporter: 1) lowering the pH of the K+ pretreatment medium to 6.7 leads to inactivation of the K+/H+ antiporter; 2) adding NH4+ to the assay medium eliminates the lag in activity induced by depleting Mg2+ in a pretreatment medium containing NH4+; 3) permitting mitochondria to respire in a tetraethylammonium(+)-containing pretreatment medium activates the K+/H+ antiporter. Each one of these procedures leads to a change in matrix pH and an effect on K+/H+ antiport which appears to require regulation of the K+/H+ antiporter by matrix protons. This finding is not only physiologically significant but also provides a useful definition of conditions required for unmasking the K+/H+ antiporter in a reproducible manner.

Evidence for two distinct chloride uniport pathways in brown adipose tissue mitochondria.

Chloride permeability of the inner membrane of brown adipose tissue mitochondria was analyzed by monitoring mitochondrial swelling in KCl salts in the presence of K+ ionophores. The results indicate that the high anion conductance observed in these mitochondria is due to the presence of two separate pathways: 1) a Cl-conducting pathway that is inhibited by guanosine 5'-diphosphate (GDP) but neither by N,N'-dicyclohexylcarbodiimide (DCCD) nor by amphiphilic amines and that is found uniquely in brown adipose tissue mitochondria and 2) an inner membrane anion uniport channel that is inhibited both by DCCD and by amphiphilic amines but not by GDP and that is opened either by depletion of matrix Mg2+ or by alkalinization of the matrix.

The mitochondrial inner-membrane anion channel possesses two mercurial-reactive regulatory sites.

The mitochondrial inner membrane anion channel catalyzes the electrophoretic transport of a wide variety of anions and is inhibited by matrix divalent cations and protons. In this paper, evidence is provided that mersalyl and p-chloromercuribenzene-sulfonate each interact with this uniporter at two distinct sites. Binding to site 1 causes a shift in the pH dependence of transport, characterized by a decrease in the pIC50 for protons from about 7.8 to about 7.3, and leads to substantial stimulation of transport in the physiological pH range. This effect is not reversed by addition of thiols such as thioglycolate. Binding of mersalyl and p-chloromercuribenzenesulfonate to site 2 inhibits the transport of most anions including Pi, citrate, malonate, sulfate and ferrocyanide. The transport of Cl- is inhibited about 60% by mersalyl, but is not inhibited by p-chloromercuribenzenesulfonate. These data suggest that inhibition is a steric effect dependent on the size of the anion and the size of the R group of the mercurial. This inhibition is reversed by thioglycolate. Dose/response curves indicate that mersalyl binds to site 1 as the dose increased from 7 to 13 nmol/mg, whereas it binds to site 2 as the dose is increased from 10 to 18 nmol/mg. Thus, at certain pH values both stimulatory and inhibitory phases can be seen in the same dose/response curve. It is suggested that these sites may contain thiol groups and that physiological regulators may exist which can effect changes in activity of the inner membrane anion uniporter similar to those exerted by mercurials.

On the regulation of the mitochondrial inner membrane anion channel by magnesium and protons.

The inner membrane of mitochondria possesses a pH-regulated anion uniporter which is activated by depletion of matrix divalent cations with A23187 (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). It is now shown that Cl- transport through this pathway is inhibited by Mg2+ and Ca2+. There appear to be two sites for inhibition by Mg2+. One has an IC50 = 38 microM at pH 7.4 and appears to be on the inside since it is only observed in the presence of A23187 (10 nmol/mg). The other has an IC50 = 440 microM at pH 7.4 and appears to be on the outside since it is observed in mitochondria pretreated with very low doses of A23187 (0.25 nmol/mg or less) and in A23187-pretreated mitochondria washed to remove A23187. Ca2+ is found to inhibit anion uniport in the presence or absence of A23187 with an IC50 of about 17 microM. In contrast to these findings Cl- uniport, activated by addition of valinomycin to respiring mitochondria without depleting endogenous Mg2+ is found to be very insensitive to exogenous Mg2+, being inhibited with an IC50 of 3.2 mM. This is explained by examination of the pH dependence of the Mg2+ IC50 in non-respiring mitochondria. The internal IC50 is found to be pH-dependent, rising to about 250 microM at pH 8.4. The external IC50 is also pH-dependent, rising to 2.5 mM or above at pH 8.4. These data are consistent with a model in which Mg2+ can only bind to the protein when it is protonated at a site with a pK of about 6.8 located in the matrix. Thus, both the intrinsic activity of the uniporter and its inhibition by Mg2+ appear to be regulated by matrix protons. This makes the rate of anion uniport much more sensitive to changes in matrix pH which is physiologically advantageous for its proposed role in volume homeostasis.

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.

On the inhibition of the mitochondrial inner membrane anion uniporter by cationic amphiphiles and other drugs.

Depleting the mitochondrial matrix of divalent cations with the ionophore A23187 activates a pH-sensitive, anion uniport pathway which can transport many anions normally regarded as impermeant (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). Addition of valinomycin to respiring mitochondria can also induce the uptake of a wide variety of anions; however, the mechanism of anion transport during this "respiration-induced" swelling is less certain. In this paper, I demonstrate that both of these processes are inhibited by a variety of cationic amphiphiles including propranolol, quinine, amiodarone, imipramine and amitriptyline, and the benzodiazepine R05-4864. Although the IC50 values for the two processes are not equal, the ratio of IC50 values for the two processes appears to be the same for all drugs. Measurements of net transmembrane proton fluxes that occur during the assays reveal that respiration-induced swelling is associated with extensive proton ejection, the peak of which coincides with the maximum rate of anion transport. Moreover, from measurements of matrix buffering power, it is estimated that the matrix pH is 3 units more alkaline during respiration-induced swelling than during A23187-induced swelling. It is also shown that the IC50 for A23187-induced transport is pH-dependent in a manner consistent with modulation of drug binding by protonation of two sites. These findings allow the difference in IC50 values for the two types of assay to be explained by the pH dependence of the binding constant for the drug. Furthermore, the pH gradient generated during respiration-induced swelling is so large that the electrical component of the proton-motive force will be negligible. Thus, despite the fact that the mitochondria are "energized," rapid electrophoretic anion influx is possible. These data provide evidence that the transport of anions in these two types of assay occurs via the same pathway.

Inhibition of the mitochondrial inner membrane anion channel by dicyclohexylcarbodiimide. Evidence for a specific transport pathway.

Electrophoretic uniport of anions through the inner mitochondrial membrane can be activated by alkaline pH or by depleting the matrix of divalent cations. It has also been suggested that, in the presence of valinomycin and potassium, respiration can also activate anion uniport. We have proposed that a single pathway is responsible for all three of these transport processes (Garlid, K. D., and Beavis, A. D. (1986) Biochim. Biophys. Acta 853, 187-204). We now present evidence that like the "pH-dependent" pore the divalent cation-regulated pore and the "respiration-induced" pore are blocked by N,N'-dicyclohexylcarbodiimide (DCCD). Moreover, the kinetics of inhibition of the latter two pathways are identical and exhibit a second order rate constant of 2.6 X 10(-3) (nmol DCCD/mg)-1.min-1. DCCD inhibits the uniport of Cl-, phosphate, malate, and other lipophobic anions completely, but it has no effect on the classical electroneutral phosphate and dicarboxylate carriers. In Mg2+-depleted mitochondria DCCD partially inhibits the transport of SCN-; however, in Mg2+-containing mitochondria and at low pH, no inhibition is observed. Furthermore, in DCCD-treated mitochondria, even following depletion of Mg2+, the transport of SCN- is independent of pH. These results lead us to conclude that two pathways for anion uniport exist: a specific, regulated pathway which can conduct a wide variety of anions and a nonregulated pathway through the lipid bilayer which only conducts lipid-soluble ions.