The mitochondrial inner membrane anion channel. Regulation by divalent cations and protons

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as Cl-, SO4(2-), and Fe(CN)6(4-) as well as physiologically important anions such as HCO3-, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with loss of matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.     

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.     

Plant inner membrane anion channel (PIMAC) function in plant mitochondria

To date, the existence of the plant inner membrane anion channel (PIMAC) has been shown only in potato mitochondria, but its physiological role remains unclear. In this study, by means of swelling experiments in K(+) and ammonium salts, we characterize a PIMAC-like anion-conducting pathway in mitochondria from durum wheat (DWM), a monocotyledonous species phylogenetically far from potato. DWM were investigated since they possess a very active potassium channel (PmitoK(ATP)), so implying a very active matching anion uniport pathway and, possibly, a coordinated function. As in potato mitochondria, the electrophoretic uptake of chloride and succinate was inhibited by matrix [H(+)], propranolol, and tributyltin, and was insensitive to Mg(2+), N,N'-dicyclohexylcarbodiimide (DCCD) and mercurials, thus showing PIMAC's existence in DWM. PIMAC actively transports dicarboxylates, oxodicarboxylates, tricarboxylates and Pi. Interestingly, a novel mechanism of swelling in ammonium salts of isolated plant mitochondria is reported, based on electrophoretic anion uptake via PIMAC and ammonium uniport via PmitoK(ATP). PIMAC is inhibited by physiological compounds, such as ATP and free fatty acids, by high electrical membrane potential (Delta Psi), but not by acyl-CoAs or reactive oxygen species. PIMAC was found to cooperate with dicarboxylate carrier by allowing succinate uptake that triggers succinate/malate exchange in isolated DWM. Similar results were obtained using mitochondria from the dicotyledonous species topinambur, so suggesting generalization of results. We propose that PIMAC is normally inactive in vivo due to ATP and Delta Psi inhibition, but activation may occur in mitochondria de-energized by PmitoK(ATP) (or other dissipative systems) to replace or integrate the operation of classical anion carriers.     

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.     

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.     

Anion regulation of active transport of protons across the membrane of the synaptic vesicles of the brain

The dependence of active transport of H+ on the presence of anions in synaptic vesicle membranes from rat brain was studied. The H+ transport was measured by monitoring the acidification of the vesicles with a permeant weak base-acridine orange. The fluorescence changes in the latter were proportional to the magnitude of artificially imposed pH gradients (delta pH). The ATP-dependent generation of delta pH was completely dependent on the presence of a permeant anion, was maximal at 150 mM Cl- and was inhibited, when the medium osmolarity was further increased by sucrose or KCl. At 150 mM only Br-, similar to Cl-, behaved as permeant anions, whereas I- was effective only at low (5-20 mM) concentrations. The anions--SCN-, ClO4-, HSO3- and I-(10-20 mM) as well as 4-acetamido-4'-isothiocyanatostilbene-2.2'-disulfonate (K0.5 = 14 microM) blocked the ATP-dependent generation of delta pH observed in the presence of Cl-, while other anions tested (F-, phosphate, bicarbonate, some organic anions) were virtually without effect and did not support the H+ transport. The dependence of the rate and extent of H+ accumulation on Cl- concentration was sigmoidal with a Hill coefficient of 2.8 and a Km value of 85-90 mM. The effects of anions point to the presence in the membrane of synaptic vesicles of an anion (chloride) channel whose conductance can regulate the H+ transport by switching it from an electrogenic to an electroneutral (coupled entry of H+ and Cl-) mode of operation.     

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.     

Reconstitution of the uncoupling protein of brown adipose tissue mitochondria. Demonstration of GDP-sensitive halide anion uniport

The fluorescent anion indicator 6-methoxy-N-(3-sulfopropyl)quinolinium was trapped in proteoliposomes reconstituted with purified 32-kDa uncoupling protein and used to detect GDP-sensitive uniports of Cl-, Br-, and I-. Transport of these halide anions was rapid and potential-dependent. F- and nitrate were found to inhibit Cl- uptake competitively, suggesting that these anions are also substrates for transport. This preparation also exhibited H+(OH-) transport, showing that the reconstituted uncoupling protein possesses both halide and H+ transport functions, as is observed in intact brown adipose tissue mitochondria. Cl- transport was inhibited to the residual level observed in liposomes without protein when GDP was present on both sides of the membrane. Cl- transport was inhibited by about 50% when GDP was present only on one side of the membrane. We infer that uncoupling protein reconstitutes into proteoliposomes with a 1:1 ratio of sidedness orientation. The Km values for Cl- uniport were 100 and 65 mM, respectively, in GDP-loaded and non-GDP-loaded vesicles. Participation of the inner membrane anion channel in the observed transport is rendered unlikely by the fact that this carrier is insensitive to GDP. A variety of additional experiments probing for inner membrane anion channel yielded uniformly negative results, confirming the absence of contamination by this protein. Our results therefore demonstrate that the uncoupling protein mediates anion translocation, a function previously reported as lacking in the reconstituted system.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.     

Glibenclamide interferes with mitochondrial bioenergetics by inducing changes on membrane ion permeability

The interference of glibenclamide, an antidiabetic sulfonylurea, with mitochondrial bioenergetics was assessed on mitochondrial ion fluxes (H+, K+, and Cl-) by passive osmotic swelling of rat liver mitochondria in K-acetate, KNO3, and KCl media, by O2 consumption, and by mitochondrial transmembrane potential (Deltapsi). Glibenclamide did not permeabilize the inner mitochondrial membrane to H+, but induced permeabilization to Cl- by opening the inner mitochondrial anion channel (IMAC). Cl- influx induced by glibenclamide facilitates K+ entry into mitochondria, thus promoting a net Cl-/K+ cotransport, Deltapsi dissipation, and stimulation of state 4 respiration rate. It was concluded that glibenclamide interferes with mitochondrial bioenergetics of rat liver by permeabilizing the inner mitochondrial membrane to Cl- and promoting a net Cl-/K+ cotransport inside mitochondria, without significant changes on membrane permeabilization to H+.     

The distribution of inorganic phosphate and malate between intra- and extramitochondrial spaces. Relationship with the transmembrane pH difference

The steady-state distribution of inorganic phosphate and malate between the intra- and extramitochondrial spaces was measured in suspensions of nonrespiring and respiring rat liver mitochondria in which the transmembrane pH difference was incrementally varied. In respiration-inhibited mitochondria, the slope of log [Pi]in/[Pi]out (ordinate) versus delta pH approached 2 by either chemical or isotopic determination of [Pi], and the slope of log [malate]in/[malate]out versus delta pH was 2.0 with an extrapolated log [Pi]in/[Pi]out value of 0.3 at delta pH = 0. We conclude that the distribution of Pi and malate for nonrespiring mitochondria were quantitatively consistent with those predicted by exchange of Pi- for OH- (or cotransport with H+) and of malate 2- for Pi2-. In respiring mitochondria using glutamate + malate as substrate, there was very little pH dependence of Pi or malate accumulation (the slopes were less than 0.5) unless n-butylmalonate (inhibitor of Pi-dicarboxylate exchange) was added before the glutamate and malate, in which case the distribution patterns at delta pH less than 0.4 were similar to those in nonrespiring mitochondria. In either case, however, after reaching a maximal value of 1.1, log [Pi]in/[Pi]out did not further increase with increasing delta pH. Thus, in normally metabolizing mitochondria, the distributions of Pi and malate are not directly correlated with the difference in pH across the membrane.     

Temperature dependence of the mitochondrial inner membrane anion channel: the relationship between temperature and inhibition by magnesium

The mitochondrial inner membrane anion channel (IMAC) carries a wide variety of anions and is postulated to be involved in mitochondrial volume homeostasis in conjunction with the K+/H+ antiporter, thus allowing the respiratory chain proton pumps to drive salt efflux. How it is regulated is uncertain; however, it is inhibited by matrix Mg2+ and matrix protons. Previously determined values for the IC50 suggested that the channel would be closed under physiological conditions. In a previous study (Liu, G., Hinch, B., Davatol-Hag, H., Lu, Y., Powers, M., and Beavis, A. D. (1996) J. Biol. Chem. 271, 19717-19723), it was demonstrated that the channel is highly temperature-dependent, and that a large component of this sensitivity resulted from an effect on the pIC50 for protons. We have now investigated the effect of temperature on the inhibition by Mg2+ and have found that it too is temperature-dependent. When the temperature is raised from 20 degrees C to 45 degrees C, the IC50 increases from 22 to 350 microm at pH 7.4 and from 80 to 1.5 mm at pH 8.4, respectively. The Arrhenius plot for the IC50 is linear with a slope = -80 kJ/mol. The IC50 is also strongly pH-dependent, and at 37 degrees C increases from 90 microm at pH 7.4 to 1230 microm at pH 8.4. In view of the extremely rapid fluxes that IMAC is capable of conducting at 37 degrees C, we conclude that inhibition by matrix Mg2+ and protons is necessary to limit its activity under physiological conditions. We conclude that the primary role of Mg2+ is to ensure IMAC is poised to allow regulation by small changes in pH in the physiological range. This control is mediated by a direct effect of H+ on the activity, in addition to an indirect effect mediated by a change in the Mg2+ IC50. The question that remains is not whether IMAC can be active at physiological concentrations of Mg2+ and H+, but what other factors might increase its sensitivity to changes in mitochondrial volume.     

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.     

New insights into mechanisms of anion uniport through the uncoupling protein of brown adipose tissue mitochondria.

GDP-sensitive Cl- uniport is a widely studied property of the uncoupling protein of brown adipose tissue mitochondria; nevertheless, little is known about its mechanism and there is even controversy over whether this protein transports Cl-. Using a fluorescent probe assay, we have demonstrated non-ohmic, electrophoretic, GDP-sensitive Cl- uniport into proteoliposomes reconstituted with purified uncoupler protein. We have also identified a large number of new anionic substrates for this porter that also inhibit Cl- uniport competitively. Anion transport, its inhibition by GDP and anion inhibition of Cl- uniport are all strongly dependent on anion hydrophobicity. These surprising results are consequential for hypotheses of common transport mechanisms in the gene family of mitochondrial anion porters.     

Anion/calcium ion ratios and proton production in some mitochondrial calcium ion uptakes.

The uptake of Ca2+ by liver mitochondria, when phosphate movement is inhibited, occurs when Co2 is present and not in its absence. Uptake of Ca2+ to form CaCO3 yields 2H+/Ca2+. Heart mitochondria, when phosphate movement is inhibited, will take up Ca2+ with the exact equivalent of hydroxybutyrate, lactate or acetate. By providing a carrier for Cl- with tributyltin, a stoicheiometric uptake of Cl- with the Ca2+ takes place. The uptakes appear to occur without significant pH change; there appears to be no CO2-dependent uptake into heart mitochondria. Oxygenation of anaerobic heart mitochondria, in the presence of an inhibitor of phosphate movement and of generation of phosphate from internal ATP, does not yield significant change of external acidity in relation to the amount of O2 added. Use of Bromothymol Blue as an indicator of the distribution of a weak acid anion confirms that the transient nature of the response of the dye distribution to Ca2+ is connected with movement of endogenous phosphate. Bromothymol Blue accumulated in response to Ca2+ is discharged when entry of the Ca2+ (in the presence of mersalyl) is mediated with nigericin. It is concluded that Ca2+ uptakes will occur alternatively with the equivalent of anions or in exchange for endogenous K+ and that proton production is connected with the changes of ionization of phosphate (unless phosphate movement is inhibited) and in liver mitochondria with the hydration of CO2.     

Oxaloacetate and malate transport by plant mitochondria

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10⁻³. From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport.     

Effects of Mg2+ and ATP on the phosphate transporter of sarcoplasmic reticulum.

The extra uptake of Ca2+ by vesicles of sarcoplasmic reticulum (SR) observed in the presence of Pi, attributable to transport of Pi by the Pi-transporter, has been studied. It has been shown that the Pi transporter is stimulated by ATP. Single channel conductance measurements have shown that the Cl- channel in the SR membrane is impermeable to Pi. It is suggested that the transporter could be an ion antiporter system. Studies of uptake as a function of pH and Mg2+ concentration suggest that transport of MgHPO4 and H2PO-4 are faster than transport of HPO2-4. For oxalate and pyrophosphate, Mg2+ binding inhibits transport. It is suggested that protonation of lysine residue(s) at the anion binding site increase the rate of transport.     

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.     

A new scheme of symbiosis: ligand- and voltage-gated anion channels in plants and animals.

Anion channels in the plasma membrane of both plant and animal cells participate in a number of important cellular functions such as volume regulation, trans-epithelial transport, stabilization of the membrane potential and excitability. Only very recently attention has turned to the presence of anion channels in higher plant cells. A dominant theme among recent discoveries is the role of Ca2+ in activating or modulating channel current involved in signal transduction. The major anion channel of stomatal guard cell protoplasts is a 32-40 pS channel which is highly selective for anions, in particular NO3-, Cl- and malate. These channels are characterized by a steep voltage dependence. Anion release is elicited upon depolarization and restricted to a narrow voltage span of -100 mV to the reversal potential of anions. During prolonged activation the current slowly inactivates. A rise in cytoplasmic calcium in the presence of nucleotides evokes activation of the anion channels. Following activation they catalyse anion currents 10-20 times higher than in the inactivated state thereby shifting the resting potential of the guard cell from a K(+)-conducting to an anion-conducting state. Patch-clamp studies have also revealed that growth hormones directly affect voltage-dependent activity of the anion channel in a dose-dependent manner. Auxin binding resulted in a shift of the activation potential towards the resting potential. Auxin-dependent gating of the anion channel is side- and hormone-specific. Its action is also channel-specific as K+ channels coexisting in the same membrane patch were insensitive to this ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

The permeability of the lysosomal membrane to small ions.

The permeability of the lysosomal membrane to small anions and cations was studied at 37 degrees C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K+ in the presence of valinomycin, and cation influx was coupled to an efflux of H+ using the protonophore 3-tert-butyl-5,2'-dichloro-4'-nitrosalicilylanilide. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases. The order of permeability of the lysosomal membrane to anions was found to be SCN- greater than I- greater than CH3COO- greater than Cl- approximately Pi greater than SO24- and that to cations Cs+ greater than K+ greater than Na+ greater than H+. These orders are largely in agreement with the lyotropic series of anions and cations. The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.