The activity of plant inner membrane anion channel (PIMAC) can be performed by a chloride channel (CLC) protein in mitochondria from seedlings of maize populations divergently selected for cold tolerance

The proteins performing the activity of the inner membrane anion channel (IMAC) and its plant counterpart (PIMAC) are still unknown. Lurin et al. (Biochem J 348: 291–295, 2000) indicated that a chloride channel (CLC) protein corresponds to PIMAC activity in tobacco seedling mitochondria. In this study, we investigated: (i) the presence of a CLC protein in maize seedling mitochondria; (ii) the involvement of this protein in plant cold tolerance; and (iii) its possible role in PIMAC activity. We validated the presence of a CLC protein (ZmCLCc) in maize mitochondria by immunoassay using a polyclonal antibody against its C-terminus. The differential expression of the ZmCLCc protein in mitochondria was measured in seedlings of maize populations divergently selected for cold tolerance and grown at different temperatures. The ZmCLCc protein level was higher in cold stressed than in non-stressed growing conditions. Moreover, the ZmCLCc level showed a direct relationship with the cold sensitivity level of the populations under both growing conditions, suggesting that selection for cold tolerance induced a constitutive change of the ZmCLCc protein amount in mitochondria. The anti-ZmCLCc antibody inhibited (about 60%) the channel-mediated anion translocations by PIMAC, whereas the same antibody did not affect the free diffusion of potassium thiocyanide through the inner mitochondrial membrane. For this reason, we conclude that the mitochondrial ZmCLCc protein can perform the PIMAC activity in maize seedlings.     

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.     

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 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.     

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.     

The giant channel of the inner mitochondrial membrane is inhibited by cyclosporin A.

In patch-clamp experiments on rat liver mitoplasts, cyclosporin A inhibited the activity of the recently described (Petronilli, V., Szabó, I., and Zoratti, M. (1989) FEBS Lett. 259, 137-143) 1.3-nanosiemens channel of the inner mitochondrial membrane at concentrations in the 10(-8)-10(-7) M range. The inhibitor acts when present on the matrix side of membrane. The Ca2(+)-dependent "permeability transition channel" of mitochondria is inhibited by cyclosporin A in the same concentration range. The results suggest therefore that the same pore is responsible for the permeabilization of the inner mitochondrial membrane and for the conduction of the high currents observed in electrophysiological experiments.     

The effect of antimycin A on mouse liver inner mitochondrial membrane channel activity.

In a patch-clamp study, we found antimycin A in low (1-2) microM concentrations decreased the open probability of the multiple conductance channel activity and the approximately 110 picosiemens channel of the inner mitochondrial membrane (for a review of mitochondrial channels see Kinnally, K. W., Antonenko, Yu. N., and Zorov, D. B. (1992) J. Bioenerg. Biomembr. 24, 99-110). Higher antimycin A concentrations (e.g. 10 microM) facilitated multiple conductance channel opening. These effects were reversible, and the binding site(s) are probably distinct from those responsible for the inhibition of the electron transport chain, since the latter are virtually irreversible. A model with two closed and two open states is presented for the approximately 110-picosiemens activity.     

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.     

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane

Background

Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.

Methods

A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out.

Results

TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization.

Conclusions

TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.

General significance

Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.     

The N-terminal cleavable extension of plant carrier proteins is responsible for efficient insertion into the inner mitochondrial membrane

A subset of mitochondrial carrier proteins from plants contain a cleavable N-terminal extension. We have used a reconstituted protein import assay system into intermembrane space-depleted mitochondria to study the role of the cleavable extension in the carrier import pathway. Insertion of carrier proteins into the inner membrane can be stimulated by the addition of a soluble intermembrane space fraction isolated from plant mitochondria. Greater stimulation of import of the adenine nucleotide carrier (ANT) and phosphate carrier (Pic), which contain N-terminal cleavable extensions, was observed compared to the import of the oxoglutarate malate carrier (OMT), which does not contain a cleavable extension. Removal of the N-terminal cleavable extension from ANT and Pic resulted in loss of stimulation of insertion into the inner membrane. Conversely, addition of the N-terminal extension from ANT or Pic to OMT resulted in significantly enhanced insertion into the inner membrane. The polytopic inner membrane proteins TIM17 and TIM23 that are imported via the carrier import pathway contain no cleavable extension, displayed high-level stimulation of insertion into the inner membrane by addition of the intermembrane space fraction. Addition of the N-terminal cleavable extension from carrier proteins to TIM23 enhanced insertion of TIM23 into the inner membrane even in the absence of the soluble intermembrane space fraction. Together, these results demonstrate that the cleavable N-terminal extensions present on carrier proteins from plants are required for efficient insertion into the inner mitochondrial membrane, and that they can stimulate insertion of any carrier-like protein into the inner membrane.     

An ABC transporter in the mitochondrial inner membrane is required for normal growth of yeast.

In an attempt to identify a mitochondrial ATP binding cassette (ABC) transporter, we have used the polymerase chain reaction to amplify 10 DNA fragments homologous to members of the ABC family from the yeast Saccharomyces cerevisiae. We disrupted five of the corresponding genes and found that one of the resulting null mutants barely grew on rich medium and failed to grow on minimal medium. This gene, termed ATM1, encodes a putative 'half-transporter' of 694 amino acids. Atm1p is synthesized with an N-terminal mitochondrial matrix-targeting signal and is located in the mitochondrial inner membrane, with its C-terminal ATPase domain exposed to the matrix. Cells lacking a functional ATM1 gene have an unstable mitochondrial genome and have white mitochondria that completely lack cytochromes. Atm1p is the first mitochondrial member of the ABC family to be identified and the only eukaryotic ABC transporter that has been shown to be necessary for normal cellular growth.     

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.     

Fusion of mitochondria in mammalian cells is dependent on the mitochondrial inner membrane potential and independent of microtubules or actin

Mitochondrial fusion is a poorly characterized process which has mainly been studied in yeast and Drosophila but is thought to occur in all eukaryotes. Until now, there was only indirect evidence to support such a process in mammalian cells. In this study, using a cell fusion system, we found that mitochondrial fusion occurs rapidly in mammalian cells and is completed in less than 24 h. We report that the fusion of mitochondria requires an intact mitochondrial inner membrane potential but is independent of a functional cytoskeleton.     

Identification and activity of superoxide-producing protein complexes of the plasma membrane of etiolated maize seedlings subjected to low positive temperature

Kinetics of superoxide anion generation by the isolated plasma membrane was determined by the rate of formazan formation from XTT in the presence of NADPH or NADH. The plasma membrane was prepared from (control) etiolated maize seedlings grown at 25°C and from (cooled) seedlings incubated at 6°C for the last day. Membrane vesicles from the control plants possessed superoxide-producing activity, and the rate of NADH oxidation was markedly higher than that of NADPH. The low-temperature incubation of the seedlings suppressed the NADPH-dependent activity, whereas the NADH-dependent one slightly increased. The solubilized by dodecyl maltoside (DDM) plasma membranes were separated into multiprotein complexes by high-resolution clear native electrophoresis (hrCN-PAGE). The aim was to find complexes exhibiting the superoxide-producing activity sensitive to inhibition by diphenylene iodonium. Several protein complexes from the plasma membrane capable of superoxide producion in the presence of NADPH or NADH were found. The maximum diphenylene iodonium-sensitive activity was found in the high-molecular weight complex, in which proteins reacting with antibodies against C-terminal peptide of phagocytic oxidase (gp91phox) were detectable. The activity of this complex was lower in the cooled than in the control seedlings and displayed higher affinity to NADPH than to NADH. To search for the cooling-induced changes in the polypeptide content of protein complexes, the two-dimensional difference gel electrophoresis (hrCN/SDS-PAGE) was used. Control and cooled samples, whose lysine had been labeled with fluorescent dyes Cy2 and Cy3, respectively, were separated by this method in one gel. Decrease in a temperature of plant growing affected the protein content of the complex so that some new proteins appeared and several polypeptides disappeared as compared with the control. There were no significant differences between the cooled and control counterparts in the content of proteins detectable with gp91phox antibodies. Therefore, the high-molecular complex containing NADPH oxidase looses proteins under low temperature that may decrease its superoxide-producing activity.     

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells

Patch–clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca²⁺-activated K⁺-selective channel K_Ca3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca²⁺] will cause mtK_Ca3.1 opening, thus linking inner membrane K⁺ permeability and transmembrane potential to Ca²⁺ signalling.     

Direct modulation of the outer mitochondrial membrane channel,voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.     

NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane studied using inside-out submitochondrial particles

Submitochondrial particles (SMP) were isolated from potato ( Solanum tuberosum L. cv. Bintje) tubers. The SMP were 91% inside-out and they were able to form a membrane potential, as monitored by oxonol VI, with succinate, NADH and NADPH. The pH dependence and kinetics of NADH and NADPH oxidation by these SMP was studied using three different electron acceptors – O₂, duroquinone and ferricyanide. In addition, the SMP were solubilized, fractionated by non-denaturing polyacrylamide gel electrophoresis, and the gels were stained for NAD(P)H dehydrogenase activity and specificity at different pH using Nitro Blue Tetrazolium. From the results we conclude that there are at least two distinct NAD(P)H dehydrogenases on the inner surface of the inner membrane: (1) Complex 1 which oxidizes NADH and deamino-NADH in a rotenone-sensitive manner, (O₂ as acceptor) with optimum activity at pH 8 and a very low K_m(NADH) of 3 μ M . It also oxidizes NADPH and deamino-NADPH in a rotenone-sensitive manner, but with a pH optimum at pH 5.8 and a very high K_m(NADPH) of more than 1 m M . This complex is found as a broad, diffuse band at the top of the gels. (2) A second dehydrogenase which oxidizes NADH in a rotenone-insensitive manner with optimum activity at pH 6.2 and a higher K_m(NADH) of 14 μ M . It also oxidizes NADPH in a rotenone-insensitive manner with an activity optimum at pH 6.8 and low K_m(NADPH) of 25 μ M . This dehydrogenase does not oxidize deamino-NAD(P)H. One of the sharp bands around the middle of the native gels may be caused by this dehydrogenase indicating that it has a relatively low molecular mass compared to Complex I. Several other NAD(P)H dehydrogenase bands were observed on the gels which we cannot yet assign.     

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.