Modulation of inner mitochondrial membrane channel activity

Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM K Cl. The 107 pS activity is slightly anion selective and voltage dependent (open with matrix positive potentials). Multiple conductance channel (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca²⁺. MCC may be responsible for the Ca²⁺-induced permeability transition observed with mitochondrial suspensions. A low conductance channel (LCC) is activated by alkaline pH and inhibited by Mg²⁺. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed.     

Hypoxia increases BK channel activity in the inner mitochondrial membrane

To explore the potential function of the BK channel in the inner mitochondrial membrane under physiological and hypoxic conditions, we used on-mitoplast and whole-mitoplast patches. Single BK channels had a conductance of 276+/-9 pS under symmetrical K(+) solutions, were Ca(2+)- and voltage-dependent and were inhibited by 0.1 microM charybdotoxin. In response to hypoxia, BK increased open probability, shifted its reversal potential (9.3+/-2.4 mV) in the positive direction and did not change its conductance. We conclude that (1) the properties at rest of this mitoplast K(+) channel are similar to those of BK channels in the plasma membrane; (2) hypoxia induces an increase, rather than a decrease (as in the plasmalemma), in the open probability of this K(+) channel, leading to K(+) efflux from the mitochondrial matrix to the outside. We speculate that this increase in K(+) efflux from mitochondria into the cytosol is important during hypoxia in maintaining cytosolic K(+).     

Calcium ion activation of the anion-conducting channel in the rat liver mitochondrial inner membrane.

Stimulation of the rat liver mitochondrial inner-membrane anion-conducting channel by aeration is dependent on the concentration of Ca2+ ions in the assay medium. Ca2+ activates anion conduction in both aerated and non-energised mitochondria but acts over a wider concentration range and produces a greater increase in anion-conductivity in aerated mitochondria. EGTA reverses Ca2+ stimulation but takes several seconds to act, indicating slow release of Ca2+ from the activation site possibly on the matrix side of the inner mitochondrial membrane. It is suggested that this channel may respond to hormone-induced changes in cytosolic Ca2+ concentration.     

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.     

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.     

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 M for DIDS, H₂-DIDS, and SITS respectively and Hill coefficients 1. In contrast, inhibition of Cl^– transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC₅₀ which is severalfold higher than that for malonate. The IC₅₀ for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC₅₀ by about 50%, but under conditions where only the matrix pH is made alkaline the IC₅₀ is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other Cl^– channels than previously thought, but also suggest that other Cl^– channels may be sensitive to some of the many regulators of IMAC which have been identified.     

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+.     

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.     

Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation (V(1/2)) of 11.4±1.1mV and an effective gating charge z(d) of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.     

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

The ATP-sensitive K(+) (K(ATP)) channels in both sarcolemmal (sarcK(ATP)) and mitochondrial inner membrane (mitoK(ATP)) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK(ATP) contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoK(ATP) remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K(ATP) into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K(ATP) channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing K(ATP) channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K(ATP) channels in mitochondria was significantly increased by PMA, as shown by K(ATP)-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K(ATP) channels was blocked by a selective PKCepsilon inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K(ATP) channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCepsilon. PKC isoform-regulated mitochondrial import of K(ATP) channels may have significant implication in cardioprotection of IPC.     

Electrophysiology of the inner mitochondrial membrane

The application of electrophysiological techniques to mitochondrial membranes has allowed the observation and partial characterization of several ion channels, including an ATP-sensitive K⁺-selective one, a high-conductance megachannel, a 107 pS anionic channel and three others studied at alkaline pH's. A reliable correlation with the results of non-electrophysiological studies has been obtained so far only for the first two cases. Activities presumed to be associated with the Ca²⁺ uniporter and with the adenine nucleotide translocator, as well as the presence of various other conductances have also been reported. The review summarizes the main properties of these pores and their possible relationship to permeation pathways identified in biochemical studies.     

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.     

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.     

Dynamic modulation of mitochondrial membrane physical properties and ATPase activity by diet lipid.

A longitudinal cross-over feeding design was used to investigate the relationship of dietary lipid composition to the membrane lipid environment and activity of mitochondrial ATPase in vivo. Rats were fed a polyunsaturated fatty-acid-rich oil (soya-bean oil) for 12 days, crossed-over to a monounsaturated fatty-acid-rich oil (rapeseed oil) for the next 11 days, then returned to soya-bean oil for 11 more days. Additional rats were fed either soya-bean oil or rapeseed oil throughout. Rats fed rapeseed oil had lower rates of ATPase-catalysed ATP/[32P]Pi exchange than rats fed soya-bean oil. Arrhenius plots showed higher transition temperature (Tt) and activation energy (Ea) for rats fed rapeseed oil. Switching from soya-bean oil to rapeseed oil was dynamically followed by changes in the thermotropic and kinetic properties of the mitochondrial ATPase exchange reaction. Returning to soya-bean oil reversed these changes. The rapid and reversible modulation of Tt caused by a change of the type of fat ingested suggests that membrane physicochemical properties are not under rigid intrinsic control but are continually modified by the profile of exogenously derived fatty acids. The studies suggest that in vivo the activity of mitochondrial ATPase is in part determined by dietary lipid via its influence on the microenvironment of the enzyme. The rapidity and ready reversibility of changes observed for this subcellular-membrane-bound enzyme suggest that dietary fatty-acid balance may be an important determinant of other membrane functions in the body.     

Prooxidants open both the mitochondrial permeability transition pore and a low-conductance channel in the inner mitochondrial membrane

Mitochondria can be induced by a variety of agents/conditions to undergo a permeability transition (MPT), which nonselectively increases the permeability of the inner membrane (i.m.) to small (<1500 Da) solutes. Prooxidants are generally considered to trigger the MPT, but some investigators suggest instead that prooxidants open a Ca(2+)-selective channel in the inner mitochondrial membrane and that the opening of this channel, when coupled with Ca(2+) cycling mediated by the Ca(2+) uniporter, leads ultimately to the observed increase in mitochondrial permeability [see, e.g., Schlegel et al. (1992) Biochem. J. 285, 65]. S. A. Novgorodov and T. I. Gudz [J. Bioenerg. Biomembr. (1996) 28, 139] propose that the i.m. contains a pore that, upon exposure to prooxidants, can open to two states, one of which conducts only H(+) and one of which is the classic MPT pore. Given the current interest in increased mitochondrial permeability as a factor in apoptotic cell death, it is important to determine whether i.m. permeability is regulated in one or multiple ways and, in the latter event, to characterize each regulatory mechanism in detail. This study examined the effects of the prooxidants diamide and t-butylhydroperoxide (t-BuOOH) on the permeability of isolated rat liver mitochondria. Under the experimental conditions used, t-BuOOH induced mitochondrial swelling only in the presence of exogenous Ca(2+) (>2 microM), whereas diamide was effective in its absence. In the absence of exogenous inorganic phosphate (P(i)), (1) both prooxidants caused a collapse of the membrane potential (DeltaPsi) that preceded the onset of mitochondrial swelling; (2) cyclosporin A eliminated the swelling induced by diamide and dramatically slowed that elicited by t-BuOOH, without altering prooxidant-induced depolarization; (3) collapse of DeltaPsi was associated with Ca(2+) efflux but not with efflux of glutathione; (4) neither Ca(2+) efflux nor DeltaPsi collapse was sensitive to ruthenium red; (5) collapse of DeltaPsi was accompanied by an increase in matrix pH; no stimulation of respiration was observed; (6) Sr(2+) was able to substitute for Ca(2+) in supporting t-BuOOH-induced i.m. depolarization, but not swelling; (7) in addition to being insensitive to CsA, the collapse of DeltaPsi was also resistant to trifluoperazine, spermine, and Mg(2+), all of which block the MPT; and (8) DeltaPsi was restored (and its collapse was inhibited) upon addition of dithiothreitol, ADP, ATP or EGTA. We suggest that these results indicate that prooxidants open two channels in the i.m.: the classic MPT and a low-conductance channel with clearly distinct properties. Opening of the low-conductance channel requires sulfhydryl group oxidation and the presence of a divalent cation; both Ca(2+) and Sr(2+) are effective. The channel permits the passage of cations, including Ca(2+), but not of protons. It is insensitive to inhibitors of the classic MPT.     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.     

Bilirubin increases mitochondrial inner membrane conductance

Bilirubin accumulates within, and induces loose coupling in, rat liver mitochondria. This state, characterized by a normal protonmotive force, but increased oxygen consumption and inner membrane conductance, could impair cellular energy metabolism. Loose coupling is observed at bilirubin concentrations (12-24 microM) attained in tissues of kernicteric animals.     

Pathways shaping the mitochondrial inner membrane

Mitochondria are complex organelles with two membranes. Their architecture is determined by characteristic folds of the inner membrane, termed cristae. Recent studies in yeast and other organisms led to the identification of four major pathways that cooperate to shape cristae membranes. These include dimer formation of the mitochondrial ATP synthase, assembly of the mitochondrial contact site and cristae organizing system (MICOS), inner membrane remodelling by a dynamin-related GTPase (Mgm1/OPA1), and modulation of the mitochondrial lipid composition. In this review, we describe the function of the evolutionarily conserved machineries involved in mitochondrial cristae biogenesis with a focus on yeast and present current models to explain how their coordinated activities establish mitochondrial membrane architecture.