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FEBS Letters 584 (2010) 2085–2092

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Review Mitochondrial chloride channels – What are they for?

Zuzana Tomaskova, Karol Ondrias *

Institute of Molecular Physiology and Genetics, Centre of Excellence for Cardiovascular Research, Slovak Academy of Sciences and Molecular Medicine Center, Slovak Academy of Sciences, 83334 Bratislava, Slovakia

article info abstract

Article history: This minireview focuses on observation of the properties, functional significance, and modulation of Received 16 November 2009 single chloride channels in the mitochondrial inner membrane using two electrophysiological Revised 11 January 2010 methods – the patch-clamp and bilayer lipid membrane methods. Measurements of parameters Accepted 19 January 2010 such as conductance, Cl/K+ selectivity, voltage or pH dependence as well as their modulation by Available online 26 January 2010 endogenous and exogenous compounds using individual mitochondrial chloride channels result Edited by Adam Szewczyk in an unexpectedly wide range of values. This paper discusses the origin of this wide variety of chan- nel parameters and the possible involvement of these channels in mitochondrial membrane poten- tial oscillations, apoptosis, carrier function, and mitochondrial fusion and fission. Keywords: Bilayer lipid membrane Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Chloride channel Mitochondria Patch-clamp Single channel property

1. Introduction myotonia, epilepsy, hyperekplexia, lysosomal storage disease, deafness, renal salt loss, kidney stones, and A chloride channel is a membrane component that, after [1,2,4–10]. Several major classes of chloride channels have been opening, allows the passing of Cl ions across the membrane. defined based on structural or functional differences: ligand- These channels are selective for Cl over cations and have chan- gated chloride channels, cystic fibrosis transmembrane conduc- nel gating properties. Chloride channels are found in the plas- tance regulators (CFTRs), the intracellular chloride channel malemma, endoplasmic reticulum, Golgi, mitochondria, (CLIC), voltage-gated chloride channels (ClCs) and calcium-acti- endosomes, lysosomes, nucleus, and cell vesicles [1–3]. They vated chloride channels [2,11–13]. regulate the movement of a major cellular anion, and thus reg- Excellent recent reviews about mitochondrial channels are ulate cellular and organellar , transport, fu- available [3,14–17]. In this paper, we focus exclusively on the sion, pH, cell cycle, cell volume, adhesion, and motility. This properties, modulation and functional significance of single chlo- functional diversity leads to the involvement of chloride chan- ride channels in the inner mitochondrial membrane (IMM) ob- nels in the regulation of blood pressure, apoptosis, free radical served using the patch-clamp and bilayer lipid membrane (BLM) release, reperfusion injury, and cardioprotection, in addition to methods. roles in various cellular pathologies [1,2,4–10]. Impaired chloride transport can cause a variety of diseases such as cystic fibrosis, 2. Electrophysiological methods

Two methods are commonly used to study the electrical prop- Abbreviations: BLM, voltage-clamp method using bilayer lipid membrane; ClC, erties of single mitochondrial chloride (mtCl) channels: the voltage-gated chloride channel; CLIC, intracellular chloride channel; DIDS, dihydro- patch-clamp method using a glass pipette and the voltage-clamp 4,40diisothiocyanostilbene-2,20-disulfonic acid; IMAC, inner mitochondrial mem- brane anion channel; IMM, inner mitochondrial membrane; mBzR, mitochondrial method using bilayer lipid membrane (BLM). The main advantage benzodiazepine receptor; mtCl channels, mitochondrial chloride channels; NPPB, 5- of the more commonly used patch-clamp method is the ability to nitro-2-(phenylpropylamino)-benzoate; phloretin, 20,40,60-trihydroxy-3-(4- study channels in their natural environment. The ‘‘attached” and hydroxyphenyl); Popen, channel open probability; PTP, permeability transition pore; ‘‘whole cell” modes are typically used, but the ‘‘excised-patch” ROS, reactive oxygen species; SITS, 4-acetamido-40-isothiocyanostilbene-2,20- mode is used as well. The ‘‘whole mitoplast” mode is particular disulfonic acid; DWm, mitochondrial membrane potential * Corresponding author. Fax: +421 2 54773666. useful, as it allows measurement of the integrated electrophysio- E-mail address: [email protected] (K. Ondrias). logical properties of the IMM under controlled conditions. In the

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.01.035 2086 Z. Tomaskova, K. Ondrias / FEBS Letters 584 (2010) 2085–2092 case of the IMM, patch-clamp experiments are typically performed 3.2. Single channel properties of channels from patched mitoplasts with mitoplasts obtained by swelling of the mitochondria, which removes the outer mitochondrial membrane. This provides the A large number of different anion-selective channels in the IMM advantage that a patched membrane may be visually observed. were observed in patched mitoplasts [36–43]. The first study of Since the membrane of the endoplasmic reticulum is in physical patched mitoplasts revealed an anion-selective 107 pS channel contact with mitochondria, it forms ‘‘mitochondria associated (150 mmol/l KCl; Cl/K+ = 4.5) that was not pH-dependent (pH membranes” [18,19] that may be difficult to eliminate completely. 6.2–9.0) [36]. A channel of 350 pS was also observed. For this reason, care must be taken to be certain that the IMM is The most studied channel is the anion-selective 108 pS channel, patched. Fluorescently tagged mitochondrial proteins and genetic which has been proposed to correspond to IMAC [42,43]. Using techniques may be useful for this purpose [20,21]. The major dis- symmetrical 150 mmol/l KCl and voltage ramps of ±40 mV at pH advantage of this method is that it can only study ion channels 7.2, a voltage-dependent Cl selective current in brown fat mito- in membranes that are accessible to the patch pipette. In mito- chondrial mitoplasts was observed. The flux of Cl into the mitop- chondria, the small, tubular crista junctions may not be accessible lasts further increased at positive voltage (with respect to the to the patch pipette [22–24]. mitoplast lumen). Contrary to the first report of Sorgato et al. In the BLM method, purified membrane vesicles are incorpo- [36], the channel was pH-dependent; at low pH the channel had rated into BLM. One advantage of this method is that one may a low open probability (Popen), whereas at alkaline pH the channel study channels from intracellular membranes that are not easily switched to long openings. Activity of this channel was not ob- accessible using the patch-clamp technique, such as intracellular served in solutions containing 1 mmol/l Mg2+. The properties of organelles, Golgi, lysosomes, endosomes, granules or mitochon- the 108 pS channel matched the flux properties of IMAC observed drial crista junctions. Such channels are incorporated into BLM by swelling assays [28], therefore, it was proposed that the 108 pS with their associated proteins and lipids, approximating the natu- channel corresponded to IMAC. The same 108 pS channel was re- ral membrane environment. This method is well-suited to the ported by Schönfeld et al. [43] in patched mitoplasts from liver study of modulation of purified channels by associated proteins, mitochondria using symmetric 150 mmol/l KCl in the presence of such as kinases or phosphatases, or lipids. In this case, the purified 0.1 lmol/l CaCl2. The channel activity was pH-dependent. Klitsch channel is incorporated into BLMs with different lipid or protein and Siemen [37] observed an anion-selective 108 pS channel in compositions. Since the molecular identities of the mtCL channels patched mitoplasts from rat brown adipose tissue in symmetric are unknown, with the exception of CLIC4 [25], this approach has 150 mmol/l KCl. The channel was partially inhibited in a reversible not yet been used. A significant disadvantage of BLM method is manner by purine , including ATP and ADP. the potential for damage of the channels during isolation. Purity Several other anion channels with different conductances have of the vesicle preparation is critical because vesicles from different also been reported. Kinnally et al., using symmetrical 150 mmol/l membranes may have different probabilities of fusion with the KCl and 0.1 lmol/l free Ca2+, observed 100 pS anion channels in BLM. Therefore, any minor contaminants with high fusion proba- heart mitochondria mitoplasts [39]. These channels were blocked bility may cause artifactual channel data. The same problem can by ligands of the mitochondrial benzodiazepine receptor (mBzR). occur when patched proteoliposomes are used. To avoid this prob- Other multiconductance channel(s) with conductances of lem, the best approach is to use a preparation of membrane vesi- 830 pS and 1.1 nS (estimated from the article figures) were also cles lacking contamination from other membranes. This is in observed. Several different channels were reported by [38] in liver many cases not possible. As an alternative, one may investigate IMM patches (symmetrical 150 mmol/l KCl and 0.1 lmol/l free the changes of the proportion of different channels in BLM at dif- Ca2+): multiconductance channels of 550 pS or 1 nS and volt- ferent stages of purification by following a specific biochemical age-dependent 92–118 pS channels. Several channels had irregular marker of the membrane under study, which increases as the puri- current fluctuations. A 15 pS anion-selective channel displayed fication proceeds while markers of the contaminating membranes many properties of IMAC (i.e. activation by matrix alkalisation decrease [26]. Use of partially purified proteins and fractions or and other properties) [44]. Channels of 220 pS (0.5 mol/l KCl) and crude membrane vesicles is unsuitable for BLM methods when 45 pS (150 mmol/l KCl) were observed at pH 8.2, but not at pH the single channel properties and pharmacological modulation of 6.8, having a selectivity of Cl/K+ = 2.2–3 [40]. Ballarin and Sorgato the channels of interest are unknown. However, using recombinant reported a 45 pS (in symmetrical 150 mmol/l KCl and 100 lmol/l proteins may be useful for the comparison of single channel prop- CaCl2) anion-selective channel in yeast IMM, which was inhibited erties of the purified fraction with and without the recombinant by ATP but insensitive to 2 mM Mg2+ [41]. The channel had a channel. Cl/K+ ratio = 3.3, and it was marginally voltage sensitive within a range of ±50 mV. Recently, De Marchi et al. characterised a ‘‘maxi” mtCl channel in 3. mtCl channels – a survey the inner membrane of colon tumour cell line mitochondria [21] (re- viewed in [17]). The channel was voltage-dependent with a conduc- 3.1. Inner mitochondrial membrane anion channel (IMAC) tance of 400 pS in symmetrical 150 mmol/l KCl, which is almost half the conductance of the permeability transition pore (PTP). It Studies of mitochondrial swelling revealed cation and anion was inhibited by several compounds including dihydro-4,40diisoth- movements into the mitochondrial matrix and provided experi- iocyanostilbene-2,20-disulfonic acid (DIDS), 4-acetamido-40-isothi- mental evidence that an IMAC was active under certain conditions ocyanostilbene-2,20-disulfonic acid (SITS), and by ATP and Mg2+ at [27–29]. IMAC fluxes of wide variety of anions ranging from small, low pH. It was proposed to be a component of the PTP. The perme- singly charged ions such as Cl , to multiply charged anions such as ability Cl/K+ ratio of this channel ranged from 7 to 18. This ‘‘maxi” citrate, superoxide and ATP, were observed [28,30,31]. These fluxes mtCl channel comigrated with the 107 pS channel. were regulated by voltage, temperature, and various compounds, 2+ and these fluxes were inhibited by matrix Mg and reduced ma- 3.3. Single channel properties observed in voltage-clamped trix pH (pH < 7.2) [28,29,32,33]. Using pharmacological and elec- proteoliposomes and vesicles in BLM trophysiological approaches, IMAC was found to contain specific 2+ binding sites for dihydropyridine (DHP) Ca antagonists unrelated Several different anion-selective channels purified as isolated 2+ to the L-type Ca channel [34,35]. mitochondrial proteins or derived from vesicles were observed in Z. Tomaskova, K. Ondrias / FEBS Letters 584 (2010) 2085–2092 2087 voltage-clamped proteoliposomes and vesicles incorporated in tissue mitochondria [56] or the pro-apoptotic Bax were reported BLM. Mitochondrial membrane contact sites isolated from rat brain to form channels selective for chloride [57]. A functionally active mitochondria were incorporated into liposomes. Several channels phosphate carrier protein was purified from yeast mitochondria, with conductances ranging from 5 pS to 1 nS, including 107 pS, reconstituted into giant liposomes and used for patch-clamp were observed in the patch-clamped proteoliposomes in symmet- experiments. In 100 mmol/l KCl, it had conductances of 40 pS rical 150 mmol/l KCl. Activity of these channels was voltage inde- and 25 pS (without and with divalent cations, respectively; Ca2+, pendent within the studied voltage range of ±70 mV [45]. Ion Mg2+) and a strong Cl selectivity [55]. Uncoupling protein (UCP) channels from sheep cardiac mitoplasts incorporated into BLM dis- from brown adipose tissue, reconstituted into giant liposomes played multisubstate anion channel conductances: 60 pS in 300/ and studied using the patch-clamp method, displayed stable 50 mmol/l choline chloride, 100 pS in symmetric 150 mmol/l KCl, 75 pS (or 2 75 pS) chloride channels (Cl/K+ 17) in symmetri- and a lower-conductance anion channel (25 or 50 pS under cal 100 mmol/l KCl and 2 mmol/l MgCl2 [56]. It was closed at high similar conditions) [46].Cl/choline+ and Cl/K+ permeabilities positive potentials on the matrix side of UCP and was reversibly were 12 and 8, respectively. The channels were not obviously inhibited by GTP, GDP, ATP, and ADP as well as blocked by DIDS. voltage-dependent, and they were unaffected by 0.5 mM SITS, Alteration of pH and the divalent ions Ca2+ and Mg2+ did not influ- 2 mM ATP, Ca2+,Mg2+ or pH (5.5–8.8) [46]. A two-substate anion ence the channel. Isolated pro-apoptotic Bax, incorporated into channel (40 pS or 85 pS in symmetric 300 or 550 mmol/l cho- BLM and studied at ±85 mV, formed channels selective for chloride line chloride, respectively) displayed Ca2+ and Mg2+ sensitive recti- (Cl/K+ = 3.3) with a minimal channel conductance of 22 pS [57]. fication [47]. Conductances of chloride channels in BLM The channel currents had at least three subconductance levels (symmetrical 450 mmol/l KCl) from rat skeletal muscle and brain and stable Bax channel had a conductance of 329 pS at pH 7.0. were 155 ± 5 pS and 120 ± 16 pS, respectively [48]. The channels were blocked by the chloride channel inhibitors SITS and DIDS 3.4. High variation of the single mtCl channel properties

[48]. A bell-shaped dependence of Popen on voltage (60 mV to +100 mV) was observed for chloride channels under asymmetrical A surprising feature of the observed mtCl channels is the broad (250/50 mmol/l KCl) and symmetrical conditions (150 mmol/l KCl, range of their single channel parameters; the presence of substates, 92 ± 14 pS). Voltages above +100 mV and below 60 mV substan- conductance ranging from 2 pS to 1 nS, variable and rather poor tially inhibited all observed anion channels. The channel was Cl/K+ permeability ratios 1.4–18 and different modulation by inhibited by DIDS from one side of the BLM only [49]. voltage, pH, Mg2+, ATP, ADP, and other endogenous and exogenous We studied mtCl channels derived from submitochondrial par- compounds. It may be assumed that the mtCl channels belong to at ticles (SMP) prepared from crude isolated mitochondria and from least three different categories of proteins: (1) the proteins that mitochondria purified through a Percoll gradient in BLM [26,50]. incidentally exhibit chloride channel properties under certain cir- The crude isolated mitochondria were contaminated with lyso- cumstances, their major physiological functions may deal with dif- somal membranes and possibly with peroxisomes [51]; addition- ferent ions, but were developed naturally for a completely distinct ally, about 10–20 times more chloride channels were observed in purpose; (2) the proteins with a main, alternative physiological BLM compared to the membrane vesicles isolated from the Per- function and an auxiliary function as a chloride channel. Some of coll-purified mitochondria [26]. No significant differences between mitochondrial carriers may belong to this category; and (3) the the single channel properties and effects of compounds (see below) proteins whose main physiological function is to serve as ‘‘real” on the channels derived from crude isolated mitochondria and Per- chloride channels. This category comprises the already annotated coll-purified mitochondria were found [26,50]. Using a 250/ genes with chloride channel properties, such as CLIC4 [25,54]. 50 mmol/l KCl gradient, the conductance and Cl/K+ permeability However, it is puzzling that the molecular identity of most of the ratio of the chloride channels varied without distinct substates mtCl channels remains unknown while the majority of plasma from 62 pS to 220 pS (mostly from 90 pS to 150 pS) and from 1.8 membrane channels have been characterised to the level of gene to 17.0, respectively. The Popen of the channels at 0 mV was greater annotation. We speculate that this is because of low channel con- than 0.6. The effect of compounds on the channels was side depen- centrations in the IMM and the possible formation of heterooligo- dent. Bongkrekic acid (BKA), atractyloside (ATR), carboxyatractylo- meric complexes having chloride permeability, which may be lost side (CAT) and ‘‘H2S” inhibited the channels from the trans side, after protein separation. whereas 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), DIDS, To classify the mtCl channels according to their single channel 20,40,60-trihydroxy-3-(4-hydroxyphenyl (phloretin) and pH 5.8 properties that may result from their molecular identity, as ob- inhibited most of the channels from the opposite cis side served by patch-clamp and BLM methods, we propose separating [26,50,52]. them into three main groups (Fig. 1): The first intracellular chloride channel identified, known as either mtCLIC or CLIC4 (p64H1, or p64 homologue 1), was localised (1) Regular/classical mtCl channels (Fig. 1A), defined as chan- to the mitochondria [25]. Isolated CLIC4 formed redox-regulated nels having stable baseline, classical stable and constant 31 pS and 57 pS channels in ‘‘tip-dip” bilayers (140 mmol/l KCl, opening and closing chloride current levels. These channels 1.5 mmol/l Mg2+, pH 6.0) [53]. Singh and Ashley [54] observed may be observed in several previously published figures poorly selective, redox-regulated CLIC4 (p64H1) ion channels [26,37,41–43,50,54,57]. incorporated in BLM. In 500/50 mmol/l TrisCl solutions (pH 7.4) (2) Ragged mtCl channels (Fig. 1B), defined as the highly fluctu- the channels had a conductance of 2.6 pS and the selectivity Cl/ ating channels with a stable baseline, but lacking stable and Tris+ = 1.8 under reducing conditions. Under oxidising and reduc- constant opening and chloride current levels. These channels ing conditions and in 500/50 mmol/l KCl solutions, the channels may be observed in the figures reported in other studies had a conductance of 8.9 pS (Cl/K+ = 1.4) and 14.8 pS (Cl/ [40,47]. K+ = 1.5), respectively. These channels displayed several substates. (3) Promiscuous mtCl channels or channel complexes (Fig. 1C), A truncated version of CLIC4 formed non-selective channels with a defined as channels that suddenly switch between Cl and reduced conductance, but they retained their redox sensitivity K+ permeability with time or under different physiological [54]. or pathological conditions (voltage, pH or oxidation status) Some mitochondrial transport proteins, such as the phosphate [58]. These channels may be observed in previously pub- carrier protein [55], the uncoupling protein from brown adipose lished figures [17,58–60]. 2088 Z. Tomaskova, K. Ondrias / FEBS Letters 584 (2010) 2085–2092

IMAC or mtCl channels were made at nearly zero potential, conditions where anions move inward [32]. Nearly all single channel studies were performed at potential differences lower than physiological, mostly in the range of ±50 mV and mostly in non-physiological solutions. Due to the insta- bility of BLM and patches at high voltage amplitudes, no sin- gle channel study has been performed at voltages between 140 mV and 220 mV. Exceptions are phosphate carrier, studied at voltage ramps of ±180 mV [55] and mtCl channels studied in BLM at approximately ±100 mV [49]. So far, mtCl single channel behaviour and activity have yet to be studied under the mitochondrial physiological conditions. The phos- phate carrier mtCl channel was not closed at ±180 mV [55], but mtCl channels were closed at >±100 mV [49]. Only some of the reported mtCl channels were voltage-dependent, and

Popen decreased or channels were closed at high negative potentials [21,49,56]. We may assume that mtCl channels are probably closed at the physiological membrane potential of the mitochondria (approximately 140 mV to 220 mV). (3) The high diversity of the mtCl channel parameters might be explained if some, or even the majority, of the different observed mtCl channels are from the family of mitochon- drial carriers. Mitochondria of eukaryotes typically contain between 35 and 55 different carriers [62]. Some of them were reported to form chloride channels after reconstitution into artificial bilayers [55,56,63]. If we suppose that most or all of them allow chloride to pass under certain circum- stances, this may explain the wide variability of the observed single channel properties. Both homodimers and threefold symmetric complexes of mitochondrial carriers in IMM were reported [62,64–67], and these features might be connected with the observed channel substates. The crys- tal structure of a human soluble chloride intracellular CLIC4, the only one which was also localised in the mitochondria [25], showed the channel to be a homotri- mer [68]. Mitochondrial proteins that are ready to pass chlo- ride as part of different mitochondrial functions, such as pro- apoptotic Bax [57], may contribute to channel diversity. (4) The observed chloride channels may form oligomeric struc- tures having different subunits, as was suggested for CLIC4 [54], voltage-dependent anion channel (VDAC) [69] or Bax [70]. (5) The observed single channel substates and several levels of Fig. 1. Representative traces of the single channel chloride current of the regular single channel currents may also be the result of cooperative (A) and the ragged (B) chloride channels. The lines on the left mark the closed state of the channels (asymmetric 250/50 mmol/l KCl, voltage = 0 mV). (C) Voltage coupling between several proteins or several mono- or het- dependence of a promiscuous channel current at asymmetric 250/50 mmol/l KCl. ero-subunits, as observed for calcium release channels [71,72]. Dashed lines indicate channel conductances. The channel had a Cl/K+ permeability ratio of 2.5 and a conductance of 712 pS at the voltages from 8 mV to +16 mV, and The molecular identities of mtCl channels remain for the most + the channel had a K /Cl permeability ratio of 4.9 and a conductance of 430 pS at part unknown. Only two chloride channels have been reported to voltages from 40 mV to 38 mV, from 16 mV to +4 mV and from +16 mV to be present in mitochondria. The first intracellular chloride channel +40 mV. It had a K+/Cl permeability ratio of 1.8 and a conductance of 145 pS at voltages from 32 mV to 22 mV (Fig. 1C with the permission from Gen. Physiol. identified, known as either mtCLIC or CLIC4 (p64H1, or p64 homo- Biophys. 2008 [56]). logue 1), was localised to the mitochondria [25]. It belongs to a new class of intracellular anion channels (CLIC). The mtCLIC chan- Why have so many different mtCl channels been reported? Gi- nel may translocate to the nucleus in response to cell stress [73], ven our current state of knowledge, we speculate on a few of the and it could be involved in mitochondrial membrane potential possibilities. generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into the mito- (1) We may suppose that not all observed ‘‘mitochondrial” chlo- chondria [74]. A pro-apoptotic role for mtCLIC in p53-mediated ride channels were derived from mitochondrial membranes, apoptosis was suggested [75]. The second chloride channel belongs and that some of them could be damaged during a sample to the ClC family, ClC-Nt. It was isolated from tobacco plants and preparation or measurement. There is also a possibility that has been proposed as a candidate for IMAC [76]. one might not distinguish between Cl and K+ channels using symmetrical KCl. 4. So many different mtCl channels – what are they for? (2) The reported physiological IMM potential is in the range of 140 mV to 220 mV [3,61]. Respiring mitochondria at The number of positive and negative charges in the mitochon- these negative potentials will tend to drive anions outward drial matrix should be very close-to-electroneutrality. This condi- through mtCl channels. However, most measurements on tion is based on the fact that any minute deviation from Z. Tomaskova, K. Ondrias / FEBS Letters 584 (2010) 2085–2092 2089 electroneutrality would result in an enormous change in the elec- mitochondrial chloride concentrations of 78 mmol/l and suggested tric field [77]. Modelling the mitochondrion as a sphere of 1 lm that lactotroph cells have a Cl mitochondrial store, from which diameter, one may calculate the amount of the electrical charge Cl was released upon stimulus. Using electron probe microanaly- (a deviation from electroneutrality) responsible for the 140 mV sis, Akar et al. reported a mitochondrial Cl concentration of membrane potential. Taking the membrane capacity of 1 lF/cm2, 76 mmol/kg dry weight (5.3 mmol/l) in left atrial appendage to produce a mitochondrial charge of 140 mV, only 2.74 10+4 cells, which was about 15% lower than in cytosol [83]. elementary charges (e.g. Cl ions) are needed, i.e. 4.57 1020 mol. After a Cl channel opens, Cl ions pass through the channel A current of 1 pA for 1 s transfers 6.242 10+6 charges through the according to the electrochemical driving force to reach equilib- mitochondrial membrane. This current transfers 227 times more rium. However, since there is no report so far of any active Cl charges per second (e.g. Cl ions) than are required to discharge transporter or Cl/X exchanger in mitochondrial membranes, it is the membrane. This implies that to discharge the membrane of not fully understood how the Cl ions are returned to the original one average mitochondrion, it is sufficient to have only one chan- Cl distribution, other than the possibility of using changes of elec- nel in IMM producing 1 pA current open for 4.4 ms. These numbers trochemical potential again. are significantly lower if mitochondrial subcompartments are con- More than half of the ClC chloride channel family members are sidered. If one takes sphere diameters of a single mitochondrial not channels, but rather secondary active catalysing cristae sack and crista junction of 200 and 50 nm, respectively, the stoichiometric exchange of two chloride ions for one proton and assumes that they form separate compartments [78], the [84,85]. In eukaryotes, the ClC Cl/H+ exchangers were found in amount of the electrical charge responsible for the 140 mV mem- internal membranes such as lysosomes, endosomes, and vacuoles brane potential is only 1098 (Cl or H+ ions; 1.829 1021 mol) [12,86,87]. However, there is no report (so far) of a Cl/H+ exchan- and 68 (Cl or H+ ions; 1.132 1022 mol), respectively. Therefore, ger found in mitochondria. to discharge a membrane having 140 mV potential, it is sufficient to have one channel in the membrane producing 1 pA current open 4.2. mtCl channels and mitochondrial carriers for 176 ls in the cristae sack and 11 ls in the crista junction. From these calculations, it follows that electrogenic transporters or As was mentioned in Section 3.4, the mitochondria of eukary- exchangers may rapidly set up and change membrane potentials otes typically contain between 35 and 55 different carriers [62]. in such small mitochondrial compartments. Some of these were reported to form chloride channels Considering the conductance of the observed mtCl channels [55,56,63]. Since the carriers form a well-defined family [88],we (10–400 pS in 150 mmol/l KCl) and other potassium and calcium speculate that some or most of them have chloride channel prop- channels found in the inner mitochondrial membranes [3,15–17], erties. However, the functional significance of their chloride chan- a question arises. Why do we have so many channels in the inner nel properties remains unresolved. mitochondrial membrane when the membrane is generally imper- meable to ions other than H+ and the electrochemical gradient 4.3. mtCl channels and mitochondrial membrane potential oscillations across the membrane is necessary as the driving force for the pro- duction of ATP from ADP? The mitochondrial membrane potential (DWm) and the passage of ions to form, maintain and modulate electrochemical gradients is 4.1. Mitochondrial ultrastructure and Cl concentration a key factor regulating mitochondrial activity. Mitochondria can

transport charged molecules and ions by utilising DWm. Changes The shapes of isolated mitochondria are now considered to be in DWm, repetitive, transient depolarisations of a single mitochon- sections of mitochondrial tubules, and it has been proposed that drion, have been observed in several types of cells [89–93]. It has a single mitochondrion consisting of a continuous mitochondrial been suggested that the transient depolarisations are triggered by reticulum exists in healthy, intact cells [78,79]. Mitochondria form Ca2+ uptake into mitochondria, Ca2+-induced opening of PTP, reac- a functional reticulum that is surrounded by two membranes: the tive oxygen species (ROS)-induced opening of IMAC and/or pH-sen- outer membrane and the inner membrane. The inner membrane is sitive and proton conductive channels [31,89–91,93–96]. A role for supposed to be composed of two subcompartments, the crista IMAC and mtCl channels in DWm oscillations is supported by obser- membrane and the inner boundary membrane [24,80]. These vations that mBzR ligands and DIDS, which are potent inhibitors of membrane domains are connected by rather small tubular struc- IMAC and mtCl channels, inhibited mitochondrial DWm oscillations tures, termed crista junctions, which are suggested to form sepa- while an mBzR agonist induced them [26,90,97]. rate compartments. They have been proposed to regulate the Ca2+ uptake and/or opening of the H+ channel and so dissipating dynamic distribution of proteins, lipids and soluble metabolites be- the mitochondrial membrane potential will change the electro- tween individual mitochondrial subcompartments [22–24,81]. chemical potential for Cl, making mtCl channels important for Whether there is electrical separation under different physiological the regulation of membrane potential. Since several mtCl channels or pathological conditions, membrane potentials, Cl concentra- were voltage-dependent and closed at high negative potential tions and mtCl channel distributions in these proposed subcom- [21,49,56], we may assume that mtCl channels play a role mostly partments remains unknown. during changes of mitochondrial potential. During the depolarisa- To assess the significance of mtCl channels in the regulation of tion phase, cations pass from the intermembrane space to the ma- Cl ion flux through IMM or membranes of mitochondrial subcom- trix and/or anions pass from the matrix. The opposite occurs during partments, one needs to consider changes of electrochemical po- the repolarisation phase. Considering the observed changes of tential across the membranes after the mtCl channel opens. This DWm and that the number of cations and anions in the matrix or depends on the concentrations of Cl ions in the matrix, the inter- intermembrane space must be close-to-electroneutrality, we may membrane space, the supposed crista junctions and other subcom- assume that Cl ions may be used as a counterion to maintain partments. The concentrations of Cl ions and other anions in these the close-to-electroneutrality conditions. compartments are unknown. The intracellular concentrations of Cl ions are in the range of 5–20 mmol/l. Using fluorescence tech- 4.4. mtCl channels and apoptosis nique, Garcia et al. reported a mean free intracellular chloride con- centration of 59.4 mmol/l in cultured rat lactotroph cells [82]. There are several indications that chloride channels play some Using radiolabeled chloride (36Cl), they observed intracellular and role in apoptosis. Ion channels in both the sarcolemmal and mito- 2090 Z. Tomaskova, K. Ondrias / FEBS Letters 584 (2010) 2085–2092 chondrial membranes have been implicated in the signal transduc- a target for pharmacological exploitation and drug development, tion cascades that regulate apoptosis [98]. A distinct role for chlo- and thus the mtCl channels are a target as well. Knowing mtCl ride in the intrinsic mitochondrial apoptotic pathway has been channel molecular identities, their remain to be suggested [99]. Electrophysiological studies indicate that the acti- discovered. The challenges are as broad and complex as the mito- vation of plasmalemmal chloride channels is associated with the chondria themselves. apoptotic process in many cell types [100,101]. This was supported by observations that chloride channel blockers inhibited apoptosis, Conflict of interest including ROS-dependent apoptosis [6,102]. The chloride channel blockers DIDS, NPPB and phloretin inhibited both H O -induced 2 2 There is no conflict of interest. apoptosis of cardiomyocytes and the activities of intracellular chlo- ride channels derived from mitochondrial and lysosomal mem- Acknowledgement branes [26,48,49]. Further evidence of a role for mtCl channels in apoptosis include a suggestion of a pro-apoptotic role of the Financial support by Slovak Science Grant Agency VEGA 2/ mtCLIC in p53-mediated apoptosis [73,75] and the formation of a 0150/10 is gratefully acknowledged. Cl selective channel (Cl/K+ = 3) by pro-apoptotic Bax protein in BLM. On the other hand, an antiapoptotic Bcl-2 protein formed References channels that were selective for K+ (K+/Cl = 3.9) [57]. It was ob- served that IMAC is superoxide permeable, and it is responsible [1] Nilius, B. and Droogmans, G. (2003) Amazing chloride channels: an overview. for the extrusion of superoxide anions from the mitochondrial ma- Acta Physiol. 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