View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 584 (2010) 2085–2092 journal homepage: www.FEBSLetters.org 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, ClÀ/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 osteopetrosis 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 ClÀ ions across the membrane. defined based on structural or functional differences: ligand- These channels are selective for ClÀ 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 membrane potential, 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; ClÀ/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 ClÀ selective current in brown fat mito- in membranes that are accessible to the patch pipette. In mito- chondrial mitoplasts was observed. The flux of ClÀ 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 nucleotides, 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