Biochemical and Biophysical Research Communications 397 (2010) 614–620

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Biochemical and Biophysical Research Communications

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Identification of a voltage-gated potassium channel in gerbilhippocampal mitochondria

Piotr Bednarczyk a,c,1,*, Joanna E. Kowalczyk b,1, Małgorzata Beresewicz b, Krzysztof Dołowy a,Adam Szewczyk c, Barbara Zabłocka b

a Department of Biophysics, Warsaw University of Life Sciences-SGGW, 159 Nowoursynowska St., 02-776 Warsaw, Polandb Molecular Biology Unit, Mossakowski Medical Research Centre, 5 Pawinskiego St., 02-106 Warsaw, Polandc Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history:Received 31 May 2010Available online 4 June 2010

Keywords:MitochondriaPotassium channelMargatoxinGerbil hippocampus

0006-291X/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.bbrc.2010.06.011

* Corresponding author at: Nencki Institute of ExpSt., 02-093 Warsaw, Poland. Fax: +48 22 8225342.

E-mail address: p.bednarczyk@nencki.gov.pl (P. Be1 These authors contributed equally to this work.

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neu-ronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mech-anism of this effect remains unclear. In this study, the single-channel activity was measured using thepatch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potas-sium-selective current with the properties of a voltage-gated Kv-type potassium channel was recordedwith mean conductance 109 ± 6 pS in a symmetrical solution. The channel was blocked at negative volt-ages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg2+ complex and Ca2+

ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potas-sium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrastto surface membrane channels, the mitochondrial voltage-gated potassium channel could have a differ-ent molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondriaand immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein inmitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassiumchannel that can influence the function of mitochondria in physiological and pathological conditionsand that has properties similar to the surface membrane Kv1.3 channel.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Mitochondria are central to the brain’s cellular response to anischemia–reperfusion insult, playing critical roles in ATP and reac-tive oxygen species (ROS) synthesis. Mitochondrial potassiumchannels are believed to contribute to the cytoprotection of injuredcardiac and neuronal tissues [1]. Several potassium channels havebeen described in the inner mitochondrial membrane: the ATP-regulated potassium channel (mitoKATP channel) [2], the large-con-ductance Ca2+-regulated potassium channel (mitoBKCa channel)[3], the intermediate-conductance Ca2+-regulated potassium chan-nel (mitoIKCa channel) [4], the voltage-gated potassium channel(mitoKv channel) [5] and the twin-pore potassium channels (mito-TASK-3 channel) [6]. A basic functional role of these channels is toallow K+ influx into the mitochondrial matrix. This phenomenoncould be involved in the mitochondrial matrix volume and mem-

ll rights reserved.

erimental Biology, 3 Pasteur

dnarczyk).

brane potential changes [7,8]. The potassium channel openers ofthe mitoBKCa channels modulate the synthesis of ROS in brainmitochondria [9]. Depending on the degree of ischemic insult,ROS are believed to be mediators of signal transduction in ischemicpreconditioning and protection [10].

The shaker-related subfamily of rat voltage-gated potassiumchannels (Kv channels) encodes delayed rectifier and rapidly inac-tivating A-type potassium channels [11]. The differential expres-sion of the specialized Kv channel subtypes in the nervoussystem reflects a wide range of functions. Immunoprecipitationsof homogenate identified a putative tetramer of Kv1.3/1.4/1.1/1.2in human CNS grey matter [12]. In rats, a differential subcellularsubunit distribution was observed in the hippocampus, withKv1.3 immunoreactivity localized in CA3 pyramidal cell dendritesand/or in the mossy fiber terminal field and in cerebellar Purkinjecells [13]. The Kv1.3 is primarily expressed in T lymphocytes, but itis also present in the kidneys [14], the epithelium [15] and the CNS[16]. In T lymphocytes, it has been shown that Kv1.3 channels playa crucial role in proliferation and volume regulation [17]. Accord-ingly, dysfunction of Kv channels causes various neuronal, immuneand cardiac disorders [18].

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The margatoxin-sensitive potassium channel Kv1.3 was identi-fied in the inner mitochondrial membrane of T lymphocytes [6]. Bio-physical, biochemical, pharmacological and genetic data haveconfirmed the functional expression of the Kv1.3 channel in lym-phocyte mitochondria. It was shown that the Kv1.3 channel is pres-ent both in the plasma and mitochondrial membranes despite a lackof the N-terminal mitochondrial targeting sequence. The mitochon-drial Kv1.3 channel probably represents an important factor inapoptotic signal transduction. It has also been shown that Bax medi-ates cytochrome c release and mitochondrial depolarization, at leastin part, via its interaction with the mitoKv1.3 channel [19].

Here, using the patch-clamp technique, we identified voltage-gated potassium channels in neuronal mitochondria. Western blotsand immunohistochemistry confirmed the expression of the Kv1.3protein in mitochondria isolated from gerbil hippocampus. Ourfindings indicate that voltage-gated potassium channels (mi-toKv1.3 channel) with properties similar to the surface membraneKv1.3 channel are present in gerbil hippocampal mitochondria.

2. Materials and methods

2.1. Sample preparation and isolation of mitochondria

Mongolian gerbils (Meriones unguiculatus) were obtained fromthe Animal House of the Mossakowski MRC. This study was per-formed in accordance with the guidelines of the Local Commissionfor the Ethics of Experiments on Animals.

All procedures were carried out at 4 �C in isotonic buffer (IB;15 mM Tris/HCl at pH = 7.5, 0.25 mM sucrose, 1 mM MgCl2, 1 mMEGTA, 2 mM EDTA, 1 mM PMSF and 1 mM DTT). Mitochondria wereisolated from the hippocampi of gerbils as described previously [20].Brain tissue was obtained by decapitation under anesthesia and wasimmediately homogenized in IB (10% w/v) followed by centrifugation(3000g, 10 min, 4 �C). The supernatant was centrifuged (11,000g,20 min, 4 �C) to obtain a crude mitochondrial fraction (P2). The puremitochondrial pellet was obtained after centrifugation of P2(100,000g, 30 min, 4 �C) with 12% Ficoll and was prepared for westernblot and patch-clamp analyses. The protein concentration was deter-mined by using a Modified Lowry Protein Assay Kit (Pierce, USA).

2.2. Electrophysiology

Patch-clamp experiments on mitoplasts were performed as de-scribed previously [21,3]. Mitoplasts were prepared from a sampleof gerbil mitochondria put into a hypotonic solution (pH = 7.2;5 mM HEPES, 100 lM CaCl2) for about 1 min to induce swellingand breakage of the outer membrane. Then, the addition of a hyper-tonic solution (pH = 7.2; 750 mM KCl, 30 mM HEPES, 100 lM CaCl2)restored the isotonicity of the medium. The patch-clamp pipette wasfilled by an isotonic solution at pH = 7.2 containing 150 mM KCl,10 mM HEPES, and 100 lM CaCl2. The isotonic solution was usedas a control solution for all presented data. The solution containingthe substance to be tested was added from the back of the patch-clamp pipette. Test solutions were pumped by a peristaltic pump-driven capillary-pipe system. The low-calcium solution (1 lMCa2+) at pH = 7.2 contained the following: 150 mM KCl, 10 mMHEPES, 1 mM EGTA and 0.752 mM CaCl2. The gradient solutionwas composed of isotonic solution at pH = 7.2 with the followingadded: 50 mM KCl, 10 mM HEPES and 100 lM CaCl2. Margatoxin,agitoxin-2 and the ATP/Mg2+ complex were added as dilutions in iso-tonic solution.

2.3. Data analysis

The currents were low-pass filtered at 1 kHz and sampled at afrequency of 100 kHz. Recordings were made in mitoplast-at-

tached single-channel mode. The pipettes made of borosilicateglass had a resistance of 10–20 MX and were pulled by a Flam-ing/Brown Puller.

The channel recording illustrations are representative of themost frequently observed conductances for the given condition.The conductance was calculated from the current–voltage relation-ship. The probability of an open channel was determined using thesingle-channel search mode of the Clampfit 10 software. Data fromthe experiments are reported as the mean value ± standard devia-tion (S.D.). ‘‘-‘‘ indicates the closed state of the channel.

2.4. SDS–PAGE and Western blot analysis

Samples containing 30 lg of protein were separated by 10%SDS–PAGE and transferred onto Hybond C Extra membranes(Amersham). The membranes were exposed to monoclonal (Alo-mone) or polyclonal (Santa Cruz Biotechnology) antibodies thatrecognize Kv1.3 protein. Specificity of the bands was confirmedby specific peptides blocking the antibodies prior to the WB. Theenhanced signal from the mitochondria-specific proteins in mito-chondria vs. crude membranes enriched in mitochondria (P2)was shown using anti-ANT and anti-VDAC (Santa Cruz Biotechnol-ogy). The purity of the mitochondrial samples was verified usingpolyclonal anti-cadherin (Abcam), monoclonal anti-PSD95 (AffinityBioReagents) and anti-InsP3R (Affinity BioReagents). Blots weredeveloped using the appropriate anti-mouse, anti-goat or anti-rab-bit antibody coupled to horseradish peroxidase (Sigma–Aldrich) inconjunction with an ECL (Amersham).

2.5. Immunohistochemistry

A double-labeling immunofluorescence were performed onfree-floating gerbil brain sections. Animals were transcardially per-fused under deep anesthesia. Next, brains were fixed in 4% parafor-maldehyde at 4 �C for 3 h, stored in 20% sucrose at 4 �C overnightand frozen at �70 �C. Cryosections (40 lm) were washed withPBS and blocked with 5% BSA in PBS containing 0.25% Triton X-100 for 60 min. The sections were incubated overnight at 4 �C witha mixture of antibodies (goat polyclonal anti-Kv1.3, 1:75, SantaCruz; mouse monoclonal anti-cytochrome oxidase subunit IV,1:200, Invitrogen) in 2% BSA in PBS with 0.25% Triton X-100. Afterwashing, the sections were incubated with a secondary antibodies[goat anti-mouse IgG2a with Alexa Fluor 488 (1:500, Invitrogen)and donkey anti-goat with Cy3 (1:800, Jackson ImmunoResearch)]for 60 min. Nuclei were stained for 15 min with Hoechst 33258(Sigma). The control staining procedure was performed withoutthe primary antibodies. Sections were analyzed by confocalmicroscopy.

3. Results

3.1. Biophysical properties of the mitochondrial voltage-gatedpotassium channel

In patch-clamp experiments with gerbil hippocampal mitop-lasts, the current characteristics for the voltage-gated potassiumchannel were observed. The single-channel current traces were re-corded at different voltages in symmetrical isotonic solutions(Fig. 1A). Fig. 1B shows the current–voltage relationship for a singlechannel opening, at different voltages, under symmetrical and gra-dient conditions. Rectification of the current was not observed.The channel conductance in symmetrical isotonic solutions calcu-lated based on the current–voltage relationship was 109 ± 6 pS.The reversal potential measured in the gradient solution was�19 mV, which indicates that the examined channel was potas-sium-selective. The distribution of open channel probabilities was

Fig. 1. Biophysical properties of the mitoKv channel from gerbil hippocampal mitochondria. (A) Single-channel recording in symmetric 150/150 mM KCl isotonic solution atdifferent voltages. (B) Current–voltage characteristics of single-channel events in symmetric 150/150 mM KCl isotonic solution (solid line, j) and in gradient 50/150 mM KClsolution (dashed line, .). (C) Open channel probability for the mitochondrial voltage-gated potassium channel at different voltages (n = 3).

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also analyzed. In Fig. 1C, we show that the probability of an openchannel in a symmetrical isotonic solution was voltage dependent.The probability of channel opening increased from 0.5 at negativevoltage to 0.75 at positive voltage.

3.2. Pharmacology of the mitochondrial voltage-gated potassiumchannel

To exclude the possibility that the observed channel activitywas caused by another type of mitochondrial potassium channel,we used 1 mM ATP/Mg2+ complex and 1 lM Ca2+. Neither calciumnor the ATP/Mg2+ complex changed the channel activity. Theseobservations indicate that the channel is not a mitochondrialATP-regulated potassium channel nor is it a mitochondrial largeconductance Ca2+-regulated potassium channel (data not shown).

Substances known to regulate voltage-gated potassium channelactivity were also used to examine ion channel properties. Fig. 2Aillustrates the activity of the channel in the control condition andafter application of 10 nM margatoxin (MgTx), a very potent inhib-itor of the Kv1.3 channel. Margatoxin inhibited the channel activ-ity, and the effect was not reversible. The probability of channelopening decreased from 0.75 to 0.05 after the application of10 nM MgTx (Fig. 2B).

Agitoxin-2 was used as a second potent blocker of voltage-gatedpotassium channels of type Kv1.x. In our study, we observed thatAgTx-2 had no effect on channel activity (Fig. 2C). This result couldindicate that the mitochondrial voltage-gated potassium channel inthe gerbil hippocampus has different properties than those of surfacemembrane Kv1.x type channels. Taken together, our data indicatethat the observed single-channel activity is similar to the mitoKv1.3channel previously reported in lymphocyte mitochondria [6].

3.3. Kv1.3. protein is present in hippocampus mitochondria

Western blot (WB) analysis confirms the expression of Kv1.3 inhippocampal mitochondria. Two anti-Kv1.3 antibodies was used:

against a peptide mapping to near the C-terminus of humanKv1.3 (Santa Cruz) and specific for aa 211-224 of the N-terminus(Alomone Labs). As demonstrated by WB, the Kv1.3 protein wasdetected in the hippocampal mitochondria (mt) and in the mito-chondria-enriched fraction (P2). To confirm that the right bandwas identified, western blots were done using a peptide thatblocked the specific antibody reaction with the antigen, and nolabeling was observed. Both antibodies detected a protein of�65 kDa in the P2 and mitochondrial fractions; the P2 gave astronger WB signal because it contained other cell membranes(Fig. 3). The co-localization of goat polyclonal anti-Kv1.3 stainingand monoclonal anti-cytochrome oxidase subunit IV (COXIV) anti-body was observed in neurons in the cortex and hippocampus(Fig. 4, arrowheads). Other localization of Kv1.3 was also detectedin neurons. However, not all of the Kv1.3 immunofluorescence wasco-localized with COXIV labeling. We observed other membranestaining (Fig. 4, arrows) and a subset of cytoplasmic Kv1.3-positivepuncta that did not overlap with the mitochondrial pattern (Fig. 4,arrows). As seen in the overlay, a minor fraction of COXIV-immu-noreactive mitochondria also appeared to not be co-localized withthe Kv1.3 protein signal.

4. Discussion

Until now, two types of potassium channels had been describedin neuronal mitochondria: the ATP-regulated potassium channeland the large-conductance Ca2+-regulated potassium channel[22]. It has been proposed that increased potassium influx intobrain mitochondria, mediated by the mitoKATP channel, affectsthe mitochondrial membrane potential and mitochondrial respira-tion. In addition, diazoxide and RP66471 increased the mitochon-drial matrix volume and induced the release of cytochrome cfrom hippocampal mitochondria [8]. Using potassium flux in pro-teoliposomes and BODIPY-FL-glyburide green fluorescent probe,it has been found that the brain mitoKATP channel is regulated by

Fig. 2. The effects of margatoxin and agitoxin-2 on mitoKv channel activity. (A) Single-channel recordings in symmetric 150/150 mM KCl solution at 40 mV under the controlconditions, after the addition of 10 nM margatoxin (+MgTx) and after perfusion with control buffer without margatoxin (�MgTx). (B) Panel shows the open channelprobability for the mitoKv channel calculated from three independent experiments (n = 3). (C) Single-channel recordings in symmetric 150/150 mM KCl solution at 30 mV incontrol and AgTx-2 and MgTx conditions as a control for identification of the mitoKv channel.

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the same ligands that regulate mitoKATP channels in the heart andliver [23]. In 1999, it was shown that the inner mitochondrialmembrane from the human glioma cell line LN229 contains amitoBKCa channel. A study of the channel activity determined thatthe probability of an open channel increased with increasing cal-cium concentrations and decreased upon application of charybdo-toxin [3]. It was reported that calcium added to isolated rat brainmitochondria induced changes in mitochondrial membrane depo-larization and increase in mitochondrial respiration. These calciumeffects were blocked by iberiotoxin and charybdotoxin. Addition-ally, NS1619, a BKCa channel opener, induced potassium flux inbrain mitochondria similar to that induced by Ca2+. These findingssuggest the presence of a large-conductance Ca2+-regulated potas-sium channel in rat brain mitochondria, which was confirmed byreconstitution of the mitochondrial inner membrane into planar li-pid bilayers and by western blot analysis [24]. Interestingly, recentstudies using high-resolution immunofluorescence and immuno-electron microscopy provided evidence of the BKCa channel’s b4subunit on the inner membrane of neuronal mitochondria in therat brain and in cultured neurons [25]. All of these findings couldsupport the notion of a neuroprotective role of mitochondrialpotassium channels in the brain. In this paper, we showed forthe first time the presence of a new voltage-gated potassium chan-

nel of the mitoKv1.3 type in the inner mitochondrial membrane ofthe gerbil hippocampus.

The Kv channels represent a class of membrane proteins thatare activated by changes in electrical potential. These channelsare involved in a number of physiological processes including neu-ronal excitability, cell proliferation, smooth muscle contractionand apoptosis [26]. The Kv1.3 channel is a predominant memberof the voltage-gated potassium channel family that is expressedin the plasma membrane in human lymphocyte T-cells. Activationof this channel is a key event in proliferation [17], and early inac-tivation seems to be important in the initiation of apoptosis [27]. Inthe rat brain, distinct combinations of Kv1 alpha subunits are co-localized in different neurons, implying that differential expres-sion, assembly and subcellular targeting of these subunits maycontribute to Kv channel diversity; this leads to pre-synaptic andpost-synaptic membrane excitability [13]. Using co-immunopre-cipitation, a putative tetramer of low abundance Kv1.3 withKv1.4/1.1/1.2 was found in human CNS grey matter, indicating re-gional variations and functional specialization of Kv1 subunit com-plexes [12]. In gerbil cerebella, strong immunoreactivity for Kv1.3was observed in the Purkinje cell bodies [28].

The presence of the Kv1.3 channel in gerbil hippocampal mito-chondria was confirmed using two antibodies, which either recog-

Fig. 3. Kv1.3 protein is expressed in gerbil brain mitochondria. Representative immunoblots showing Kv1.3 in mitochondria isolated from gerbil hippocampus. The proteinwas detected using two polyclonal antibodies: (i) against a peptide mapping near the C-terminus of Kv1.3 and (ii) specific for aa 211-224 of the N-terminus of human Kv1.3.Additional WB was performed using a peptides blocking the antibody reaction with the antigen. The crude (P2) and pure mitochondria (mt) (30 lg) were immunoblottedwith anti-VDAC and anti-ANT to show the specificity for mitochondria. The purity of mitochondria was verified by WB using anti-cadherin, -PSD 95 and -InsP3R.Immunoblots are representative of at least three separate experiments.

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nize peptides near the C-terminus or are specific for aa 211-224 ofthe N-terminus of human plasma membrane Kv1.3, in two comple-mentary methods: western blotting and immunohistochemistry.Mitochondrial localization of the voltage-dependent potassiumchannel may be related to a pro- or anti-apoptotic function ofmitochondria. Previously, the mitoKv1.3 channel was reported tobe present in the mitochondrial inner membrane of T lymphocytecells. This channel, with a conductance of �17 pS, was detected inmitoplasts and inhibited by margatoxin, a selective inhibitor of theKv1.3 channel. This channel had properties similar to those of plas-ma membrane Kv1.3 channels, which are also present in T lympho-cytes [5]. Later, it was suggested that Bax mediates cytochrome crelease and mitochondrial depolarization in lymphocytes at leastin part via its interaction with the mitoKv1.3 [19].

Our studies, performed with patch-clamp and immunodetec-tions, reveal the functional presence of a mitoKv1.3 channel in ger-bil hippocampal mitochondria. This channel has the followingproperties:

– the conductance of the channel is �109 pS, and its reversalpotential is �19 mV, indicating that the examined channel ispotassium-selective;

– the open channel probability increases from 0.5 at negativevoltage to 0.75 at positive voltage;

– the channel is blocked by MgTx;– the channel is not inhibited by the ATP/Mg2+ complex and is not

regulated by Ca2+ ions;– the channel is not blocked by agitoxin-2, a potent inhibitor of

voltage-gated potassium channels.

This last observation suggests that the mitochondrial voltage-gated potassium channel could, in contrast to surface membranechannels, have a different molecular structure with no affinity forAgTx-2, possibly implying different function and/or modulation.Potassium channels discovered in mitochondria display a dualstructure, function and localization in the cell. Recent data suggestthat not only the plasma membrane Kv channel but also the mito-chondrial Kv1.3 channel contributes to the stimulation of pro-grammed cell death [29,30].

In olfactory bulb plasma membranes, the shaker ion channelKv1.3 was reported to be suppressed by an insulin receptor kinasevia tyrosine phosphorylation of critical N- and C-terminal residues[31]. It was reported that adaptor protein alteration of kinase-in-duced plasma membrane Kv1.3 channel modulation is related tothe degree of direct protein–protein association, and that the chan-nel itself can reciprocally modulate receptor-linked tyrosine kinaseexpression and activity [32]. In addition, phorbol esters suppressKv1.3 currents recorded in Xenopus oocytes expressing Kv1.3 pro-tein [33]. In line with the data showing the co-existence of PKC acti-vation, ROS production and mitochondrial potassium channelactivation in heart and neuron preconditioning [34,35], we assumedthat a massive, post-ischemic translocation of PKCbb to mitochon-dria in the ischemia-resistant sector of gerbil hippocampus [20]might be related to a yet-unknown endogenous mechanism of neu-roprotection that involves mitochondrial ion channels. However,our electrophysiological experiments, did not show changes inKv1.3 channel activity in the presence of recombinant PKCb1 protein(data not shown).

Fig. 4. (A) Triple-labeled immunofluorescence staining of gerbil hippocampus sections (CA3 region) with Kv1.3 channel subunit antibody (red), mitochondrial marker COXIV(green) and cell nuclei visualizer Hoechst 33258 (blue). Yellow indicates the co-localization of Kv1.3 and COXIV on the mitochondrial membrane. Arrows show Kv1.3localized on the plasma membrane or other membranes not containing mitochondrial marker COXIV, arrowheads point to the co-localization of Kv1.3 and COXIV on themitochondrial membrane. Similar close co-localizations were observed in the gerbil cortex. Results shown are representative of five independent experiments. (B–D)Magnification of the boxed area shown in a pointing to the detailed pattern of Kv1.3 channel subunit immunoreactivity (B) with COXIV (C). Closely co-localizedagglomerations in D (yellow).

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In summary, a new voltage-gated potassium channel, similar toa Kv1.3 channel, was found in the inner membrane of gerbil hippo-campal mitochondria. Immunocytochemical studies suggest thatKv1.3 subunit expression is restricted to a subpopulation of mito-chondria. The role of this protein in normal and especially in ische-mia-challenged brains remains to be elucidated.

Acknowledgments

This study was supported by the Nencki Institute of Experimen-tal Biology, a grant from the MSHE (P-N/31/2006) and MitoNet.pl.P.B. would like to acknowledge Prof. Detlef Siemen for teaching thepatch-clamp technique and for discussions during a postdoc sup-ported by EMBO (2006) and DAAD (2008).

References

[1] A. Szewczyk, W. Jarmuszkiewicz, W.S. Kunz, Mitochondrial potassiumchannels, IUBMB Life 61 (2009) 134–143.

[2] I. Inoue, H. Nagase, K. Kishi, T. Higuti, ATP-sensitive K+ channel in themitochondrial inner membrane, Nature 352 (1991) 244–247.

[3] D. Siemen, C. Loupatatzis, J. Borecky, E. Gulbins, F. Lang, Ca2+-activated Kchannel of the BK-type in the inner mitochondrial membrane of a humanglioma cell line, Biochem. Biophys. Res. Commun. 257 (1999) 549–554.

[4] U. De Marchi, N. Sassi, B. Fioretti, L. Catacuzzeno, G.M. Cereghetti, I. Szabo, M.Zoratti, Intermediate conductance Ca2+-activated potassium channel (KCa3.1)in the inner mitochondrial membrane of human colon cancer cells, CellCalcium 45 (2009) 509–516.

[5] I. Szabo, J. Bock, A. Jekle, M. Soddemann, C. Adams, F. Lang, M. Zoratti, E.Gulbins, A novel potassium channel in lymphocyte mitochondria, J. Biol. Chem.280 (2005) 12790–12798.

[6] Z. Rusznak, G. Bakondi, L. Kosztka, K. Pocsai, B. Dienes, J. Fodor, A. Telek, M.Gonczi, G. Szucs, L. Csernoch, Mitochondrial expression of the two-poredomain TASK-3 channels in malignantly transformed and non-malignanthuman cells, Virchows Arch. 452 (2008) 415–426.

[7] A.P. Halestrap, Regulation of mitochondrial metabolism through changes inmatrix volume, Biochem. Soc. Trans. 22 (1994) 522–529.

[8] G. Debska, R. May, A. Kicinska, A. Szewczyk, C.E. Elger, W.S. Kunz, Potassiumchannel openers depolarize hippocampal mitochondria, Brain Res. 892 (2001)42–50.

[9] B. Kulawiak, A.P. Kudin, A. Szewczyk, W.S. Kunz, BK channel openers inhibitROS production of isolated rat brain mitochondria, Exp. Neurol. 212 (2008)543–547.

[10] M.A. Perez-Pinzon, K.R. Dave, A.P. Raval, Role of reactive oxygen species andprotein kinase C in ischemic tolerance in the brain, Antioxid. Redox. Signal. 7(2005) 1150–1157.

[11] G.A. Gutman, K.G. Chandy, S. Grissmer, M. Lazdunski, D. McKinnon, L.A.Pardo, G.A. Robertson, B. Rudy, M.C. Sanguinetti, W. Stühmer, X. Wang,International Union of Pharmacology. LIII. Nomenclature and molecularrelationships of voltage-gated potassium channels, Pharmacol. Rev. 57(2005) 473–508.

[12] S.K. Coleman, J. Newcombe, J. Pryke, J.O. Dolly, Subunit composition of Kv1channels in human CNS, J. Neurochem. 73 (1999) 849–858.

[13] R.W. Veh, R. Lichtinghagen, S. Sewing, F. Wunder, I.M. Grumbach, O. Pongs,Immunohistochemical localization of five members of the Kv1 channelsubunits: contrasting subcellular locations and neuron-specific co-localizations in rat brain, Eur. J. Neurosci. 7 (1995) 2189–2205.

[14] X. Yao, A.Y. Chang, E.L. Boulpaep, A.S. Segal, G.V. Desir, Molecular cloning of aglibenclamide-sensitive, voltage-gated potassium channel expressed in rabbitkidney, J. Clin. Invest. 97 (1996) 2525–2533.

[15] M. Grunnet, H.B. Rasmussen, A. Hay-Schmidt, D.A. Klaerke, The voltage-gatedpotassium channel subunit, Kv1.3 is expressed in epithelia, Biochim. Biophys.Acta 1616 (2003) 85–94.

[16] C. Mourre, M.N. Chernova, M.F. Martin-Eauclaire, R. Bessone, G. Jacquet, M.Gola, S.L. Alper, M. Crest, Distribution in rat brain of binding sites of kaliotoxin,a blocker of Kv1.1 and Kv1.3 alpha-subunits, J. Pharmacol. Exp. Ther. 291(1999) 943–952.

620 P. Bednarczyk et al. / Biochemical and Biophysical Research Communications 397 (2010) 614–620

[17] K.G. Chandy, H. Wulff, C. Beeton, M. Pennington, G.A. Gutman, M.D. Cahalan, K+

channels as targets for specific immunomodulation, Trends Pharmacol. Sci. 25(2004) 280–289.

[18] C.C. Shieh, M. Coghlan, J.P. Sullivan, M. Gopalakrishnan, Potassium channels:molecular defects, diseases, and therapeutic opportunities, Pharmacol. Rev. 52(2000) 557–594.

[19] I. Szabo, J. Bock, H. Grassme, M. Soddemann, B. Wilker, F. Lang, M. Zoratti, E.Gulbins, Mitochondrial potassium channel Kv1.3 mediates Bax-inducedapoptosis in lymphocytes, Proc. Natl. Acad. Sci. USA 105 (2008) 14861–14866.

[20] J.E. Kowalczyk, M. Beresewicz, B. Gajkowska, B. Zablocka, Association ofprotein kinase C delta and phospholipid scramblase 3 in hippocampalmitochondria correlates with neuronal vulnerability to brain ischemia,Neurochem. Int. 55 (2009) 157–163.

[21] Y. Cheng, X.Q. Gu, P. Bednarczyk, F.R. Wiedemann, G.G. Haddad, D. Siemen,Hypoxia increases activity of the BK-channel in the inner mitochondrialmembrane and reduces activity of the permeability transition pore, CellPhysiol. Biochem. 22 (2008) 127–136.

[22] P. Bednarczyk, Potassium channels in brain mitochondria, Acta Biochim. Pol.56 (2009) 385–392.

[23] R. Bajgar, S. Seetharaman, A.J. Kowaltowski, K.D. Garlid, P. Paucek,Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain, J. Biol. Chem. 276 (2001) 33369–33374.

[24] J. Skalska, P. Bednarczyk, M. Piwonska, B. Kulawiak, G. Wilczynski, K. Dołowy,A.P. Kudin, W.S. Kunz, A. Szewczyk, Calcium ions regulate K+ uptake into brainmitochondria: the evidence for a novel potassium channel, Int. J. Mol. Sci. 10(2009) 1104–1120.

[25] M. Piwonska, E. Wilczek, A. Szewczyk, G.M. Wilczynski, Differentialdistribution of Ca2+-activated potassium channel beta4 subunit in rat bra:immunolocalization in neuronal mitochondria, Neuroscience 153 (2008) 446–460.

[26] C. Miller, An overview of the potassium channel family, Genome Biol. 1 (2000).REVIEWS0004.

[27] E. Gulbins, I. Szabo, K. Baltzer, F. Lang, Ceramide-induced inhibition of Tlymphocyte voltage-gated potassium channel is mediated by tyrosine kinases,Proc. Natl. Acad. Sci. USA 94 (1997) 7661–7666.

[28] Y.H. Chung, K.M. Joo, R.H. Nam, Y.S. Kim, W.B. Lee, C.I. Cha,Immunohistochemical study on the distribution of the voltage-gatedpotassium channels in the gerbil cerebellum, Neurosci. Lett. 374 (2005) 58–62.

[29] E. Gulbins, N. Sassi, H. Grassmè, M. Zoratti, I. Szabò, Role of Kv1.3mitochondrial potassium channel in apoptotic signalling in lymphocytes,Biochim. Biophys. Acta, doi: 10.1016/j.bbabio.2010.01.018.

[30] I. Szabò, M. Zoratti, E. Gulbins, Contribution of voltage-gated potassiumchannels to the regulation of apoptosis, FEBS Lett. 584 (2010) 2049–2056.

[31] D.R. Marks, K. Tucker, M.A. Cavallin, T.G. Mast, D.A. Fadool, Awake intranasalinsulin delivery modifies protein complexes and alters memory, anxiety, andolfactory behaviors, J. Neurosci. 29 (2009) 6734–6751.

[32] B.S. Colley, M.A. Cavallin, K. Biju, D.R. Marks, D.A. Fadool, Brain-derivedneurotrophic factor modulation of Kv1.3 channel is disregulated by adaptorproteins Grb10 and nShc, BMC Neurosci. 10 (2009) 8.

[33] J. Aiyar, S. Grissmer, K.G. Chandy, Full-length and truncated Kv1.3 K+ channelsare modulated by 5-HT1c receptor activation and independently by PKC, Am. J.Physiol. 265 (1993) C1571–C1578.

[34] G. Lebuffe, P.T. Schumacker, Z.H. Shao, T. Anderson, H. Iwase, T.L. Vanden Hoek,ROS and NO trigger early preconditioning: relationship to mitochondrial KATP

channel, Am. J. Physiol. Heart Circ. Physiol. 284 (2003) H299–H308.[35] D. Liu, C. Lu, R. Wan, W.W. Auyeung, M.P. Mattson, Activation of mitochondrial

ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocationand cytochrome c release, J. Cereb. Blood Flow Metab. 22 (2002) 431–443.