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Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria
Biochemical and Biophysical Research Communications 397 (2010) 614–620
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Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria Piotr Bednarczyk a,c,1,*, Joanna E. Kowalczyk b,1, Małgorzata Bere˛sewicz b, Krzysztof Dołowy a, Adam Szewczyk c, Barbara Zabłocka b a b c
Department of Biophysics, Warsaw University of Life Sciences-SGGW, 159 Nowoursynowska St., 02-776 Warsaw, Poland ´ skiego St., 02-106 Warsaw, Poland Molecular Biology Unit, Mossakowski Medical Research Centre, 5 Pawin Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland
a r t i c l e
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Article history: Received 31 May 2010 Available online 4 June 2010 Keywords: Mitochondria Potassium channel Margatoxin Gerbil hippocampus
a b s t r a c t Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109 ± 6 pS in a symmetrical solution. The channel was blocked at negative voltages 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 potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Mitochondria are central to the brain’s cellular response to an ischemia–reperfusion insult, playing critical roles in ATP and reactive oxygen species (ROS) synthesis. Mitochondrial potassium channels are believed to contribute to the cytoprotection of injured cardiac and neuronal tissues [1]. Several potassium channels have been described in the inner mitochondrial membrane: the ATPregulated potassium channel (mitoKATP channel) [2], the large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) [3], the intermediate-conductance Ca2+-regulated potassium channel (mitoIKCa channel) [4], the voltage-gated potassium channel (mitoKv channel) [5] and the twin-pore potassium channels (mitoTASK-3 channel) [6]. A basic functional role of these channels is to allow K+ influx into the mitochondrial matrix. This phenomenon could be involved in the mitochondrial matrix volume and mem-
* Corresponding author at: Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland. Fax: +48 22 8225342. E-mail address: [email protected] (P. Bednarczyk). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.011
brane potential changes [7,8]. The potassium channel openers of the mitoBKCa channels modulate the synthesis of ROS in brain mitochondria [9]. Depending on the degree of ischemic insult, ROS are believed to be mediators of signal transduction in ischemic preconditioning and protection [10]. The shaker-related subfamily of rat voltage-gated potassium channels (Kv channels) encodes delayed rectifier and rapidly inactivating A-type potassium channels [11]. The differential expression of the specialized Kv channel subtypes in the nervous system reflects a wide range of functions. Immunoprecipitations of homogenate identified a putative tetramer of Kv1.3/1.4/1.1/1.2 in human CNS grey matter [12]. In rats, a differential subcellular subunit distribution was observed in the hippocampus, with Kv1.3 immunoreactivity localized in CA3 pyramidal cell dendrites and/or in the mossy fiber terminal field and in cerebellar Purkinje cells [13]. The Kv1.3 is primarily expressed in T lymphocytes, but it is also present in the kidneys [14], the epithelium [15] and the CNS [16]. In T lymphocytes, it has been shown that Kv1.3 channels play a crucial role in proliferation and volume regulation [17]. Accordingly, dysfunction of Kv channels causes various neuronal, immune and cardiac disorders [18].
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The margatoxin-sensitive potassium channel Kv1.3 was identified in the inner mitochondrial membrane of T lymphocytes [6]. Biophysical, biochemical, pharmacological and genetic data have confirmed the functional expression of the Kv1.3 channel in lymphocyte mitochondria. It was shown that the Kv1.3 channel is present both in the plasma and mitochondrial membranes despite a lack of the N-terminal mitochondrial targeting sequence. The mitochondrial Kv1.3 channel probably represents an important factor in apoptotic signal transduction. It has also been shown that Bax mediates cytochrome c release and mitochondrial depolarization, at least in part, via its interaction with the mitoKv1.3 channel [19]. Here, using the patch-clamp technique, we identified voltagegated potassium channels in neuronal mitochondria. Western blots and immunohistochemistry confirmed the expression of the Kv1.3 protein in mitochondria isolated from gerbil hippocampus. Our findings indicate that voltage-gated potassium channels (mitoKv1.3 channel) with properties similar to the surface membrane Kv1.3 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 from the Animal House of the Mossakowski MRC. This study was performed in accordance with the guidelines of the Local Commission for 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 mM EGTA, 2 mM EDTA, 1 mM PMSF and 1 mM DTT). Mitochondria were isolated from the hippocampi of gerbils as described previously [20]. Brain tissue was obtained by decapitation under anesthesia and was immediately 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 pure mitochondrial pellet was obtained after centrifugation of P2 (100,000g, 30 min, 4 °C) with 12% Ficoll and was prepared for western blot and patch-clamp analyses. The protein concentration was determined by using a Modified Lowry Protein Assay Kit (Pierce, USA). 2.2. Electrophysiology Patch-clamp experiments on mitoplasts were performed as described previously [21,3]. Mitoplasts were prepared from a sample of gerbil mitochondria put into a hypotonic solution (pH = 7.2; 5 mM HEPES, 100 lM CaCl2) for about 1 min to induce swelling and breakage of the outer membrane. Then, the addition of a hypertonic solution (pH = 7.2; 750 mM KCl, 30 mM HEPES, 100 lM CaCl2) restored the isotonicity of the medium. The patch-clamp pipette was filled by an isotonic solution at pH = 7.2 containing 150 mM KCl, 10 mM HEPES, and 100 lM CaCl2. The isotonic solution was used as a control solution for all presented data. The solution containing the substance to be tested was added from the back of the patchclamp pipette. Test solutions were pumped by a peristaltic pumpdriven capillary-pipe system. The low-calcium solution (1 lM Ca2+) at pH = 7.2 contained the following: 150 mM KCl, 10 mM HEPES, 1 mM EGTA and 0.752 mM CaCl2. The gradient solution was composed of isotonic solution at pH = 7.2 with the following added: 50 mM KCl, 10 mM HEPES and 100 lM CaCl2. Margatoxin, agitoxin-2 and the ATP/Mg2+ complex were added as dilutions in isotonic solution. 2.3. Data analysis The currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. Recordings were made in mitoplast-at-
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tached single-channel mode. The pipettes made of borosilicate glass had a resistance of 10–20 MX and were pulled by a Flaming/Brown Puller. The channel recording illustrations are representative of the most frequently observed conductances for the given condition. The conductance was calculated from the current–voltage relationship. The probability of an open channel was determined using the single-channel search mode of the Clampfit 10 software. Data from the experiments are reported as the mean value ± standard deviation (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 (Alomone) or polyclonal (Santa Cruz Biotechnology) antibodies that recognize Kv1.3 protein. Specificity of the bands was confirmed by specific peptides blocking the antibodies prior to the WB. The enhanced signal from the mitochondria-specific proteins in mitochondria vs. crude membranes enriched in mitochondria (P2) was shown using anti-ANT and anti-VDAC (Santa Cruz Biotechnology). The purity of the mitochondrial samples was verified using polyclonal anti-cadherin (Abcam), monoclonal anti-PSD95 (Affinity BioReagents) and anti-InsP3R (Affinity BioReagents). Blots were developed using the appropriate anti-mouse, anti-goat or anti-rabbit antibody coupled to horseradish peroxidase (Sigma–Aldrich) in conjunction with an ECL (Amersham). 2.5. Immunohistochemistry A double-labeling immunofluorescence were performed on free-floating gerbil brain sections. Animals were transcardially perfused under deep anesthesia. Next, brains were fixed in 4% paraformaldehyde at 4 °C for 3 h, stored in 20% sucrose at 4 °C overnight and frozen at 70 °C. Cryosections (40 lm) were washed with PBS and blocked with 5% BSA in PBS containing 0.25% Triton X100 for 60 min. The sections were incubated overnight at 4 °C with a mixture of antibodies (goat polyclonal anti-Kv1.3, 1:75, Santa Cruz; mouse monoclonal anti-cytochrome oxidase subunit IV, 1:200, Invitrogen) in 2% BSA in PBS with 0.25% Triton X-100. After washing, 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 without the primary antibodies. Sections were analyzed by confocal microscopy. 3. Results 3.1. Biophysical properties of the mitochondrial voltage-gated potassium channel In patch-clamp experiments with gerbil hippocampal mitoplasts, the current characteristics for the voltage-gated potassium channel were observed. The single-channel current traces were recorded at different voltages in symmetrical isotonic solutions (Fig. 1A). Fig. 1B shows the current–voltage relationship for a single channel opening, at different voltages, under symmetrical and gradient conditions. Rectification of the current was not observed. The channel conductance in symmetrical isotonic solutions calculated 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 potassium-selective. The distribution of open channel probabilities was
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Fig. 1. Biophysical properties of the mitoKv channel from gerbil hippocampal mitochondria. (A) Single-channel recording in symmetric 150/150 mM KCl isotonic solution at different 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 KCl solution (dashed line, .). (C) Open channel probability for the mitochondrial voltage-gated potassium channel at different voltages (n = 3).
also analyzed. In Fig. 1C, we show that the probability of an open channel in a symmetrical isotonic solution was voltage dependent. The probability of channel opening increased from 0.5 at negative voltage to 0.75 at positive voltage. 3.2. Pharmacology of the mitochondrial voltage-gated potassium channel To exclude the possibility that the observed channel activity was caused by another type of mitochondrial potassium channel, we used 1 mM ATP/Mg2+ complex and 1 lM Ca2+. Neither calcium nor the ATP/Mg2+ complex changed the channel activity. These observations indicate that the channel is not a mitochondrial ATP-regulated potassium channel nor is it a mitochondrial large conductance Ca2+-regulated potassium channel (data not shown). Substances known to regulate voltage-gated potassium channel activity were also used to examine ion channel properties. Fig. 2A illustrates the activity of the channel in the control condition and after application of 10 nM margatoxin (MgTx), a very potent inhibitor of the Kv1.3 channel. Margatoxin inhibited the channel activity, and the effect was not reversible. The probability of channel opening decreased from 0.75 to 0.05 after the application of 10 nM MgTx (Fig. 2B). Agitoxin-2 was used as a second potent blocker of voltage-gated potassium channels of type Kv1.x. In our study, we observed that AgTx-2 had no effect on channel activity (Fig. 2C). This result could indicate that the mitochondrial voltage-gated potassium channel in the gerbil hippocampus has different properties than those of surface membrane Kv1.x type channels. Taken together, our data indicate that the observed single-channel activity is similar to the mitoKv1.3 channel 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 in hippocampal mitochondria. Two anti-Kv1.3 antibodies was used:
against a peptide mapping to near the C-terminus of human Kv1.3 (Santa Cruz) and specific for aa 211-224 of the N-terminus (Alomone Labs). As demonstrated by WB, the Kv1.3 protein was detected in the hippocampal mitochondria (mt) and in the mitochondria-enriched fraction (P2). To confirm that the right band was identified, western blots were done using a peptide that blocked the specific antibody reaction with the antigen, and no labeling was observed. Both antibodies detected a protein of 65 kDa in the P2 and mitochondrial fractions; the P2 gave a stronger WB signal because it contained other cell membranes (Fig. 3). The co-localization of goat polyclonal anti-Kv1.3 staining and monoclonal anti-cytochrome oxidase subunit IV (COXIV) antibody was observed in neurons in the cortex and hippocampus (Fig. 4, arrowheads). Other localization of Kv1.3 was also detected in neurons. However, not all of the Kv1.3 immunofluorescence was co-localized with COXIV labeling. We observed other membrane staining (Fig. 4, arrows) and a subset of cytoplasmic Kv1.3-positive puncta that did not overlap with the mitochondrial pattern (Fig. 4, arrows). As seen in the overlay, a minor fraction of COXIV-immunoreactive mitochondria also appeared to not be co-localized with the Kv1.3 protein signal.
4. Discussion Until now, two types of potassium channels had been described in neuronal mitochondria: the ATP-regulated potassium channel and the large-conductance Ca2+-regulated potassium channel [22]. It has been proposed that increased potassium influx into brain mitochondria, mediated by the mitoKATP channel, affects the mitochondrial membrane potential and mitochondrial respiration. In addition, diazoxide and RP66471 increased the mitochondrial matrix volume and induced the release of cytochrome c from hippocampal mitochondria [8]. Using potassium flux in proteoliposomes and BODIPY-FL-glyburide green fluorescent probe, it has been found that the brain mitoKATP channel is regulated by
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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 control conditions, after the addition of 10 nM margatoxin (+MgTx) and after perfusion with control buffer without margatoxin ( MgTx). (B) Panel shows the open channel probability 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 in control and AgTx-2 and MgTx conditions as a control for identification of the mitoKv channel.
the same ligands that regulate mitoKATP channels in the heart and liver [23]. In 1999, it was shown that the inner mitochondrial membrane from the human glioma cell line LN229 contains a mitoBKCa channel. A study of the channel activity determined that the probability of an open channel increased with increasing calcium concentrations and decreased upon application of charybdotoxin [3]. It was reported that calcium added to isolated rat brain mitochondria induced changes in mitochondrial membrane depolarization and increase in mitochondrial respiration. These calcium effects were blocked by iberiotoxin and charybdotoxin. Additionally, NS1619, a BKCa channel opener, induced potassium flux in brain mitochondria similar to that induced by Ca2+. These findings suggest the presence of a large-conductance Ca2+-regulated potassium channel in rat brain mitochondria, which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers and by western blot analysis [24]. Interestingly, recent studies using high-resolution immunofluorescence and immunoelectron microscopy provided evidence of the BKCa channel’s b4 subunit on the inner membrane of neuronal mitochondria in the rat brain and in cultured neurons [25]. All of these findings could support the notion of a neuroprotective role of mitochondrial potassium channels in the brain. In this paper, we showed for the first time the presence of a new voltage-gated potassium chan-
nel of the mitoKv1.3 type in the inner mitochondrial membrane of the gerbil hippocampus. The Kv channels represent a class of membrane proteins that are activated by changes in electrical potential. These channels are involved in a number of physiological processes including neuronal excitability, cell proliferation, smooth muscle contraction and apoptosis [26]. The Kv1.3 channel is a predominant member of the voltage-gated potassium channel family that is expressed in the plasma membrane in human lymphocyte T-cells. Activation of this channel is a key event in proliferation [17], and early inactivation seems to be important in the initiation of apoptosis [27]. In the rat brain, distinct combinations of Kv1 alpha subunits are colocalized in different neurons, implying that differential expression, assembly and subcellular targeting of these subunits may contribute to Kv channel diversity; this leads to pre-synaptic and post-synaptic membrane excitability [13]. Using co-immunoprecipitation, a putative tetramer of low abundance Kv1.3 with Kv1.4/1.1/1.2 was found in human CNS grey matter, indicating regional variations and functional specialization of Kv1 subunit complexes [12]. In gerbil cerebella, strong immunoreactivity for Kv1.3 was observed in the Purkinje cell bodies [28]. The presence of the Kv1.3 channel in gerbil hippocampal mitochondria was confirmed using two antibodies, which either recog-
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Fig. 3. Kv1.3 protein is expressed in gerbil brain mitochondria. Representative immunoblots showing Kv1.3 in mitochondria isolated from gerbil hippocampus. The protein was 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 immunoblotted with 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.
nize peptides near the C-terminus or are specific for aa 211-224 of the N-terminus of human plasma membrane Kv1.3, in two complementary methods: western blotting and immunohistochemistry. Mitochondrial localization of the voltage-dependent potassium channel may be related to a pro- or anti-apoptotic function of mitochondria. Previously, the mitoKv1.3 channel was reported to be present in the mitochondrial inner membrane of T lymphocyte cells. This channel, with a conductance of 17 pS, was detected in mitoplasts and inhibited by margatoxin, a selective inhibitor of the Kv1.3 channel. This channel had properties similar to those of plasma membrane Kv1.3 channels, which are also present in T lymphocytes [5]. Later, it was suggested that Bax mediates cytochrome c release and mitochondrial depolarization in lymphocytes at least in part via its interaction with the mitoKv1.3 [19]. Our studies, performed with patch-clamp and immunodetections, reveal the functional presence of a mitoKv1.3 channel in gerbil hippocampal mitochondria. This channel has the following properties: – the conductance of the channel is 109 pS, and its reversal potential is 19 mV, indicating that the examined channel is potassium-selective; – the open channel probability increases from 0.5 at negative voltage 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 voltagegated potassium channel could, in contrast to surface membrane channels, have a different molecular structure with no affinity for AgTx-2, possibly implying different function and/or modulation. Potassium channels discovered in mitochondria display a dual structure, function and localization in the cell. Recent data suggest that not only the plasma membrane Kv channel but also the mitochondrial Kv1.3 channel contributes to the stimulation of programmed cell death [29,30]. In olfactory bulb plasma membranes, the shaker ion channel Kv1.3 was reported to be suppressed by an insulin receptor kinase via tyrosine phosphorylation of critical N- and C-terminal residues [31]. It was reported that adaptor protein alteration of kinase-induced plasma membrane Kv1.3 channel modulation is related to the degree of direct protein–protein association, and that the channel itself can reciprocally modulate receptor-linked tyrosine kinase expression and activity [32]. In addition, phorbol esters suppress Kv1.3 currents recorded in Xenopus oocytes expressing Kv1.3 protein [33]. In line with the data showing the co-existence of PKC activation, ROS production and mitochondrial potassium channel activation in heart and neuron preconditioning [34,35], we assumed that a massive, post-ischemic translocation of PKCbb to mitochondria in the ischemia-resistant sector of gerbil hippocampus [20] might be related to a yet-unknown endogenous mechanism of neuroprotection that involves mitochondrial ion channels. However, our electrophysiological experiments, did not show changes in Kv1.3 channel activity in the presence of recombinant PKCb1 protein (data not shown).
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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 Kv1.3 localized on the plasma membrane or other membranes not containing mitochondrial marker COXIV, arrowheads point to the co-localization of Kv1.3 and COXIV on the mitochondrial 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-localized agglomerations in D (yellow).
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Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria Piotr Bednarczyk a,c,1,*, Joanna E. Kowalczyk b,1, Małgorzata Bere˛sewicz b, Krzysztof Dołowy a, Adam Szewczyk c, Barbara Zabłocka b a b c
Department of Biophysics, Warsaw University of Life Sciences-SGGW, 159 Nowoursynowska St., 02-776 Warsaw, Poland ´ skiego St., 02-106 Warsaw, Poland Molecular Biology Unit, Mossakowski Medical Research Centre, 5 Pawin 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
Article history: Received 31 May 2010 Available online 4 June 2010 Keywords: Mitochondria Potassium channel Margatoxin Gerbil hippocampus
a b s t r a c t Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109 ± 6 pS in a symmetrical solution. The channel was blocked at negative voltages 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 potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Mitochondria are central to the brain’s cellular response to an ischemia–reperfusion insult, playing critical roles in ATP and reactive oxygen species (ROS) synthesis. Mitochondrial potassium channels are believed to contribute to the cytoprotection of injured cardiac and neuronal tissues [1]. Several potassium channels have been described in the inner mitochondrial membrane: the ATPregulated potassium channel (mitoKATP channel) [2], the large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) [3], the intermediate-conductance Ca2+-regulated potassium channel (mitoIKCa channel) [4], the voltage-gated potassium channel (mitoKv channel) [5] and the twin-pore potassium channels (mitoTASK-3 channel) [6]. A basic functional role of these channels is to allow K+ influx into the mitochondrial matrix. This phenomenon could be involved in the mitochondrial matrix volume and mem-
* Corresponding author at: Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland. Fax: +48 22 8225342. E-mail address: [email protected] (P. Bednarczyk). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.011
brane potential changes [7,8]. The potassium channel openers of the mitoBKCa channels modulate the synthesis of ROS in brain mitochondria [9]. Depending on the degree of ischemic insult, ROS are believed to be mediators of signal transduction in ischemic preconditioning and protection [10]. The shaker-related subfamily of rat voltage-gated potassium channels (Kv channels) encodes delayed rectifier and rapidly inactivating A-type potassium channels [11]. The differential expression of the specialized Kv channel subtypes in the nervous system reflects a wide range of functions. Immunoprecipitations of homogenate identified a putative tetramer of Kv1.3/1.4/1.1/1.2 in human CNS grey matter [12]. In rats, a differential subcellular subunit distribution was observed in the hippocampus, with Kv1.3 immunoreactivity localized in CA3 pyramidal cell dendrites and/or in the mossy fiber terminal field and in cerebellar Purkinje cells [13]. The Kv1.3 is primarily expressed in T lymphocytes, but it is also present in the kidneys [14], the epithelium [15] and the CNS [16]. In T lymphocytes, it has been shown that Kv1.3 channels play a crucial role in proliferation and volume regulation [17]. Accordingly, dysfunction of Kv channels causes various neuronal, immune and cardiac disorders [18].
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The margatoxin-sensitive potassium channel Kv1.3 was identified in the inner mitochondrial membrane of T lymphocytes [6]. Biophysical, biochemical, pharmacological and genetic data have confirmed the functional expression of the Kv1.3 channel in lymphocyte mitochondria. It was shown that the Kv1.3 channel is present both in the plasma and mitochondrial membranes despite a lack of the N-terminal mitochondrial targeting sequence. The mitochondrial Kv1.3 channel probably represents an important factor in apoptotic signal transduction. It has also been shown that Bax mediates cytochrome c release and mitochondrial depolarization, at least in part, via its interaction with the mitoKv1.3 channel [19]. Here, using the patch-clamp technique, we identified voltagegated potassium channels in neuronal mitochondria. Western blots and immunohistochemistry confirmed the expression of the Kv1.3 protein in mitochondria isolated from gerbil hippocampus. Our findings indicate that voltage-gated potassium channels (mitoKv1.3 channel) with properties similar to the surface membrane Kv1.3 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 from the Animal House of the Mossakowski MRC. This study was performed in accordance with the guidelines of the Local Commission for 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 mM EGTA, 2 mM EDTA, 1 mM PMSF and 1 mM DTT). Mitochondria were isolated from the hippocampi of gerbils as described previously [20]. Brain tissue was obtained by decapitation under anesthesia and was immediately 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 pure mitochondrial pellet was obtained after centrifugation of P2 (100,000g, 30 min, 4 °C) with 12% Ficoll and was prepared for western blot and patch-clamp analyses. The protein concentration was determined by using a Modified Lowry Protein Assay Kit (Pierce, USA). 2.2. Electrophysiology Patch-clamp experiments on mitoplasts were performed as described previously [21,3]. Mitoplasts were prepared from a sample of gerbil mitochondria put into a hypotonic solution (pH = 7.2; 5 mM HEPES, 100 lM CaCl2) for about 1 min to induce swelling and breakage of the outer membrane. Then, the addition of a hypertonic solution (pH = 7.2; 750 mM KCl, 30 mM HEPES, 100 lM CaCl2) restored the isotonicity of the medium. The patch-clamp pipette was filled by an isotonic solution at pH = 7.2 containing 150 mM KCl, 10 mM HEPES, and 100 lM CaCl2. The isotonic solution was used as a control solution for all presented data. The solution containing the substance to be tested was added from the back of the patchclamp pipette. Test solutions were pumped by a peristaltic pumpdriven capillary-pipe system. The low-calcium solution (1 lM Ca2+) at pH = 7.2 contained the following: 150 mM KCl, 10 mM HEPES, 1 mM EGTA and 0.752 mM CaCl2. The gradient solution was composed of isotonic solution at pH = 7.2 with the following added: 50 mM KCl, 10 mM HEPES and 100 lM CaCl2. Margatoxin, agitoxin-2 and the ATP/Mg2+ complex were added as dilutions in isotonic solution. 2.3. Data analysis The currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. Recordings were made in mitoplast-at-
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tached single-channel mode. The pipettes made of borosilicate glass had a resistance of 10–20 MX and were pulled by a Flaming/Brown Puller. The channel recording illustrations are representative of the most frequently observed conductances for the given condition. The conductance was calculated from the current–voltage relationship. The probability of an open channel was determined using the single-channel search mode of the Clampfit 10 software. Data from the experiments are reported as the mean value ± standard deviation (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 (Alomone) or polyclonal (Santa Cruz Biotechnology) antibodies that recognize Kv1.3 protein. Specificity of the bands was confirmed by specific peptides blocking the antibodies prior to the WB. The enhanced signal from the mitochondria-specific proteins in mitochondria vs. crude membranes enriched in mitochondria (P2) was shown using anti-ANT and anti-VDAC (Santa Cruz Biotechnology). The purity of the mitochondrial samples was verified using polyclonal anti-cadherin (Abcam), monoclonal anti-PSD95 (Affinity BioReagents) and anti-InsP3R (Affinity BioReagents). Blots were developed using the appropriate anti-mouse, anti-goat or anti-rabbit antibody coupled to horseradish peroxidase (Sigma–Aldrich) in conjunction with an ECL (Amersham). 2.5. Immunohistochemistry A double-labeling immunofluorescence were performed on free-floating gerbil brain sections. Animals were transcardially perfused under deep anesthesia. Next, brains were fixed in 4% paraformaldehyde at 4 °C for 3 h, stored in 20% sucrose at 4 °C overnight and frozen at 70 °C. Cryosections (40 lm) were washed with PBS and blocked with 5% BSA in PBS containing 0.25% Triton X100 for 60 min. The sections were incubated overnight at 4 °C with a mixture of antibodies (goat polyclonal anti-Kv1.3, 1:75, Santa Cruz; mouse monoclonal anti-cytochrome oxidase subunit IV, 1:200, Invitrogen) in 2% BSA in PBS with 0.25% Triton X-100. After washing, 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 without the primary antibodies. Sections were analyzed by confocal microscopy. 3. Results 3.1. Biophysical properties of the mitochondrial voltage-gated potassium channel In patch-clamp experiments with gerbil hippocampal mitoplasts, the current characteristics for the voltage-gated potassium channel were observed. The single-channel current traces were recorded at different voltages in symmetrical isotonic solutions (Fig. 1A). Fig. 1B shows the current–voltage relationship for a single channel opening, at different voltages, under symmetrical and gradient conditions. Rectification of the current was not observed. The channel conductance in symmetrical isotonic solutions calculated 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 potassium-selective. The distribution of open channel probabilities was
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Fig. 1. Biophysical properties of the mitoKv channel from gerbil hippocampal mitochondria. (A) Single-channel recording in symmetric 150/150 mM KCl isotonic solution at different 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 KCl solution (dashed line, .). (C) Open channel probability for the mitochondrial voltage-gated potassium channel at different voltages (n = 3).
also analyzed. In Fig. 1C, we show that the probability of an open channel in a symmetrical isotonic solution was voltage dependent. The probability of channel opening increased from 0.5 at negative voltage to 0.75 at positive voltage. 3.2. Pharmacology of the mitochondrial voltage-gated potassium channel To exclude the possibility that the observed channel activity was caused by another type of mitochondrial potassium channel, we used 1 mM ATP/Mg2+ complex and 1 lM Ca2+. Neither calcium nor the ATP/Mg2+ complex changed the channel activity. These observations indicate that the channel is not a mitochondrial ATP-regulated potassium channel nor is it a mitochondrial large conductance Ca2+-regulated potassium channel (data not shown). Substances known to regulate voltage-gated potassium channel activity were also used to examine ion channel properties. Fig. 2A illustrates the activity of the channel in the control condition and after application of 10 nM margatoxin (MgTx), a very potent inhibitor of the Kv1.3 channel. Margatoxin inhibited the channel activity, and the effect was not reversible. The probability of channel opening decreased from 0.75 to 0.05 after the application of 10 nM MgTx (Fig. 2B). Agitoxin-2 was used as a second potent blocker of voltage-gated potassium channels of type Kv1.x. In our study, we observed that AgTx-2 had no effect on channel activity (Fig. 2C). This result could indicate that the mitochondrial voltage-gated potassium channel in the gerbil hippocampus has different properties than those of surface membrane Kv1.x type channels. Taken together, our data indicate that the observed single-channel activity is similar to the mitoKv1.3 channel 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 in hippocampal mitochondria. Two anti-Kv1.3 antibodies was used:
against a peptide mapping to near the C-terminus of human Kv1.3 (Santa Cruz) and specific for aa 211-224 of the N-terminus (Alomone Labs). As demonstrated by WB, the Kv1.3 protein was detected in the hippocampal mitochondria (mt) and in the mitochondria-enriched fraction (P2). To confirm that the right band was identified, western blots were done using a peptide that blocked the specific antibody reaction with the antigen, and no labeling was observed. Both antibodies detected a protein of 65 kDa in the P2 and mitochondrial fractions; the P2 gave a stronger WB signal because it contained other cell membranes (Fig. 3). The co-localization of goat polyclonal anti-Kv1.3 staining and monoclonal anti-cytochrome oxidase subunit IV (COXIV) antibody was observed in neurons in the cortex and hippocampus (Fig. 4, arrowheads). Other localization of Kv1.3 was also detected in neurons. However, not all of the Kv1.3 immunofluorescence was co-localized with COXIV labeling. We observed other membrane staining (Fig. 4, arrows) and a subset of cytoplasmic Kv1.3-positive puncta that did not overlap with the mitochondrial pattern (Fig. 4, arrows). As seen in the overlay, a minor fraction of COXIV-immunoreactive mitochondria also appeared to not be co-localized with the Kv1.3 protein signal.
4. Discussion Until now, two types of potassium channels had been described in neuronal mitochondria: the ATP-regulated potassium channel and the large-conductance Ca2+-regulated potassium channel [22]. It has been proposed that increased potassium influx into brain mitochondria, mediated by the mitoKATP channel, affects the mitochondrial membrane potential and mitochondrial respiration. In addition, diazoxide and RP66471 increased the mitochondrial matrix volume and induced the release of cytochrome c from hippocampal mitochondria [8]. Using potassium flux in proteoliposomes and BODIPY-FL-glyburide green fluorescent probe, it has been found that the brain mitoKATP channel is regulated by
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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 control conditions, after the addition of 10 nM margatoxin (+MgTx) and after perfusion with control buffer without margatoxin ( MgTx). (B) Panel shows the open channel probability 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 in control and AgTx-2 and MgTx conditions as a control for identification of the mitoKv channel.
the same ligands that regulate mitoKATP channels in the heart and liver [23]. In 1999, it was shown that the inner mitochondrial membrane from the human glioma cell line LN229 contains a mitoBKCa channel. A study of the channel activity determined that the probability of an open channel increased with increasing calcium concentrations and decreased upon application of charybdotoxin [3]. It was reported that calcium added to isolated rat brain mitochondria induced changes in mitochondrial membrane depolarization and increase in mitochondrial respiration. These calcium effects were blocked by iberiotoxin and charybdotoxin. Additionally, NS1619, a BKCa channel opener, induced potassium flux in brain mitochondria similar to that induced by Ca2+. These findings suggest the presence of a large-conductance Ca2+-regulated potassium channel in rat brain mitochondria, which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers and by western blot analysis [24]. Interestingly, recent studies using high-resolution immunofluorescence and immunoelectron microscopy provided evidence of the BKCa channel’s b4 subunit on the inner membrane of neuronal mitochondria in the rat brain and in cultured neurons [25]. All of these findings could support the notion of a neuroprotective role of mitochondrial potassium channels in the brain. In this paper, we showed for the first time the presence of a new voltage-gated potassium chan-
nel of the mitoKv1.3 type in the inner mitochondrial membrane of the gerbil hippocampus. The Kv channels represent a class of membrane proteins that are activated by changes in electrical potential. These channels are involved in a number of physiological processes including neuronal excitability, cell proliferation, smooth muscle contraction and apoptosis [26]. The Kv1.3 channel is a predominant member of the voltage-gated potassium channel family that is expressed in the plasma membrane in human lymphocyte T-cells. Activation of this channel is a key event in proliferation [17], and early inactivation seems to be important in the initiation of apoptosis [27]. In the rat brain, distinct combinations of Kv1 alpha subunits are colocalized in different neurons, implying that differential expression, assembly and subcellular targeting of these subunits may contribute to Kv channel diversity; this leads to pre-synaptic and post-synaptic membrane excitability [13]. Using co-immunoprecipitation, a putative tetramer of low abundance Kv1.3 with Kv1.4/1.1/1.2 was found in human CNS grey matter, indicating regional variations and functional specialization of Kv1 subunit complexes [12]. In gerbil cerebella, strong immunoreactivity for Kv1.3 was observed in the Purkinje cell bodies [28]. The presence of the Kv1.3 channel in gerbil hippocampal mitochondria was confirmed using two antibodies, which either recog-
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Fig. 3. Kv1.3 protein is expressed in gerbil brain mitochondria. Representative immunoblots showing Kv1.3 in mitochondria isolated from gerbil hippocampus. The protein was 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 immunoblotted with 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.
nize peptides near the C-terminus or are specific for aa 211-224 of the N-terminus of human plasma membrane Kv1.3, in two complementary methods: western blotting and immunohistochemistry. Mitochondrial localization of the voltage-dependent potassium channel may be related to a pro- or anti-apoptotic function of mitochondria. Previously, the mitoKv1.3 channel was reported to be present in the mitochondrial inner membrane of T lymphocyte cells. This channel, with a conductance of 17 pS, was detected in mitoplasts and inhibited by margatoxin, a selective inhibitor of the Kv1.3 channel. This channel had properties similar to those of plasma membrane Kv1.3 channels, which are also present in T lymphocytes [5]. Later, it was suggested that Bax mediates cytochrome c release and mitochondrial depolarization in lymphocytes at least in part via its interaction with the mitoKv1.3 [19]. Our studies, performed with patch-clamp and immunodetections, reveal the functional presence of a mitoKv1.3 channel in gerbil hippocampal mitochondria. This channel has the following properties: – the conductance of the channel is 109 pS, and its reversal potential is 19 mV, indicating that the examined channel is potassium-selective; – the open channel probability increases from 0.5 at negative voltage 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 voltagegated potassium channel could, in contrast to surface membrane channels, have a different molecular structure with no affinity for AgTx-2, possibly implying different function and/or modulation. Potassium channels discovered in mitochondria display a dual structure, function and localization in the cell. Recent data suggest that not only the plasma membrane Kv channel but also the mitochondrial Kv1.3 channel contributes to the stimulation of programmed cell death [29,30]. In olfactory bulb plasma membranes, the shaker ion channel Kv1.3 was reported to be suppressed by an insulin receptor kinase via tyrosine phosphorylation of critical N- and C-terminal residues [31]. It was reported that adaptor protein alteration of kinase-induced plasma membrane Kv1.3 channel modulation is related to the degree of direct protein–protein association, and that the channel itself can reciprocally modulate receptor-linked tyrosine kinase expression and activity [32]. In addition, phorbol esters suppress Kv1.3 currents recorded in Xenopus oocytes expressing Kv1.3 protein [33]. In line with the data showing the co-existence of PKC activation, ROS production and mitochondrial potassium channel activation in heart and neuron preconditioning [34,35], we assumed that a massive, post-ischemic translocation of PKCbb to mitochondria in the ischemia-resistant sector of gerbil hippocampus [20] might be related to a yet-unknown endogenous mechanism of neuroprotection that involves mitochondrial ion channels. However, our electrophysiological experiments, did not show changes in Kv1.3 channel activity in the presence of recombinant PKCb1 protein (data not shown).
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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 Kv1.3 localized on the plasma membrane or other membranes not containing mitochondrial marker COXIV, arrowheads point to the co-localization of Kv1.3 and COXIV on the mitochondrial 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-localized agglomerations in D (yellow).
In summary, a new voltage-gated potassium channel, similar to a Kv1.3 channel, was found in the inner membrane of gerbil hippocampal mitochondria. Immunocytochemical studies suggest that Kv1.3 subunit expression is restricted to a subpopulation of mitochondria. The role of this protein in normal and especially in ischemia-challenged brains remains to be elucidated. Acknowledgments This study was supported by the Nencki Institute of Experimental Biology, a grant from the MSHE (P-N/31/2006) and MitoNet.pl. P.B. would like to acknowledge Prof. Detlef Siemen for teaching the patch-clamp technique and for discussions during a postdoc supported by EMBO (2006) and DAAD (2008). References [1] A. Szewczyk, W. Jarmuszkiewicz, W.S. Kunz, Mitochondrial potassium channels, IUBMB Life 61 (2009) 134–143. [2] I. Inoue, H. Nagase, K. Kishi, T. Higuti, ATP-sensitive K+ channel in the mitochondrial inner membrane, Nature 352 (1991) 244–247. [3] D. Siemen, C. Loupatatzis, J. Borecky, E. Gulbins, F. Lang, Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma 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, Cell Calcium 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.
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