Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2006 Sep 7;577(Pt 1):17–29. doi: 10.1113/jphysiol.2006.118299

Targeted expression of Kir6.2 in mitochondria confers protection against hypoxic stress

Marko Ljubkovic 1,2, Jasna Marinovic 1,2, Andreas Fuchs 1,4, Zeljko J Bosnjak 1,2, Martin Bienengraeber 1,3
PMCID: PMC2000685  PMID: 16959852

Abstract

Selective K+ transport in the inner mitochondrial membrane has been attributed to at least three different channel types: ATP-sensitive, Ca2+-regulated and voltage-dependent K+ channels. Studies utilizing their selective modulators have suggested that an increased activity of these channels plays an important role in the cellular protection from metabolic stress. However, direct evidence for this effect is largely absent, and recent findings on the lack of specificity for several channel openers and blockers have questioned the actual contribution of the mitochondrial K+ channels in the preservation of cellular viability. In order to directly investigate the role of enhanced mitochondrial K+ uptake in cellular protection, we selectively expressed the inward rectifying K+ channel Kir6.2 in the mitochondria of HEK293 and HL-1 cells. Targeted Kir6.2 expression was achieved by cloning the Kir6.2 gene in pCMV/mito/GFP vector and the proper trafficking to mitochondria was confirmed by colocalization studies and Western blot. An increased K+ influx to mitochondria overexpressing Kir6.2, as evidenced by using the K+-sensitive PBFI AM fluorescent dye, substantially improved the cellular viability after hypoxic stress, which was assessed by lactate dehydrogenase (LDH) release. In parallel, monitoring of mitochondrial Ca2+ during stress, via the specific indicator rhod-2, revealed a significant attenuation of Ca2+ accumulation in mitochondria overexpressing K+ channels. This effect was abolished in mitochondria expressing an inactive mutant of Kir6.2. Mitochondria expressing Kir6.2 K+ channel also exhibited a significant degree of depolarization that became even more pronounced during the stress. In conclusion, this study provides the first non-pharmacological evidence that an increased K+ influx to mitochondria protects against hypoxic stress by preventing detrimental effects of Ca2+ overload.

Mitochondria are at the core of cellular energy metabolism as the site of the most ATP production. In addition, they play an important role in regulating ionic homeostasis of the cell. Mitochondrial Ca2+ (mitoCa2+) overload, that follows an increase in cytosolic Ca2+ during ischaemia and reperfusion, has a detrimental effect on mitochondrial function and cell survival, and is a key event in cardiac injury (Stone et al. 1989; Allen et al. 1993; Duchen, 2000; Brookes et al. 2004). Prevention of the mitoCa2+ overload would provide an important cardioprotective strategy against hypoxic stress that occurs in ischaemic heart disease. Ion channels, the regulators of ionic homeostasis, are well-positioned candidates for this purpose. A case in point are the ATP-sensitive potassium (KATP) channels that have been shown to play an important role in protecting the heart from ischaemia/reperfusion injury (Seino, 1999; Zingman et al. 2002). These channels, originally discovered in cardiac plasma membrane (Noma, 1983), are formed by association of a pore-forming inwardly rectifying K+ channel (Kir6.1 or Kir6.2) (Inagaki et al. 1995a, 1995b) and regulatory sulphonylurea receptor (SUR2A/B) (Aguilar-Bryan et al. 1995; Inagaki et al. 1996), an ABC transporter family member. Evidence has been provided that metabolic enzymes may associate with the cardiac KATP channel (Carrasco et al. 2001; Crawford et al. 2002a, 2002b; Jovanovic et al. 2005). Subsequently, ATP-sensitive K+ channel activity was also reported in mitochondria (Inoue et al. 1991), and more recently Ca2+-activated and voltage-regulated potassium channels (mitoBKCa and mitoKv1.3) have been described (Inoue et al. 1991; Siemen et al. 1999; Xu et al. 2002; Szabo et al. 2005). The increased activity of all of these mitochondrial K+ channels has been linked to cellular protection during stress, and there is a growing amount of data supporting the protective efficacy of mitochondrial K+ influx (Garlid et al. 1997; Fryer et al. 2001; Bock et al. 2002; Sato et al. 2005). However, these results were mostly obtained by pharmacological means, using putative activators and inhibitors of the mitochondrial K+ channels, while direct evidence for K+ flux and its protective mechanism is largely missing. In addition, the molecular identity of these channels is still unknown, and some investigators even question their very existence (Hanley et al. 2002; Brustovetsky et al. 2005; Drose et al. 2006).

Therefore, the goal of this investigation was to assess the protective mechanism of mitochondrial K+ flux independently from pharmacological modulators. In the present study, we overexpressed Kir6.2 K+ channel in the mitochondria of HL-1 and HEK293 cell lines, and tested the hypothesis that an increased K+ influx to mitochondria confers protection against metabolic stress.

Methods

Cloning, mutagenesis and expression of Kir6.2

The cDNA for Kir6.2 was amplified with Pwo DNA polymerase (Roche, Basel, Switzerland) from mouse Quick-Clone cDNA (Clontech, Mountain View, CA, USA) using 5′-ATAGAATGCGGCCGCACTGTCCCGAAAGGGCATTATCC-3′ as forward and 5′-TAAGACTGCGGCCGCATCAGGACAAGGAATCTGGAGAG-3′ as reverse primer, and inserted into the NotI site of the pCMV/myc/mito/GFP vector (Invitrogen, Carlsbad, CA, USA), in frame with the mitochondrial targeting sequence and green fluorescence protein (GFP). Kir6.2 cDNA was also inserted into the NotI site of pCMV/myc/mito in order to express Kir6.2 in the absence of GFP (myc/mito–Kir6.2). Point mutations (132GFG134 to AAA) were introduced in the K+-selectivity filter of Kir6.2 to create inactive mito/GFP–Kir6.2AAA (Koster et al. 2002) by performing PCR with complementary primers with the desired amino acid changes (QuickChange, Stratagene, La Jolla, CA, USA). The following forward primer was used (mutations are marked in bold): 5′-GAGGTCCAGGTGACCATTGCTGCCGCCGGACGCATGGTGACAGAG-3′. The reverse primer was complementary. The proper identity and orientation of the constructs were confirmed by DNA sequencing.

HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). HL-1 cells, a murine cardiac muscle cell line (Claycomb et al. 1998), were cultured in Claycomb-Medium (JRH Biosciences, Lenexa, KS, USA) supplemented with 10% FBS, 4 mm glutamine, 10 μm noradrenaline and 1% penicillin/streptomycin on gelatine/fibronectin-coated flasks (White et al. 2004). Cells were maintained in a humidified 5% CO2 incubator at 37°C and passaged the day before transfection at about 50% confluence in 35 mm tissue culture dishes. Transfection was performed in serum-free DMEM medium containing 6 μl Fugene reagent (Roche) and 2 μg plasmid DNA encoding the desired construct. The cells were cultured for an additional 48 h before conducting the experiments. For stable transfection, HL-1 or HEK293 cells were grown in the presence of 600 μg ml−1 neomycin, and colonies were selected to test for expression by GFP fluorescence and Western blot.

Western blotting

The microsomal and mitochondrial fractions were isolated from transfected HEK293 and HL-1 cells by differential centrifugation in 0.3 m mannitol, 0.1% bovine serum albumin, 2 mm EDTA, 10 mm Hepes, pH 7.4. After cell homogenization with a glass homogenizer, the suspension was centrifuged for 10 min at 1000 g at 4°C (the supernatant represents the cellular fraction), followed by supernatant centrifugation at 14 000 g for 15 min at 4°C. The supernatant was centrifuged at 100 000 g for 60 min to pellet the microsomal fraction. The resulting pellets were washed twice with cold isolation buffer, and the protein content was determined using the Lowry method (BIO-RAD, Hercules, CA, USA). Equivalent amounts of protein samples (50 μg) were separated on a 4–20% polyacrylamide gel, and then Western blotting performed as described (Chiari et al. 2005) using a 1: 200 dilution of a rabbit-raised antibody against Kir6.2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were stripped and reprobed with an antibody against subunit I of cytochrome c oxidase (Invitrogen) as a marker for mitochondria.

Laser-scanning confocal microscopy

HEK293 and HL-1 cells were visualized using an inverted laser-scanning confocal microscope (Eclipse TE2000-U, Nikon Inc., Japan) with a ×40/1.3 oil-immersion objective. Probes were excited at 488 nm with an argon laser, and at 543 nm with a Green HeNe laser. The scanning speed was set to a minimal pixel dwell time of 1.92 μs, and a set of filters (ND4 and ND8) was used in order to minimize dye bleaching. Each 512 × 512-pixel image was averaged twice via software-selected repeated line scan mode to ameliorate signal-to-noise ratio. Data were analysed using Metamorph 6.1 software (Universal Imaging, West Chester, PA, USA).

Mitochondrial staining with MitoFluor Red and colocalization analysis

Cells were loaded with mitochondrial marker MitoFluor Red589 (MFR, Invitrogen) for 10 min (200 nm), and washed three times before confocal analysis. For colocalization analysis, GFP fluorescence was monitored through a 515 nm barrier filter (excitation by argon laser), and MFR fluorescence through a 590 nm filter (excitation by Green HeNe). Each fluorescent wavelength was recorded independently and then combined to create a composite image.

Mitochondrial potassium uptake

Cultured HEK293 cells were loaded with a K+-sensitive fluorescent indicator PBFI AM in a loading protocol that allowed exclusive labelling of the mitochondria (Zoeteweij et al. 1994; Xu et al. 2002). In order to determine the rate of K+ uptake to the mitochondrial matrix, the bath solution was rapidly switched from 0 to 50 mm K+, and net K+ influx was recorded as the change in PBFI fluorescence ratio from two excitation wavelengths (340/380 nm, emission at 510 ± 20 nm). To ensure that the response was evoked through K+-selective channels, the rate of K+ influx was also monitored in the presence of the K+ channel blocker Ba2+ (1 mm). A more detailed description of mitochondrial K+ uptake measurements is provided in the Supplemental material.

Hypoxic stress and assessment of cell damage

To render HL-1 and HEK293 cells hypoxic, serum- and glucose-free DMEM was saturated with 5% CO2 and 95% N2, in the presence of 2-deoxyglucose (10 mm). Culture dishes were put in an airtight chamber in the incubator and flushed with the hypoxic gas mixture. The cells were exposed to hypoxia for 4 h, after which the hypoxic medium was replaced by DMEM containing glucose and 10% FBS, and cells were allowed to reoxygenate for 12 h. The cells that underwent such treatment were compared to control cells that were kept in normal DMEM containing 10% FBS, without hypoxia. In order to assess the degree of cellular damage, LDH release was measured in the supernatant of culture medium, according to the manufacturer's instructions (Diagnostic Chemicals Limited, Oxford, CT, USA) at a wavelength of 340 nm and expressed as percentage change over control (normoxia). In parallel, the cell viability was assessed using the MTT assay. Formazan formation was quantified spectrophotometrically at a wavelength of 570 nm with background subtraction at 650 nm.

Monitoring of mitochondrial Ca2+ during metabolic inhibition

The cells were incubated with Ca2+-sensitive fluorescent indicator rhod-2 AM (10 μm, Invitrogen) in a two-step cold/warm loading protocol (60 min at 4°C and 120 min at 37°C) (Trollinger et al. 2000). After loading, the cells were superfused with a normal external solution (mm: 140 NaCl, 5 KCl, 1.5 CaCl2, 1 MgSO4, 10 Hepes, and 5.5 glucose, pH 7.4). Metabolic inhibition was accomplished by switching to a solution containing (mm): 135 NaCl, 5 KCl, 1.5 CaCl2, 1 MgSO4, 10 Hepes, 10 2-deoxyglucose, and 2.5 NaCN, pH 7.4. Rhod-2 fluorescence was acquired at 590 nm (excitation with Green HeNe). Only the cells that expressed a similar degree of initial rhod-2 loading were analysed. Details on monitoring of mitoCa2+ are provided in the Supplemental material.

Analysis of mitochondrial membrane potential

HEK293 or HL-1 cells were incubated with the mitochondrial membrane potential (ΔΨm) indicator tetramethylrhodamine (TMRE, 100 nm, Invitrogen) for 30 min in the culture medium. TMRE fluorescence was obtained through a 590 nm barrier filter upon excitation by Green HeNe. All experiments were performed using identical image settings (gain, pinhole size, objective, filters) and conditions of TMRE incubation. Membrane potential was monitored under control conditions and during cell exposure to various drugs, as well as during metabolic inhibition. Throughout the experiments, TMRE was included in superfusing solutions.

Statistical analysis

Data are presented as mean ± s.e.m. and the number of cells or experiments is shown as n. Statistical comparisons were performed using one-way analysis of variance with Bonferroni's post hoc test. Differences at P < 0.05 were considered significant.

Results

Targeted expression of Kir6.2 in mitochondria

The cDNA encoding for Kir6.2 was amplified by PCR and subcloned into the vector pCMV/mito/GFP in frame and downstream from GFP and the mitochondrial targeting sequence (Fig. 1A). HEK293 cells, lacking expression of endogenous KATP channels (Giblin et al. 1999), were transfected with the generated construct (pCMV/mito/GFP–Kir6.2) and loaded with the mitochondrial marker MFR. GFP fluorescence revealed the intracellular distribution of GFP–Kir6.2 (green, Fig. 1B), and MFR fluorescence displayed the mitochondrial pattern within the cells (red). Colocalization was confirmed by merging the two images (yellow, overlay) and it verified mitochondrial localization of Kir6.2 in transfected cells.

Figure 1. Mitochondrial targeting of Kir6.2.

Figure 1

Open in a new tab

A, expression construct was generated by inserting the Kir6.2 gene in frame and downstream of the mitochondrial targeting sequence and green fluorescence protein (GFP) into the NotI restriction enzyme recognition site. B, confocal fluorescence imaging of HEK293 cells transfected with generated construct (green) and loaded with MFR, a mitochondrial-specific marker (red). GFP–Kir6.2 traffics to mitochondria, as seen by overlaying the green and red images (yellow). C, immunoblotting of cellular (cell), mitochondrial (mito) and microsomal (micro) fractions isolated from transfected HEK293 cells with Kir6.2 and cytochrome c oxidase (cyt-ox) (subunit I) antibody. Expression of GFP–Kir6.2 was detected in mitochondria of mito/GFP–Kir6.2 cells, but was not found in their plasma membrane. Mitochondrial expression was also confirmed in the cells transfected with the inactive form of Kir6.2 (mito/GFP–Kir6.2AAA). D, immunoblotting of mitochondrial fraction isolated from transfected HL-1 cells with the Kir6.2 antibody revealed a single band of ∼70 kDa in pCMV/mito/GFP–Kir6.2 transfected cells. Representative blots of three different experiments are shown.

Expression of mito/GFP–Kir6.2 was confirmed in transfected HEK293 cells by immunoblotting the total cellular and mitochondrial fraction with anti-Kir6.2 antibody. A single band, absent in pCMV/mito/GFP-transfected cells, was detected at ∼70 kDa in the cells transfected with pCMV/mito/GFP–Kir6.2 construct (Fig. 1C), which corresponds well with the estimated molecular weight of the GFP–Kir6.2 complex. Mitochondrial enrichment was confirmed by probing against subunit I of cytochrome c oxidase. Lack of GFP–Kir6.2 expression in the plasma membrane was verified by probing the microsomal fraction with Kir6.2 antibody, as well as with patch-clamp recordings (Supplemental material). Mitochondrial Kir6.2 expression was also confirmed in HEK293 cells transfected with inactive mito/GFP–Kir6.2AAA construct, stably transfected HL-1 cardiomyocytes (Fig. 1D) and in cells transfected with myc/mito–Kir6.2 (not shown).

The rate of K+ uptake is increased in mitochondria overexpressing Kir6.2

In order to determine whether mitochondrially expressed Kir6.2 acts as a functional K+ channel, we directly measured K+ uptake into mitochondria using the K+-selective fluorescent indicator PBFI AM (Zoeteweij et al. 1994; Xu et al. 2002). A loading protocol that favoured mitochondrial localization of the dye was applied (Fig. 2A). The rate of K+ flux into mitochondria was determined as the change in PBFI fluorescence ratio from two excitation wavelengths (340/380 nm) after increasing the external K+ concentration. As shown in Fig. 2B (representative recordings), and C (data summary) the rate of K+ influx was significantly higher in mitochondria overexpressing Kir6.2, compared to mitochondria expressing only GFP (n = 20 cells). The measured K+ influx was sensitive to the K+ channel blocker Ba2+. These experiments confirmed that mitochondrial K+ uptake is enhanced in mitochondria overexpressing Kir6.2.

Figure 2. Increased K+ uptake to mitochondria overexpressing Kir6.2.

Figure 2

Open in a new tab

A, wide-field fluorescence imaging of cultured cells loaded with PBFI and mitochondrial marker MFR. A mitochondrial pattern of PBFI fluorescence after digitonin permeabilization was apparent. B, representative recordings of the change in mitochondrial PBFI 340/380 nm ratio with time for mito/GFP–Kir6.2- and mito/GFP-transfected cells. The bathing solution was rapidly switched from 0 to 50 mm K+ and the rate of K+ influx was determined through linear fitting of the initial rising phase of the PBFI ratio. C, The rate of change of mitochondrial PBFI ratio (340/380 nm) was greater in mitochondria overexpressing Kir6.2 (mito/GFP–Kir6.2) than in control cells (mito/GFP). K+ uptake was sensitive to K+-channel blocker Ba2+. *P < 0.05 versus mito/GFP + Ba2+, #P < 0.05 versus mito/GFP and mito/GFP-Kir6.2 + Ba2+. Error bars represent s.e.m.

Mitochondrial Kir6.2 overexpression improves cellular survival during hypoxic stress

To investigate the influence of mitochondrial K+ channel overexpression on cellular viability, HL-1 cardiomyocytes and HEK293 cells which stably expressed either mito/GFP–Kir6.2 or mito/GFP were subjected to hypoxia/reoxygenation. The relative increase in LDH release after hypoxia/reoxygenation was less in mito/GFP–Kir6.2 than in mito/GFP transfected cells (162.4 ± 7.4% versus 239.6 ± 15.5% for HL-1 cells and 139.6 ± 9.2% versus 184.7 ± 10.1% for HEK293 cells, n = 14 dishes, P < 0.05), as shown in Fig. 3A. Moreover, addition of 5-hydroxydecanoate (5-HD, 200 μm), a reported mitoKATP inhibitor, during hypoxia/reoxygenation did not significantly change the extent of cellular damage in either group (Fig. 3A). These findings were confirmed by the MTT assay (Fig. 3B). Viability was preserved more effectively in the cells with mitochondria overexpressing Kir6.2 (61.4 ± 2.6% for HL-1 cells and 75.3 ± 3.1% for HEK293 cells), when compared to the cells expressing only mito/GFP (45.7 ± 2.5% for HL-1 cells and 59.4 ± 3.4% for HEK293 cells, n = 14 dishes, P < 0.05). Therefore, the overexpression of Kir6.2 K+ channel in mitochondria protects cells against hypoxic stress. However, this protection is not reversed in the presence of 5-HD.

Figure 3. Cellular viability after hypoxia/reoxygenation is improved by mitochondrial Kir6.2 overexpression.

Figure 3

Open in a new tab

A, LDH release is expressed relative to control level (CON) determined in cells that were kept under normoxic conditions. HL-1 cells with mitochondrial Kir6.2 overexpression (mito/GFP–Kir6.2) suffer less damage when exposed to hypoxic stress, as indicated by reduced release of LDH, compared to mito/GFP-transfected cells. Addition of 5-HD (200 μm) during stress did not change significantly the degree of cellular damage in any cell group. B, the formation of formazan in the MTT assay, as an indicator of cell viability, is better maintained in mito/GFP–Kir6.2 compared to mito/GFP-expressing cells. The viability of the cells that did not undergo hypoxia/reoxygenation is set as 100%. *P < 0.05 versus CON, #P < 0.05 versus mito/GFP. Error bars represent s.e.m.

Mitochondrial Kir6.2 overexpression blunts mitochondrial Ca2+ overload during metabolic inhibition

To monitor the changes in mitoCa2+ during stress, transfected HEK293 cells were loaded with rhod-2 AM, a mitoCa2+-sensitive fluorescent indicator (Hajnoczky et al. 1995). Representative images showing rhod-2 fluorescence in mito/GFP and mito/GFP–Kir6.2-expressing cells recorded at baseline and during exposure to metabolic inhibitors are shown in Fig. 4A (red). GFP fluorescent signal originating from mitochondria of transfected cells (green) verified the mitochondrial localization of rhod-2. As presented in Fig. 4B, an increase in mitochondrial rhod-2 fluorescence observed during metabolic inhibition in mito/GFP transfected cells was substantially blunted in mito/GFP–Kir6.2 transfected cells (241 ± 24% of control level versus 161 ± 12%, respectively, n = 30 cells). MitoCa2+ accumulation was similarly reduced in cells expressing Kir6.2 lacking GFP tag (myc/mito–Kir6.2), while this effect was not observed when cells were transfected with an inactive form of Kir6.2 (mito/GFP–Kir6.2AAA, n = 30 cells). Addition of 5-HD did not affect significantly the extent of rhod-2 fluorescence changes during stress (Fig. 4C).

Figure 4. Mitochondrial Ca2+ accumulation is attenuated during metabolic inhibition in mito/GFP–Kir6.2-transfected cells.

Figure 4

Open in a new tab

A, HEK293 cells expressing mito/GFP (upper panel) or mito/GFP–Kir6.2 (lower panel) were loaded with mitochondrial Ca2+ indicator rhod-2 and subjected to metabolic inhibition (MI). During stress conditions, rhod-2 fluorescence was increased to a lesser extent in the cells expressing Kir6.2 K+ channels in their mitochondria. B, time course of the relative rhod-2 fluorescence changes in cells expressing mito/GFP, mito/GFP–Kir6.2, myc/mito–Kir6.2 or mito/GFP–Kir6.2AAA constructs. Relative rhod-2 fluorescence is expressed as a percentage of control level (CON), which was recorded before exposure to metabolic inhibition. C, the presence of 5-HD (200 μm) did not affect significantly the extent of relative rhod-2 fluorescence changes during metabolic inhibition. *P < 0.05 versus CON, #P < 0.05 versus mito/GFP and mito/GFP–Kir6.2AAA (after 20 min of metabolic inhibition). Error bars represent s.e.m.

Mitochondria overexpressing Kir6.2 are partially depolarized

Influx of potassium ions through mitochondrial K+-selective channels causes depolarization of the highly negative mitochondrial membrane potential (Debska et al. 2001; Murata et al. 2001). To assess the effect of mitochondrial Kir6.2 expression on ΔΨm, we loaded transfected HL-1 and HEK293 cells with TMRE, a cationic fluorescent indicator that accumulates in mitochondria proportionally to ΔΨm. Figure 5A, upper panel shows that in HEK293 cells transfected with mito/GFP (arrows), TMRE accumulated to a similar extent as in untransfected cells (arrowheads). However, the cells that were successfully transfected with mito/GFP–Kir6.2 (lower panel, arrows) exhibited less TMRE fluorescence in mitochondria under identical conditions, indicating their partially depolarized state. The data are summarized in Fig. 5B. The intensity of mitochondrial TMRE fluorescence in mito/GFP–Kir6.2-expressing cells is significantly lower compared to mito/GFP-expressing cells (1034 ± 43 arbitrary units (au) versus 2875 ± 158 a.u. for HL-1 cells and 894 ± 91 a.u. versus 2854 ± 137 a.u. for HEK293 cells, n = 30 cells, P < 0.05). However, mitochondrial expression of the dominant negative form of Kir6.2 (mito/GFP–Kir6.2AAA) did not result in significantly reduced TMRE accumulation in mitochondria (n = 30 cells). It should be noted that change in ΔΨm is proportional to the log of TMRE fluorescence intensity change (O'Reilly et al. 2003). Treatment with reported mitoKATP modulators pinacidil (100 μM, activator) or glibenclamide (50 μm, inhibitor) did not affect the intensity of TMRE fluorescence in mitochondria expressing mito/GFP–Kir6.2. During metabolic inhibition, a decrease in TMRE fluorescence relative to control conditions was detected in both cell groups, indicating mitochondrial depolarization (Fig. 5D). Interestingly, this decrease was more pronounced in mitochondria overexpressing Kir6.2 compared to those expressing only GFP, even though they initially accumulated less TMRE.

Figure 5. Membrane potential is partially depolarized in Kir6.2-overexpressing mitochondria.

Figure 5

Open in a new tab

A, HEK293 cells were transfected with mito/GFP (green). Accumulation of TMRE potentiometric dye (red) in mitochondria of the cells successfully transfected with mito/GFP (upper panel, arrows) is not different from untransfected cells (arrowheads). Cells expressing mito/GFP–Kir6.2 accumulate less TMRE (lower panel, arrows) than untransfected cells (arrowheads). B, summarized data for mitochondrial TMRE fluorescence of transfected HEK293 and HL-1 cells. Mitochondrial expression of inactive mito/GFP–Kir6.2AAA construct did not reduce significantly the level of TMRE fluorescence. D, relative TMRE fluorescence intensity during metabolic inhibition is expressed as a percentage of control level (CON). *P < 0.05 versus CON, #P < 0.05 versus mito/GFP, a.u. = arbitrary units, PIN = pinacidil, GLIB = glibenclamide.

Discussion

In the present study we overexpressed Kir6.2, the pore-forming subunit of the sarcolemmal KATP channel, in the mitochondria of cultured cells. The main goal of our research was to directly investigate the effect of increased mitochondrial K+ influx on mitochondrial functional parameters and cellular viability during stress conditions. Our results show that an enhanced mitochondrial K+ uptake elicits cellular protection from hypoxic injury and reduces the accumulation of mitochondrial Ca2+ in cells exposed to metabolic stress.

Since the initial discovery of K+-selective transport in liver mitochondria (Inoue et al. 1991), K+ conductance has been detected in the mitochondria of different tissues (Paucek et al. 1992; Bajgar et al. 2001; Debska et al. 2002; Dahlem et al. 2004). To date, the existence of at least three different K+ channels has been reported in the inner mitochondrial membrane (mitoKATP, mitoBKCa and mitoKv1.3), and their electrophysiological and pharmacological properties have been studied in various models including mitoplasts (Inoue et al. 1991; Xu et al. 2002; Szabo et al. 2005), proteosomes (Paucek et al. 1992), planar lipid bilayers (Zhang et al. 2001; Nakae et al. 2003), and intact cells (Kohro et al. 2001). Evidence has been provided that modulation of activity of these channels could contribute to the cellular protection against hypoxic injury (Garlid et al. 1997; Liu et al. 1998; Sato et al. 2005), and numerous attempts have been made to decipher their exact role in mitochondrial and cellular pathophysiology. However, the molecular structure and the mechanism of mitochondrial K+ channels' protective action remain a topic of discussion.

In order to investigate the impact of alterations in K+ homeostasis on mitochondrial parameters and cellular viability, we selectively expressed the Kir6.2 K+ channel in the mitochondria of cultured cardiomyocytes and HEK293 cells. Mitochondrial expression was accomplished by the addition of cytochrome c oxidase targeting presequence, which was used previously by others to direct various proteins to mitochondria (Rizzuto et al. 1992). Patch-clamp and Western blot experiments confirmed that no additional K+ current was present in the plasma membrane. The orientation and the mechanism whereby Kir6.2, fused to the hydrophilic GFP, inserts into the inner mitochondrial membrane, is unknown. It may be possible that the connecting loop between the two transmembrane helices is translocated through the membrane utilizing the TIM22 complex (Rehling et al. 2003). The finding that Kir6.2 targeted to mitochondria increases K+ uptake and partially depolarizes ΔΨm suggests its functional incorporation into the inner mitochondrial membrane, as well as some spontaneous channel opening even under normoxic conditions (John et al. 1998). The increase of K+ flux into mitochondria overexpressing Kir6.2, as assessed with the K+-sensitive fluorescent indicator PBFI AM, demonstrated that the detected changes in the membrane potential could be directly correlated with an enhanced K+ conductance of the mitochondrial membrane. Nevertheless, these changes in ΔΨm had no detrimental effects on the cellular viability in mito/GFP–Kir6.2-expressing cells. Rather, the cellular tolerance to stress was substantially enhanced, as documented by the improved survival of mito/GFP–Kir6.2 cells exposed to hypoxia/reoxygenation. During the metabolic inhibition, mitochondrial depolarization was more pronounced in the cells expressing mito/GFP–Kir6.2 than in control cells, indicating that Kir6.2 over-expressed in mitochondria retains its ability to act as a metabolic sensor. Additional control experiments in which Kir6.2 was expressed without the attached GFP tag (myc/mito–Kir6.2) confirmed that GFP is not altering the properties of Kir6.2 in the mitochondrial membrane. Furthermore, in order to exclude the non-specific effects of the Kir6.2 protein inserted into the inner mitochondrial membrane, we generated a construct for the expression of Kir6.2 that does not exhibit K+ transport activity (mito/GFP–Kir6.2AAA). This was accomplished by introducing 132GFG134 to AAA amino acid mutations in the K+-selectivity filter of the pore (Supplemental material, Koster et al. 2002; Tong et al. 2006). When mito/GFP–Kir6.2AAA was expressed in HEK293 cells, mitochondrial membrane potential and Ca2+ accumulation during stress did not differ from the cells expressing only mito/GFP. This finding indicates that the effects we observed pertaining to mitochondrial function can indeed be ascribed to K+ influx via mitoKir6.2.

Activation of the putative mitochondrial K+ channels (mitoKATP and mitoBKCa) mediates cellular protection against metabolic stress in a variety of tissues (Bajgar et al. 2001; Oldenburg et al. 2002; Hai et al. 2005), and is considered a central event in the mechanism of ischaemic preconditioning (Liu et al. 1998; Gross & Fryer, 1999; Shintani et al. 2004; Sato et al. 2005). However, the mechanism whereby an increased K+ inflow to mitochondria elicits cellular protection remains controversial. Increased activity of the mitochondrial K+ channels depolarizes ΔΨm and attenuates mitochondrial Ca2+ accumulation during ischaemia, by decreasing a driving force for the Ca2+ uptake (Ishida et al. 2001; Murata et al. 2001). A reduced Ca2+ uptake would prevent the opening of the mitochondrial permeability transition pore (mPTP), cytochrome c release and apoptosis/necrosis (Crompton, 1999; Halestrap, 1999). Others have suggested that the K+ influx is accompanied by the alkalinization of the matrix, an increase in reactive oxygen species (ROS) and a mild swelling of mitochondria, which are all essential for protection (Kowaltowski et al. 2001; Andrukhiv et al. 2006; Costa et al. 2006). Expansion of the mitochondrial matrix under hypoxic stress could protect critical mitochondrial energetic functions such as fatty acid oxidation, respiration and ATP production (Halestrap, 1989; Dos Santos et al. 2002).

Most of the previous studies utilized various pharmacological activators and inhibitors of mitochondrial K+ channels in order to investigate the link between mitochondrial K+ homeostasis and cellular protection. However, recent findings revealed that many of these agents have additional mitochondrial and cellular targets. This led to the assumption that their mechanism of action may not be based on modulation of K+ fluxes, but could involve different metabolic pathways (Hanley et al. 2002; Das et al. 2003). Our results indicate that increased mitochondrial K+ influx indeed has the potential to protect cell from hypoxic stress. The viability of cardiomyocytes and HEK293 cells that underwent hypoxia/reoxygenation was substantially improved by the mitochondrial overexpression of Kir6.2. Monitoring of mitochondrial Ca2+ during simulated ischaemia revealed that Ca2+ accumulation was both diminished and delayed in these mitochondria. In addition, we also observed an increase in ROS production (Supplemental material), which has been shown to participate in the triggering phase of cardioprotection by preconditioning.

Subunits of the sarcolemmal KATP channel have been found in mitochondria in several studies (Lacza et al. 2003a, 2003b; Singh et al. 2003). Recently, immunogold labelling revealed that both Kir6.1 and Kir6.2, but not SUR, are present in heart mitochondria (Lacza et al. 2003b). This study suggested the possibility that an inwardly rectifying Kir6.x may form the mitoKATP channel. Conversely, an earlier study showed that flavoprotein fluorescence, an indicator of mitochondrial K+ channels' activity, was unaffected after disruption of the Kir6.2 gene in a mouse model (Suzuki et al. 2002). Further, non-targeted expression of Kir6.2 did not result in improved cellular tolerance to stress without addition of the KATP channel opener (Jovanovic et al. 1998). In the present study, we explored the effectiveness of the reported mitoKATP channel modulators pinacidil, glibenclamide and 5-HD on mitochondrially expressed Kir6.2. These drugs did not affect mitoKir6.2-evoked changes in ΔΨm, cell survival or mitochondrial Ca2+. The absence of effect of the mitoKATP channel regulators in our model could indicate that Kir6.2 in the mitochondria does not resemble the mitoKATP channel. However, this could also be attributed to the absence of a regulatory subunit that is required to complement Kir6.2. Both glibenclamide and pinacidil modulate sarcolemmal KATP channel activity via an interaction with the SUR subunit. Although a SUR-like protein with a smaller molecular mass than SUR has been reported in mitochondria (Lacza et al. 2003b), and mitochondrial ATP-binding cassette protein 1 has been recently associated with an increased resistance against oxidative stress (Ardehali et al. 2005), no specific interaction of these proteins with the K+ channel openers or sulphonylureas has been reported until now. Similarly, the site of 5-HD action is also unknown. The specificity of 5-HD for the mitoKATP channel became questionable due to the fact that it can enter the fatty acid β-oxidation metabolic pathway in mitochondria (Hanley et al. 2005). Similar non-specific targets have been also reported for pinacidil and glibenclamide (Cook, 1987; Tominaga et al. 1995; Hanley et al. 2002). However, at least HL-1 cardiomyocytes potentially express regulatory proteins involved in mitoKATP channel regulation. Possibly the presence of the GFP group affects the proper assembly of Kir6.2 with the eventual subunits, or alters its sensitivity to different modulators.

While our data do not confirm or rule out the existence of mitoKATP or other mitochondrial K+ channels, this study demonstrates that mitochondrially expressed Kir6.2 acts as a functional channel, and provides direct evidence that an increased K+ flux into mitochondria elicits cellular protection against hypoxia/reoxygenation injury. Heterologous expression studies, possibly combined with deletion/silencing, of other candidate proteins for mitochondrial K+ channels might prove a valuable tool in investigating the function and composition of endogenous channels responsible for cytoprotection, independently from partially non-specific pharmacological drugs.

Acknowledgments

The authors thank Dr Anna Stadnicka and Dr Wai-Meng Kwok for helpful discussions and Ms Chiaki Kwok for technical assistance. This study was supported in part by NIH (HL-34708 and GM-066730 to Z.J.B.), Advancing a Healthier Wisconsin Program (to M.B.), American Heart Association Predoctoral Grant (0515460Z to MLj) and the Department of Anaesthesiology, Medical College of Wisconsin.

Supplementary material

Supplemental material contains a detailed description on measurements of mitochondrial K+ uptake and monitoring of mitoCa2+, aswell as electrophysiological recordings and analysis of ROS production.

The online version of this paper can be accessed at:

DOI: 10.1113/jphysiol.2006.118299

http://jp.physoc.org/cgi/content/full/jphysiol.2006.118299/DC1 and contains supplemental material.

This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com

Supplemental data
jphysiol_2006.118299_index.html (640B, html)

References

  1. Aguilar-Bryan L, Nichols CG, Wechsler SW, Clement JPT, Boyd AE, 3rd, Gonzalez G, Herrera-Sosa H, Nguy K, Bryan J, Nelson DA. Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science. 1995;268:423–426. doi: 10.1126/science.7716547. [DOI] [PubMed] [Google Scholar]
  2. Allen SP, Darley-Usmar VM, McCormack JG, Stone D. Changes in mitochondrial matrix free calcium in perfused rat hearts subjected to hypoxia-reoxygenation. J Mol Cell Cardiol. 1993;25:949–958. doi: 10.1006/jmcc.1993.1107. [DOI] [PubMed] [Google Scholar]
  3. Andrukhiv A, Costa AD, West IC, Garlid KD. Am J Physiol Heart Circ Physiol. 2006. Opening mitoKATP increases superoxide generation from Complex I of the electron transport chain. in press. [DOI] [PubMed] [Google Scholar]
  4. Ardehali H, O'Rourke B, Marban E. Cardioprotective role of the mitochondrial ATP-binding cassette protein 1. Circ Res. 2005;97:740–742. doi: 10.1161/01.RES.0000186277.12336.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bajgar R, Seetharaman S, Kowaltowski AJ, Garlid KD, Paucek P. Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain. J Biol Chem. 2001;276:33369–33374. doi: 10.1074/jbc.M103320200. [DOI] [PubMed] [Google Scholar]
  6. Bock J, Szabo I, Jekle A, Gulbins E. Actinomycin D-induced apoptosis involves the potassium channel Kv1.3. Biochem Biophys Res Commun. 2002;295:526–531. doi: 10.1016/s0006-291x(02)00695-2. [DOI] [PubMed] [Google Scholar]
  7. Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol. 2004;287:C817–C833. doi: 10.1152/ajpcell.00139.2004. [DOI] [PubMed] [Google Scholar]
  8. Brustovetsky T, Shalbuyeva N, Brustovetsky N. Lack of manifestations of diazoxide/5-hydroxydecanoate-sensitive KATP channel in rat brain non-synaptosomal mitochondria. J Physiol. 2005;568:47–59. doi: 10.1113/jphysiol.2005.091199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carrasco AJ, Dzeja PP, Alekseev AE, Pucar D, Zingman LV, Abraham MR, Hodgson D, Bienengraeber M, Puceat M, Janssen E, Wieringa B, Terzic A. Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels. Proc Natl Acad Sci U S A. 2001;98:7623–7628. doi: 10.1073/pnas.121038198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chiari PC, Bienengraeber MW, Pagel PS, Krolikowski JG, Kersten JR, Warltier DC. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits. Anesthesiology. 2005;102:102–109. doi: 10.1097/00000542-200501000-00018. [DOI] [PubMed] [Google Scholar]
  11. Claycomb WC, Lanson NA, Jr, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ., Jr HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A. 1998;95:2979–2984. doi: 10.1073/pnas.95.6.2979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cook GA. The hypoglycemic sulfonylureas glyburide and tolbutamide inhibit fatty acid oxidation by inhibiting carnitine palmitoyltransferase. J Biol Chem. 1987;262:4968–4972. [PubMed] [Google Scholar]
  13. Costa AD, Quinlan CL, Andrukhiv A, West IC, Jaburek M, Garlid KD. The direct physiological effects of mitoK (ATP) opening on heart mitochondria. Am J Physiol Heart Circ Physiol. 2006;290:H406–H415. doi: 10.1152/ajpheart.00794.2005. [DOI] [PubMed] [Google Scholar]
  14. Crawford RM, Budas GR, Jovanovic S, Ranki HJ, Wilson TJ, Davies AM, Jovanovic A. M-LDH serves as a sarcolemmal K (ATP) channel subunit essential for cell protection against ischemia. EMBO J. 2002a;21:3936–3948. doi: 10.1093/emboj/cdf388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Crawford RM, Ranki HJ, Botting CH, Budas GR, Jovanovic A. Creatine kinase is physically associated with the cardiac ATP-sensitive K+ channel in vivo. FASEB J. 2002b;16:102–104. doi: 10.1096/fj.01-0466fje. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J. 1999;341:233–249. [PMC free article] [PubMed] [Google Scholar]
  17. Dahlem YA, Horn TF, Buntinas L, Gonoi T, Wolf G, Siemen D. The human mitochondrial KATP channel is modulated by calcium and nitric oxide: a patch-clamp approach. Biochim Biophys Acta. 2004;1656:46–56. doi: 10.1016/j.bbabio.2004.01.003. [DOI] [PubMed] [Google Scholar]
  18. Das M, Parker JE, Halestrap AP. Matrix Volume measurements challenge the existence of diazoxide/glibencamide-sensitive KATP channels in rat mitochondria. J Physiol. 2003;547:893–902. doi: 10.1113/jphysiol.2002.035006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Debska G, Kicinska A, Skalska J, Szewczyk A, May R, Elger CE, Kunz WS. Opening of potassium channels modulates mitochondrial function in rat skeletal muscle. Biochim Biophys Acta. 2002;1556:97–105. doi: 10.1016/s0005-2728(02)00340-7. [DOI] [PubMed] [Google Scholar]
  20. Debska G, May R, Kicinska A, Szewczyk A, Elger CE, Kunz WS. Potassium channel openers depolarize hippocampal mitochondria. Brain Res. 2001;892:42–50. doi: 10.1016/s0006-8993(00)03187-5. [DOI] [PubMed] [Google Scholar]
  21. Dos Santos P, Kowaltowski AJ, Laclau MN, Seetharaman S, Paucek P, Boudina S, Thambo JB, Tariosse L, Garlid KD. Mechanisms by which opening the mitochondrial ATP-sensitive K+ channel protects the ischemic heart. Am J Physiol Heart Circ Physiol. 2002;283:H284–H295. doi: 10.1152/ajpheart.00034.2002. [DOI] [PubMed] [Google Scholar]
  22. Drose S, Brandt U, Hanley PJ. K+-independent actions of diazoxide question the role of inner membrane KATP channels in mitochondrial cytoprotective signaling. J Biol Chem. 2006;281:23733–23739. doi: 10.1074/jbc.M602570200. [DOI] [PubMed] [Google Scholar]
  23. Duchen MR. Mitochondria and Ca2+ in cell physiology and pathophysiology. Cell Calcium. 2000;28:339–348. doi: 10.1054/ceca.2000.0170. [DOI] [PubMed] [Google Scholar]
  24. Fryer RM, Hsu AK, Gross GJ. Mitochondrial K (ATP) channel opening is important during index ischemia and following myocardial reperfusion in ischemic preconditioned rat hearts. J Mol Cell Cardiol. 2001;33:831–834. doi: 10.1006/jmcc.2001.1350. [DOI] [PubMed] [Google Scholar]
  25. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D'Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ Res. 1997;81:1072–1082. doi: 10.1161/01.res.81.6.1072. [DOI] [PubMed] [Google Scholar]
  26. Giblin JP, Leaney JL, Tinker A. The molecular assembly of ATP-sensitive potassium channels. Determinants on the pore forming subunit. J Biol Chem. 1999;274:22652–22659. doi: 10.1074/jbc.274.32.22652. [DOI] [PubMed] [Google Scholar]
  27. Gross GJ, Fryer RM. Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning. Circ Res. 1999;84:973–979. doi: 10.1161/01.res.84.9.973. [DOI] [PubMed] [Google Scholar]
  28. Hai S, Takemura S, Minamiyama Y, Yamasaki K, Yamamoto S, Kodai S, Tanaka S, Hirohashi K, Suehiro S. Mitochondrial K(ATP) channel opener prevents ischemia–reperfusion injury in rat liver. Transplant Proc. 2005;37:428–431. doi: 10.1016/j.transproceed.2004.12.112. [DOI] [PubMed] [Google Scholar]
  29. Hajnoczky G, Robb-Gaspers LD, Seitz MB, Thomas AP. Decoding of cytosolic calcium oscillations in the mitochondria. Cell. 1995;82:415–424. doi: 10.1016/0092-8674(95)90430-1. [DOI] [PubMed] [Google Scholar]
  30. Halestrap AP. The regulation of the matrix Volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism. Biochim Biophys Acta. 1989;973:355–382. doi: 10.1016/s0005-2728(89)80378-0. [DOI] [PubMed] [Google Scholar]
  31. Halestrap AP. The mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury. Biochem Soc Symp. 1999;66:181–203. doi: 10.1042/bss0660181. [DOI] [PubMed] [Google Scholar]
  32. Hanley PJ, Drose S, Brandt U, Lareau RA, Banerjee AL, Srivastava DK, Banaszak LJ, Barycki JJ, Van Veldhoven PP, Daut J. 5-Hydroxydecanoate is metabolised in mitochondria and creates a rate-limiting bottleneck for beta-oxidation of fatty acids. J Physiol. 2005;562:307–318. doi: 10.1113/jphysiol.2004.073932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hanley PJ, Mickel M, Loffler M, Brandt U, Daut J. KATP channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart. J Physiol. 2002;542:735–741. doi: 10.1113/jphysiol.2002.023960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Inagaki N, Gonoi T, Clement JPT, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science. 1995a;270:1166–1170. doi: 10.1126/science.270.5239.1166. [DOI] [PubMed] [Google Scholar]
  35. Inagaki N, Gonoi T, Clement JP, Wang CZ, Aguilar-Bryan L, Bryan J, Seino S. A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron. 1996;16:1011–1017. doi: 10.1016/s0896-6273(00)80124-5. [DOI] [PubMed] [Google Scholar]
  36. Inagaki N, Tsuura Y, Namba N, Masuda K, Gonoi T, Horie M, Seino Y, Mizuta M, Seino S. Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart. J Biol Chem. 1995b;270:5691–5694. doi: 10.1074/jbc.270.11.5691. [DOI] [PubMed] [Google Scholar]
  37. Inoue I, Nagase H, Kishi K, Higuti T. ATP-sensitive K+ channel in the mitochondrial inner membrane. Nature. 1991;352:244–247. doi: 10.1038/352244a0. [DOI] [PubMed] [Google Scholar]
  38. Ishida H, Hirota Y, Genka C, Nakazawa H, Nakaya H, Sato T. Opening of mitochondrial KATP channels attenuates the ouabain-induced calcium overload in mitochondria. Circ Res. 2001;89:856–858. doi: 10.1161/hh2201.100341. [DOI] [PubMed] [Google Scholar]
  39. John SA, Monck JR, Weiss JN, Ribalet B. The sulphonylurea receptor SUR1 regulates ATP-sensitive mouse Kir6.2 K+ channels linked to the green fluorescent protein in human embryonic kidney cells (HEK 293) J Physiol. 1998;510:333–345. doi: 10.1111/j.1469-7793.1998.333bk.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Jovanovic A, Jovanovic S, Lorenz E, Terzic A. Recombinant cardiac ATP-sensitive K+ channel subunits confer resistance to chemical hypoxia–reoxygenation injury. Circulation. 1998;98:1548–1555. doi: 10.1161/01.cir.98.15.1548. [DOI] [PubMed] [Google Scholar]
  41. Jovanovic S, Du Q, Crawford RM, Budas GR, Stagljar I, Jovanovic A. Glyceraldehyde 3-phosphate dehydrogenase serves as an accessory protein of the cardiac sarcolemmal K(ATP) channel. EMBO Rep. 2005;6:848–852. doi: 10.1038/sj.embor.7400489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kohro S, Hogan QH, Nakae Y, Yamakage M, Bosnjak ZJ. Anesthetic effects on mitochondrial ATP-sensitive K channel. Anesthesiology. 2001;95:1435–1340. doi: 10.1097/00000542-200112000-00024. [DOI] [PubMed] [Google Scholar]
  43. Koster JC, Remedi MS, Flagg TP, Johnson JD, Markova KP, Marshall BA, Nichols CG. Hyperinsulinism induced by targeted suppression of beta cell KATP channels. Proc Natl Acad Sci U S A. 2002;99:16992–16997. doi: 10.1073/pnas.012479199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Kowaltowski AJ, Seetharaman S, Paucek P, Garlid KD. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria. Am J Physiol Heart Circ Physiol. 2001;280:H649–H657. doi: 10.1152/ajpheart.2001.280.2.H649. [DOI] [PubMed] [Google Scholar]
  45. Lacza Z, Snipes JA, Kis B, Szabo C, Grover G, Busija DW. Investigation of the subunit composition and the pharmacology of the mitochondrial ATP-dependent K+ channel in the brain. Brain Res. 2003a;994:27–36. doi: 10.1016/j.brainres.2003.09.046. [DOI] [PubMed] [Google Scholar]
  46. Lacza Z, Snipes JA, Miller AW, Szabo C, Grover G, Busija DW. Heart mitochondria contain functional ATP-dependent K+ channels. J Mol Cell Cardiol. 2003b;35:1339–1347. doi: 10.1016/s0022-2828(03)00249-9. [DOI] [PubMed] [Google Scholar]
  47. Liu Y, Sato T, O'Rourke B, Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation. 1998;97:2463–2469. doi: 10.1161/01.cir.97.24.2463. [DOI] [PubMed] [Google Scholar]
  48. Murata M, Akao M, O'Rourke B, Marban E. Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca2+ overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection. Circ Res. 2001;89:891–898. doi: 10.1161/hh2201.100205. [DOI] [PubMed] [Google Scholar]
  49. Nakae Y, Kwok WM, Bosnjak ZJ, Jiang MT. Isoflurane activates rat mitochondrial ATP-sensitive K+ channels reconstituted in lipid bilayers. Am J Physiol Heart Circ Physiol. 2003;284:H1865–H1871. doi: 10.1152/ajpheart.01031.2002. [DOI] [PubMed] [Google Scholar]
  50. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature. 1983;305:147–148. doi: 10.1038/305147a0. [DOI] [PubMed] [Google Scholar]
  51. O'Reilly CM, Fogarty KE, Drummond RM, Tuft RA, Walsh JV., Jr Quantitative analysis of spontaneous mitochondrial depolarizations. Biophys J. 2003;85:3350–3357. doi: 10.1016/S0006-3495(03)74754-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Oldenburg O, Cohen MV, Yellon DM, Downey JM. Mitochondrial KATP channels: role in cardioprotection. Cardiovasc Res. 2002;55:429–437. doi: 10.1016/s0008-6363(02)00439-x. [DOI] [PubMed] [Google Scholar]
  53. Paucek P, Mironova G, Mahdi F, Beavis AD, Woldegiorgis G, Garlid KD. Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria. J Biol Chem. 1992;267:26062–26069. [PubMed] [Google Scholar]
  54. Rehling P, Model K, Brandner K, Kovermann P, Sickmann A, Meyer HE, Kuhlbrandt W, Wagner R, Truscott KN, Pfanner N. Protein insertion into the mitochondrial inner membrane by a twin-pore translocase. Science. 2003;299:1747–1751. doi: 10.1126/science.1080945. [DOI] [PubMed] [Google Scholar]
  55. Rizzuto R, Simpson AW, Brini M, Pozzan T. Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature. 1992;358:325–327. doi: 10.1038/358325a0. [DOI] [PubMed] [Google Scholar]
  56. Sato T, Saito T, Saegusa N, Nakaya H. Mitochondrial Ca2+-activated K+ channels in cardiac myocytes: a mechanism of the cardioprotective effect and modulation by protein kinase A. Circulation. 2005;111:198–203. doi: 10.1161/01.CIR.0000151099.15706.B1. [DOI] [PubMed] [Google Scholar]
  57. Seino S. ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol. 1999;61:337–362. doi: 10.1146/annurev.physiol.61.1.337. [DOI] [PubMed] [Google Scholar]
  58. Shintani Y, Node K, Asanuma H, Sanada S, Takashima S, Asano Y, Liao Y, Fujita M, Hirata A, Shinozaki Y, Fukushima T, Nagamachi Y, Okuda H, Kim J, Tomoike H, Hori M, Kitakaze M. Opening of Ca2+-activated K+ channels is involved in ischemic preconditioning in canine hearts. J Mol Cell Cardiol. 2004;37:1213–1218. doi: 10.1016/j.yjmcc.2004.09.012. [DOI] [PubMed] [Google Scholar]
  59. Siemen D, Loupatatzis C, Borecky J, Gulbins E, Lang F. Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line. Biochem Biophys Res Commun. 1999;257:549–554. doi: 10.1006/bbrc.1999.0496. [DOI] [PubMed] [Google Scholar]
  60. Singh H, Hudman D, Lawrence CL, Rainbow RD, Lodwick D, Norman RI. Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes. J Mol Cell Cardiol. 2003;35:445–459. doi: 10.1016/s0022-2828(03)00041-5. [DOI] [PubMed] [Google Scholar]
  61. Stone D, Darley-Usmar V, Smith DR, O'Leary V. Hypoxia-reoxygenation induced increase in cellular Ca2+ in myocytes and perfused hearts: the role of mitochondria. J Mol Cell Cardiol. 1989;21:963–973. doi: 10.1016/0022-2828(89)90795-5. [DOI] [PubMed] [Google Scholar]
  62. Suzuki M, Sasaki N, Miki T, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, Seino S, Marban E, Nakaya H. Role of sarcolemmal KATP channels in cardioprotection against ischemia/reperfusion injury in mice. J Clin Invest. 2002;109:509–516. doi: 10.1172/JCI14270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Szabo I, Bock J, Jekle A, Soddemann M, Adams C, Lang F, Zoratti M, Gulbins E. A novel potassium channel in lymphocyte mitochondria. J Biol Chem. 2005;280:12790–12798. doi: 10.1074/jbc.M413548200. [DOI] [PubMed] [Google Scholar]
  64. Tominaga M, Horie M, Sasayama S, Okada Y. Glibenclamide, an ATP-sensitive K+ channel blocker, inhibits cardiac cAMP-activated Cl− conductance. Circ Res. 1995;77:417–423. doi: 10.1161/01.res.77.2.417. [DOI] [PubMed] [Google Scholar]
  65. Tong X, Porter LM, Liu G, Dhar-Chowdhury P, Srivastava S, Pountney DJ, Yoshida H, Artman M, Fishman GI, Yu C, Iyer R, Morley GE, Gutstein DE, Coetzee WA. Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits. Am J Physiol Heart Circ Physiol. 2006;291:H543–H551. doi: 10.1152/ajpheart.00051.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Trollinger DR, Cascio WE, Lemasters JJ. Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca2+-indicating fluorophores. Biophys J. 2000;79:39–50. doi: 10.1016/S0006-3495(00)76272-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. White SM, Constantin PE, Claycomb WC. Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function. Am J Physiol Heart Circ Physiol. 2004;286:H823–H829. doi: 10.1152/ajpheart.00986.2003. [DOI] [PubMed] [Google Scholar]
  68. Xu W, Liu Y, Wang S, McDonald T, Van Eyk JE, Sidor A, O'Rourke B. Cytoprotective role of Ca2+- activated K+ channels in the cardiac inner mitochondrial membrane. Science. 2002;298:1029–1033. doi: 10.1126/science.1074360. [DOI] [PubMed] [Google Scholar]
  69. Zhang DX, Chen YF, Campbell WB, Zou AP, Gross GJ, Li PL. Characteristics and superoxide-induced activation of reconstituted myocardial mitochondrial ATP-sensitive potassium channels. Circ Res. 2001;89:1177–1183. doi: 10.1161/hh2401.101752. [DOI] [PubMed] [Google Scholar]
  70. Zingman LV, Hodgson DM, Bast PH, Kane GC, Perez-Terzic C, Gumina RJ, Pucar D, Bienengraeber M, Dzeja PP, Miki T, Seino S, Alekseev AE, Terzic A. Kir6.2 is required for adaptation to stress. Proc Natl Acad Sci U S A. 2002;99:13278–13283. doi: 10.1073/pnas.212315199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Zoeteweij JP, Van De Water B, De Bont HJ, Nagelkerke JF. Mitochondrial K+ as modulator of Ca2+-dependent cytotoxicity in hepatocytes. Novel application of the K+-sensitive dye PBFI (K+-binding benzofuran isophthalate) to assess free mitochondrial K+ concentrations. Biochem J. 1994;299(2):539–543. doi: 10.1042/bj2990539. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental data
jphysiol_2006.118299_index.html (640B, html)
jphysiol_2006.118299_1.pdf (351.3KB, pdf)

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES