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Matrix Mg2+ regulates mitochondrial ATP-dependent potassium channel from heart
FEBS 29352
FEBS Letters 579 (2005) 1625–1632
Matrix Mg2+ regulates mitochondrial ATP-dependent potassium channel from heart Piotr Bednarczyka,b, Krzysztof Dołowyb, Adam Szewczyka,* a
Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland b Department of Biophysics, Agricultural University SGGW, 159 Nowoursynowska St., 02-776 Warsaw, Poland Received 2 December 2004; revised 11 January 2005; accepted 31 January 2005 Available online 18 February 2005 Edited by Peter Brzezinski
Abstract Mitochondrial ATP-regulated potassium (mitoKATP) channels play an important role in cardioprotection. Single channel activity was measured after reconstitution of inner mitochondrial membranes from bovine myocardium into a planar lipid bilayer. After incorporation, the potassium channel was recorded with a mean conductance of 103 ± 9 pS. The channel activity was inhibited by ATP/Mg and activated by GDP. Magnesium ions alone affected, in a dose dependent manner, both the channel conductance and the open probability. Magnesium ions regulated the mitoKATP channel only when added to the trans compartment. We conclude that Mg2+ regulates the cardiac mitoKATP channel from the matrix site by affecting both the channel conductance and gating. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Heart mitochondria; Potassium channel; ATP; Magnesium; Black lipid membrane
1. Introduction Potassium selective channels are present in the inner mitochondrial membrane [1,2]. In cardiac mitochondria an ATPregulated potassium channel (mitoKATP channel) [3] and a large conductance Ca2+-activated potassium channel (mitoBKCa channel) [4] have been identified. MitoKATP channels were also found in liver [5], brain [6,7], skeletal muscle [8], and human T-lymphocyte mitochondria [9]. Both the mitoKATP and mitoBKCa channels are regulated by potassium channel openers [10–12], of which diazoxide seems to be an especially potent activator of the mitoKATP channel [13]. The molecular identity of the mitoKATP channel is unknown [1]. Several observations on the pharmacological profile and immunoreactivity with specific antibodies may suggest that the mitoKATP channel belongs to the inward rectifier K+ channel family – Kir6.x [14]. Using specific antibodies, Kir6.1, Kir6.2, and sulfonylurea receptor (SUR2A) subunits were demonstrated in ventricular myocyte mitochondria [15]. Recently, it was hypothesized that a complex of five proteins in * Corresponding author. Fax: +4822 8225342. E-mail address: [email protected] (A. Szewczyk).
Abbreviations: mitoKATP channel, mitochondrial ATP-regulated potassium channel; 5-HD, 5-hydroxydecanoic acid; SMP, submitochondrial particles
the mitochondrial inner membrane is capable of transporting K+ with characteristics similar to those of the mitoKATP channel [16,17]. Similar to the plasma membrane KATP channel, mitoKATP is probably composed of sulfonylurea receptor (mitoSUR). The usage of 125I-glibenclamide lead to labeling of 28 kDa protein in bovine heart mitochondria [18,19]. Fluorescent probe BODIPY-glibenclamide labeled a 64 kDa protein in brain mitochondria [7]. The primary function of the mitoKATP channel is to allow K+ transport into the mitochondrial matrix. This phenomenon could be involved in mitochondrial volume homeostasis [20]. The mitoKATP channel activity is probably coupled to the cellular energetic state via its inhibition by ATP and ADP [21]. Additionally, the channel is blocked by palmitoyl- or oleylCoA [37] and activated by GTP and GDP [37]. The cardiac mitoKATP channel plays an important role in cardioprotection (for reviews, see [11,20,22–24]). Cytoprotective action of potassium channel openers acting on mitochondria was also observed in a neuronal system [25]. The majority of observations concerning regulation of the mitoKATP channel is based on modulation by channel inhibitors and potassium channel openers of mitochondrial function such as matrix volume, mitochondrial potential, or oxygen consumption [3,6,8]. Another approach is based on K+ flux measurements with the use of potassium specific fluorescent dyes [3,7,13] or radioactive 86Rb+ isotope, a K+ analogue [26]. Recently, reconstitution of the mitoKATP channel allowing single channel recordings was applied successfully to study new properties of the mitoKATP channel [26–28]. Addition of xanthine/xanthine oxidase, an O 2 -generating system, resulted in a marked activation of the mitoKATP channel, further abolished by 5-hydroxydecanoic acid (5-HD) or a sulfhydryl alkylating compound, N-ethylmaleimide [27]. Moreover, a direct activation of the mitoKATP channel by the anesthetic isoflurane was observed [28]. Recently, inhibition of the cardiac mitoKATP channel by quinine was described [26]. Magnesium ions have been known for many years to affect cation transporting pathways in mitochondria [29]. These include both K+ and Na+ uniporters [29] and K+/H+ exchanger [30]. Mitochondrial potassium transport plays an essential role in maintaining mitochondrial matrix volume, due to matrix swelling or contraction, and mitochondrial cell signaling by means of reactive oxygen species or cytochrome c release [30]. In our experiments, we used purified mitochondrial inner membrane reconstituted into a planar lipid bilayer in order to study the effects of Mg2+ of the cardiac mitoKATP channel activity. Here, we show that Mg2+ interact with the mitoKATP channel affecting both the channel conductance and the open
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.01.077
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probability. Our data strongly suggest that the cardiac mitoKATP channel is regulated by magnesium from the matrix side.
2. Materials and methods 2.1. Materials L -a-Phosphatidyl-choline (asolectin), n-decane and protease (Subtilisin A, P5380) from Bacillus licheniformis were from Sigma–Aldrich, Germany. All other chemicals were of the highest purity available commercially. 2.2. Isolation of mitochondria Bovine heart mitochondria were isolated at 4 C as described earlier [27]. Briefly, a fragment of bovine heart muscle was minced in isolation buffer (200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 5 mM MOPS, 0.1% BSA, and 1 mM EGTA, pH 7.15) and homogenized with 25 U protease/gram tissue using a teflon pestle. The homogenate was then centrifuged at 8000 · g for 10 min to remove the protease. The pellet was resuspended in the isolation buffer and centrifuged again at 700 · g, for 10 min. to remove cellular debris. The supernatant was centrifuged at 8000 · g for 10 min. at 4 C to pellet the mitochondria. The mitochondria were then washed and suspended in the isolation buffer without EGTA. The suspension was loaded on top of Percoll solution (30% Percoll, 0.25 M sucrose, 1 mM EDTA, and 10 mM HEPES, pH 7.4) and centrifuged at 35 000 · g for 30 min. The mitochondrial fraction was then collected and washed twice with the isolation buffer without EGTA and resuspended at 10–20 mg of protein/ml. 2.3. Preparation of submitochondrial particles Freshly prepared mitochondria were sonicated 8 · 15 s and centrifuged at 16 000 · g for 15 min to pellet unbroken mitochondria. The supernatant was again centrifuged at 140 000 · g for 35 min and submitochondrial particles (SMP) were resuspended in the isolation buffer without EGTA at 5 mg of protein/ml. The polarity of SMP vesicles was estimated to be 40–50% outside-out (depending on the preparation), using cytochrome c oxidase activity measurements. 2.4. Black lipid membrane measurements Black lipid membranes (BLMs) were formed in a 250 lm diameter hole drilled in a Delrin cup (Warner Instrument Corp., Hamden, CT, USA), which separated two chambers (cis and trans 1 ml internal volume). The chambers contained 50/150 mM KCl (cis/trans), 20 mM Tris–HCl, pH 7.2. The outline of the aperture was coated with a lipid solution and N2 dried prior to bilayer formation to improve membrane stability. BLMs were painted using asolectin in n-decane at a final concentration of 25 mg lipid/ml. Bovine heart SMP (1–5 lg of protein) was added to the 1 ml trans compartment (Fig. 1D). Incorporation of the mitoKATP channel into the BLM was usually observed within few minutes. All measurements were carried out at air conditioned room (24 C). Formation and thinning of the bilayer was monitored by capacitance measurements and optical observations. The final accepted capacitance values ranged from 110 to 180 pF. Electrical connections were made by Ag/AgCl electrodes and agar salt bridges (3 M KCl) to minimize liquid junction potentials. Voltage was applied to the cis compartment of the chamber and the trans compartment was grounded. The current was measured using a bilayer membrane amplifier (BLM-120, BioLogic). For removing the solutions from the cis or trans compartments we used a perfusion system containing a holder with glass tip, perfusion pump (sp260p syringe pump, USA), glass syringe (FORTUNA OPTIMA Glasspritze, Aldrich) and teflon tubing (C-FLEX TUBING, Sigma). 2.5. Data analysis Signals were filtered at 500 Hz. The current were digitized at a sampling rate of 100 kHz (A/D converter PowerLab 2/20, ADInstruments) and transferred to a PC or digital tape recorder (DTR-1204, BioLogic) for off-line analysis by Chart v4.2.3 (PowerLab ADInstruments) and pCLAMP8 (Axon Laboratory). The pCLAMP8 software package was used for data processing. The channel recordings illustrated are representative of the most frequently observed conductances under
P. Bednarczyk et al. / FEBS Letters 579 (2005) 1625–1632 the given conditions. The conductance was calculated from the current–voltage relationship. The probability of channel opening (P(open)) was calculated with an automatic interval setting. The channel open (sopen) and closed (sclosed) lifetimes were calculated from logarithmic binning mode using the Marquardt-LSQ fitting method, order one, without weighting. n denotes the number of experiments, and N the number of events. sopen, sclosed, P(open) were calculated from segments of continuous recordings lasting 60 s and with N P 1000 events. Data from the experiments are reported as mean value or values ± S.E. (standard error) or S.D. (standard deviation). The permeability ratios for K+ and Cl were calculated according to the Goldman–Hodgkin–Katz voltage equation [31]: Erev ¼
RT ½Clcis þ ðP K =P Cl Þ½Ktrans ln ; zF ½Cltrans þ ðP K =P Cl Þ½Kcis
ð1Þ
where Erev is the potential at which the current is zero, R is the gas constant, T is temperature in Kelvin, z = 1 (chloride anion charge), F is FaradayÕs constant, Pion is the permeability of the ion, and [ion] is the respective concentration of the ion in the cis and trans chambers. We can rewrite Eq. (1) as P K =P Cl ¼
n½Cltrans ½Clcis ; ½Ktrans n½Clcis
ð2Þ
where zFErev n ¼ exp : RT
2.6. Protein concentration assay Protein concentration of SMP particles preparations were measured the Bio-Rad Protein Assay kit according to the instruction of the manufacturer.
3. Results 3.1. Single channel properties of the mitoKATP channel A preparation of the inner mitochondrial membrane was reconstituted into BLM and current changes characteristic for an ion channel were observed (n = 23). Fig. 1A single channel recording in a 50/150 mM KCl (cis/trans) gradient at 0 mV before and after channel incorporation (arrow) into BLM are shown. Current–time traces were recorded in the 50/150 mM KCl (cis/trans) gradient at different voltages (Fig. 1B). Fig. 1C shows current–voltage relationships for single channel opening at different voltages under gradient conditions. The reversal potential measured in the 50/150 mM KCl gradient was equal to 25 mV and this proved that the examined channel was cation-selective. The mean reversal potential calculated after curve fitting to the experimental data was equal to 25.9 ± 2.5 mV allowing calculation of the examined channel permeability PK/PCl at 19.3 (see Section 2). Substances known to modulate the mitoKATP channel activity were also used to examine the properties of the ion channel studied. Addition of 500 lM ATP/Mg2+ inhibited the channel activity (Fig. 2A). The effect was reversed by addition of a potassium channel opener – diazoxide; moreover, potassium channel inhibitors such as 5-HD and glibenclamide inhibited the channel activity (data not shown). Addition of HMR 1098, an inhibitor specific for plasma membrane KATP channels [32,33] had no influence on the channel activity (data not shown). We have recently described the pharmacological profile of the potassium channel activity under investigation [26]. The effect of ATP/Mg2+ complex and ATP on single channel activity was examined. Fig. 2A shows single chan-
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Fig. 1. Single channel recordings of bovine heart mitoKATP channel in planar lipid bilayer. (A) Single channel recordings in 50/150 mM KCl (cis/ trans) gradient before and after reconstitution of SMP vesicles (arrow) at 0 mV. – indicates the closed state. (B) Single channel recordings in 50/ 150 mM KCl (cis/trans) gradient at different voltages (n = 23). (C) Current–voltage characteristics of single channel events in 50/150 mM KCl gradient. (D) Configuration of the cis and trans compartments used in the experiments. Reconstitution of the inner mitochondrial membrane into a planar lipid bilayer was performed as described under Section 2.
nel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV before and after addition of 1 mM Mg2+ + 500 lM ATP to the cis compartment. The addition of ATP/Mg2+ inhibited the channel activity only in cis compartment within 10 min. The channel activity was not influenced by ATP/Mg2+ added to the trans side. The inhibitory effect of ATP–Mg2+was further reversed by addition of 600 lM GDP (cis). In the lower panel, the effects are also seen from the amplitude histograms fitted with Gaussian curves calculated from the same single channel recordings. Interestingly, the channel activity was not affected by application of 500 lM ATP (without the presence of magnesium ions) to the cis compartment (Fig. 2B). The experiments described above proved that the potassium channel observed after incorporation of SMP vesicles in BLM was in fact the mitoKATP channel. In further experiments the effects of Mg2+ were studied. Fig. 3A shows single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and
after addition of 1 mM Mg2+. After addition of 1 mM Mg2+ to the trans compartment were observed changes both in the current amplitude and open probability. Addition of 1 mM Mg2+ to the cis compartment was without effect on the mitoKATP channel activity (Fig. 3A). The open probability (P(open)) of the mitoKATP channel in 50/150 mM KCl (cis/ trans) gradient at 30 mV under control conditions, after addition of 1 mM Mg2+ and after perfusion in the trans compartment are shown in Fig. 3B, left panel. We observed that P(open) increased from 0.71 ± 0.14 to 0.94 ± 0.15 in the presence of 1 mM MgCl2 in the trans compartment. The righthand panel of Fig. 3B shows the current amplitude of the mitoKATP channel in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions, after addition of 1 mM Mg2+ and after perfusion in the trans compartment. The amplitude decreased from 5.4 ± 0.6 pA to 2.2 ± 0.8 pA after 1 mM MgCl2 was added to the trans compartment. Fig. 3C shows the mean open and closed time distribution of the mitoKATP channel in 50/150 mM KCl (cis/trans) gradient at
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Fig. 2. ATP, ATP/Mg2+ and GDP/Mg2+ affect the activity of the mitoKATP channel. (A) Single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and after addition of 1 mM Mg2+ + 500 lM ATP (cis) as well as in the presence of 1 mM Mg2+, 500 lM ATP (cis) and after addition of 600 lM GDP (cis) (n = 5). Shown below are amplitude histograms fitted with superimposed Gaussian curves. (B) Single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and after addition of 500 lM ATP (cis) (n = 5). Shown below are amplitude histograms fitted with superimposed Gaussian curves. – indicates the closed state of the channel. O, open state; C, closed state.
30 mV. The mean open time increased from 32 ± 2 to 172 ± 5 ms, and the mean closed time decreased from 12 ± 2 to 5 ± 2 ms upon addition of 1 mM Mg2+ to the trans compartment. Fig. 4A presents single channel recordings in 50/150 mM KCl (cis/trans) gradient at different voltages under control conditions and after addition of 1 mM Mg2+ to the trans compartment (Fig. 4A). Fig. 4B shows the current–voltage characteristics of single channel events in 50/150 mM KCl gradient under control conditions and after addition of 1 mM Mg2+ to the trans compartment. It shows that the K+ current amplitude was changed by Mg2+ addition only at negative voltages.
In the presence of 1 mM MgCl2 in the trans compartment, the mitoKATP channel conductance decreased from 110 ± 6 to 60 ± 5 pS in 50/150 mM KCl gradient only at negative voltages. Fig. 5A presents single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and after addition of 10 M, 1 mM and 10 mM MgCl2 (trans). The dose–response curve of the K+ current amplitude after MgCl2 addition to (trans) is shown in Fig. 5B. The K+ current amplitude of the mitoKATP channel decreases from 5.2 ± 0.6 pA at the control conditions to 0.0 ± 0.5 pA in the presence
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Fig. 3. Mg2+ affects the activity of the mitoKATP channel. (A) Single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control condition, after addition of 1 mM Mg2+, and after subsequent perfusion in two other experiments. MgCl2 was added only to the cis (n = 5) and or only to the trans (n = 10) compartment. – indicates the closed state. (B) Left panel shows open probability (P(open)) of mitoKATP channel in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions, after addition of 1 mM Mg2+ to the trans compartment and after perfusion. The results are presented as mean ± S.D. (n = 3). *P < 0.01 vs. control. At right panel were shown current amplitude of mitoKATP channel in gradient 50/150 mM KCl (cis/trans) solution at 30 mV under control condition, after addition of 1 mM Mg2+ to the trans compartment and after perfusion. The results are presented as mean ± S.D. (n = 3). **P < 0.001 vs. control. (C) Open and closed dwell time distribution in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions, after addition of 1 mM Mg2+ and after perfusion in trans compartment. The results are presented as means ± S.E. (n = 3). *P < 0.05 vs. control.
of 10 mM Mg2+. It was calculated that MgCl2 inhibits the K+ current amplitude of the mitoKATP channel with an EC50 value of 0.99 ± 0.02 mM.
4. Discussion Magnesium ions regulate the cation transporting systems present in the inner mitochondrial membrane [29]. Reduction of surface-bound magnesium induces Na+ uniport
[34], while depletion of intramitochondrial magnesium unmasks K+ uniport and K+/H+ antiport [30]. Regulation of K+ traffic through the inner mitochondrial membrane controls changes of mitochondrial matrix volume which, in turn, play a regulatory role in mitochondrial metabolism (for a review see [35]). There are strong indications that the mitoKATP channel may be responsible, at least partially, for the K+ uniport activity playing a role in mitochondrial volume homeostasis [36]. Hence, we have decided to study the effects of magnesium on channel activity of cardiac mitoKATP.
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Fig. 4. Single channel recordings of the mitoKATP channel activity in planar lipid bilayer at different voltages. (A) Single channel recordings in 50/150 mM KCl (cis/trans) gradient at different voltages under control and after addition of 1 mM Mg2+ in trans compartment (n = 3). (B) Current–voltage characteristics of single channel events in 50/150 mM KCl gradient under control condition (solid lines, d) and after addition of 1 mM Mg2+ in the trans compartment (solid lines, m). – indicates the closed state.
That Mg2+ interacts with the mitoKATP channel was suggested by previous observations based on flux and binding measurements [3,18,37]. First, with the use of proteoliposomes and flux assay it was shown that Mg2+ was required for ATP inhibition [3]. Also inhibition of the mitoKATP channel by long-chain acyl-CoA esters, like inhibition by ATP, exhibited an absolute requirement for Mg2+ [38]. Second, magnesium reduced the inhibitory potency of glibenclamide to block the cardiac mitoKATP channel [3]. Third, it was shown that Mg2+ caused also an increase in the total and specific binding of [3H]glibenclamide to the inner mitochondrial membrane [19]. In summary, magnesium alone had no effect in the absence of ATP on the mitoKATP channel activity measured with the use of proteoliposomes and the fluorescent, potassium selective, probe PBFI. The lack of an effect of Mg2+ on the channel activity described by Paucek et al. [3] differs from the conclusions drawn from studies in intact mitochondria showing that the K+ electrogenic uptake via K+ uniport is blocked by magnesium ions [38,39]. Measuring single channel activity with the BLM technique gave us a unique opportunity to study the regulation of mitoKATP from both sides of the membrane (cis or trans). In this paper, we have shown that Mg2+ directly affects the mitoKATP channel activity. First, it increases, in the milimolar concentra-
P. Bednarczyk et al. / FEBS Letters 579 (2005) 1625–1632
Fig. 5. Effect of Mg2+ on the activity of mitoKATP channel. (A) Single channel recording in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and after addition of 10 lM, 1 mM and 10 mM MgCl2 to trans compartment (n = 3). – indicates the closed state. (B) The dose–response curve for Mg2+ inhibition of K+ current amplitude. Magnesium was added to the trans compartment.
tion range, the open probability of the mitoKATP channel. Second, it decreases the K+ current amplitude so that at high doses of Mg2+, we were not able to detect any K+ current. The observed effects were reversed upon removal of Mg2+. Interestingly, the Mg2+ effects were asymmetric, i.e., they were observed only upon application of Mg2+ from the trans side. Because the effect of magnesium was observed only from the trans side of the BLM system it was likely that the magnesium regulatory site of the mitoKATP channel exhibits polarity, i.e., it faces the matrix or ‘‘cytosolic’’ side. Previously, it was shown that the inhibitory site for ATP/Mg2+ faces the ‘‘cytosolic’’ side of mitochondria [40]. Using the BLM system we have observed ATP/Mg inhibition [3] and GDP activation [37] of the mitoKATP channel only from the cis side. Hence, we assumed that the cis side represents the ‘‘cytosolic’’ side of mitochondria [40]. The regulation of the mitoKATP channel by magnesium was observed only from the side opposite to that of ATP/Mg action, i.e., from the trans side. This strongly suggests that magnesium ion regulates the mitoKATP channel from the matrix side of mitochondria (Fig. 6). This is in agreement with the observations that Mg2+ matrix (but not surface) depletion induces a K+-selective electrogenic transport in the inner mitochondrial membrane [38,39]. However, rigorous proofs of our postulate will require confirmation from studies on intact mitochondria. The above assumption on the site of Mg2+ action will also lead to the conclusion that submilimolar concentration of free Mg2+ would cause rectification of mitochondrial potassium current: the outward (out of matrix) potassium current will be lower than the inward one. Additional to the lowering of the channel conductance by Mg2+, an increase in the open probability of the mitoKATP
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Fig. 6. Schematic representation of the regulation of the mitoKATP channel by ATP/Mg2+ and Mg2+. Activation is indicated as ¯ and inhibition as §.
channel was observed. Similar results were previously observed by measuring the activity of plasma membrane potassium channels. For example, magnesium ions increased the open probability and reduced the current amplitude of retinal glial cell Ca2+-activated K+ channels [41]. Recently, modulation of the mitoKATP channel by Ca2+ in lymphocytes was described [9]. The calcium ions alone influenced neither open probability nor the burst frequency of the mitoKATP channel. Only the presence of ATP and the initial calcium (before ATP application), modulated the behavior of the mitoKATP channel was observed [9]. This conclusion suggest that the mitoKATP channel posses calcium binding site, similar as the mitoBKCa channel described in cardiac [4] and glioma cells mitochondria [42]. Mitochondria can take up and extrude Mg2+, for example via a cAMP-dependent pathway for fast release of mitochondrial Mg2+ that might be hormonally regulated [43]. In conclusion, the results of our investigations support the concept that magnesium ions interfere with the mitoKATP channel by affecting its conductance and open probability. This poses the question regarding the role that the magnesium regulation of the mitoKATP channel plays in metabolic regulation via mitochondrial matrix volume changes. Acknowledgements: This study was supported in part by Polish State Committee for Scientific Research Grant No. 6P04A01019. This study was also supported partially by NATO collaborative Grant LST.CLG.979217 and by a grant from SGGW No. 50406020013.
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1632 [25] Mattson, M.P. and Liu, D. (2003) Mitochondrial potassium channels and uncoupling proteins in synaptic plasticity and neuronal cell death. Biochem. Biophys. Res. Commun. 304, 539– 549. [26] Bednarczyk, P., Kicinska, A., Kominkova, V., Ondrias, K., Dolowy, K. and Szewczyk, A. (2004) Quinine inhibits mitochondrial ATP-regulated potassium channel from bovine heart. J. Membr. Biol. 199, 63–72. [27] Zhang, D.X., Chen, Y.F., Campbell, W.B., Zou, A.P., Gross, G.J. and Li, P.L. (2001) Characteristics and superoxide-induced activation of reconstituted myocardial mitochondrial ATP-sensitive potassium channels. Circ. Res. 89, 1177–1183. [28] Nakae, Y., Kwok, W.M., Bosnjak, Z.J. and Jiang, M.T. (2003) Isoflurane activates rat mitochondrial ATP-sensitive K+ channels reconstituted in lipid bilayers. Am. J. Physiol. 284, H1865–H1871. [29] Bernardi, P. (1999) Mitochondrial transport of cations: channels, exchangers and permeability transition. Physiol. Rev. 79, 1127– 1155. [30] Garlid, K.D. and Paucek, P. (2003) Mitochondrial potassium transport: the K+ cycle. Biochim. Biophys. Acta. 1606, 23–41. [31] Hille, B. (2001) Selective permeability: independenceIon Channel of Excitable Membranes, pp. 441–470, Sinauer Associates Inc., Sunderland, USA. [32] Gogelein, H., Ruetten, H., Albus, U., Englert, H.C. and Busch, A.E. (2001) Effects of the cardioselective KATP channel blocker HMR1098 on cardiac function in isolated perfused working rat hearts and in anesthetized rats during ischemia and reperfusion. Naunyn Schmiedebergs Arch. Pharmacol. 364, 33–41. [33] Liu, Y., Ren, G., OÕRourke, B., Marban, E. and Seharaseyon, J. (2001) Pharmacological comparison of native mitochondrial KATP channels with molecularly defined surface KATP channels. Mol. Pharmacol. 59, 225–230.
P. Bednarczyk et al. / FEBS Letters 579 (2005) 1625–1632 [34] Brierley, G.P., Baysal, K. and Jung, D. (1994) Cation transport systems in mitochondria: Na+ and K+ uniports and exchangers. J. Bioenerg. Biomembr. 26, 519–526. [35] Halestrap, A.P. (1994) Regulation of mitochondrial metabolism through changes in matrix volume. Biochem. Soc. Trans. 22, 522– 529. [36] Szewczyk, A., Pikula, S., Wojcik, G. and Nalecz, M.J. (1996) Glibenclamide inhibits mitochondrial K+ and Na+ uniports induced by magnesium depletion. Int. J. Biochem. Cell. Biol. 28, 863–971. [37] Paucek, P., Yarov-Yarovoy, V., Sun, X. and Garlid, K.D. (1996) Inhibition of the mitochondrial KATP channel by long-chain acyl-CoA esters and activation by guanine nucleotides. J. Biol. Chem. 271, 32084–32088. [38] Bernardi, P., Angrilli, A., Ambrosin, V. and Azzone, G.F. (1989) Activation of latent K+ uniport in mitochondria treated with ionophore A23187. J. Biol. Chem. 234, 18902–18906. [39] Nicolli, A., Redetti, A. and Bernardi, P. (1991) The K+ conductance of the inner mitochondrial membrane. A study of the inducible uniport for monovalent cations. J. Biol. Chem. 269, 465–470. [40] Yarov-Yarovoy, V., Paucek, P., Jaburek, M. and Garlid, K.D. (1997) The nucleotide regulatory sites on the mitochondrial KATP channel face the cytosol. Biochim. Biophys. Acta 1321, 128–136. [41] Bringmann, A., Faude, F. and Reichenbach, A. (1997) Mammalian retinal glial (Muller) cells express large-conductance Ca2+activated K+ channels that are modulated by Mg2+ and pH and activated by protein kinase A. Glia 19, 311–323. [42] Siemen, D., Loupatatzis, C., Borecky, J., Gulbins, E. and Lang, F. (1999) Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line. Biochem. Biophys. Res. Commun. 257, 549–554. [43] Jung, D.W. and Brierley, G.P. (1994) Magnesium transport by mitochondria. J. Bioenerg. Biomembr. 26, 527–535.
FEBS Letters 579 (2005) 1625–1632
Matrix Mg2+ regulates mitochondrial ATP-dependent potassium channel from heart Piotr Bednarczyka,b, Krzysztof Dołowyb, Adam Szewczyka,* a
Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland b Department of Biophysics, Agricultural University SGGW, 159 Nowoursynowska St., 02-776 Warsaw, Poland Received 2 December 2004; revised 11 January 2005; accepted 31 January 2005 Available online 18 February 2005 Edited by Peter Brzezinski
Abstract Mitochondrial ATP-regulated potassium (mitoKATP) channels play an important role in cardioprotection. Single channel activity was measured after reconstitution of inner mitochondrial membranes from bovine myocardium into a planar lipid bilayer. After incorporation, the potassium channel was recorded with a mean conductance of 103 ± 9 pS. The channel activity was inhibited by ATP/Mg and activated by GDP. Magnesium ions alone affected, in a dose dependent manner, both the channel conductance and the open probability. Magnesium ions regulated the mitoKATP channel only when added to the trans compartment. We conclude that Mg2+ regulates the cardiac mitoKATP channel from the matrix site by affecting both the channel conductance and gating. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Heart mitochondria; Potassium channel; ATP; Magnesium; Black lipid membrane
1. Introduction Potassium selective channels are present in the inner mitochondrial membrane [1,2]. In cardiac mitochondria an ATPregulated potassium channel (mitoKATP channel) [3] and a large conductance Ca2+-activated potassium channel (mitoBKCa channel) [4] have been identified. MitoKATP channels were also found in liver [5], brain [6,7], skeletal muscle [8], and human T-lymphocyte mitochondria [9]. Both the mitoKATP and mitoBKCa channels are regulated by potassium channel openers [10–12], of which diazoxide seems to be an especially potent activator of the mitoKATP channel [13]. The molecular identity of the mitoKATP channel is unknown [1]. Several observations on the pharmacological profile and immunoreactivity with specific antibodies may suggest that the mitoKATP channel belongs to the inward rectifier K+ channel family – Kir6.x [14]. Using specific antibodies, Kir6.1, Kir6.2, and sulfonylurea receptor (SUR2A) subunits were demonstrated in ventricular myocyte mitochondria [15]. Recently, it was hypothesized that a complex of five proteins in * Corresponding author. Fax: +4822 8225342. E-mail address: [email protected] (A. Szewczyk).
Abbreviations: mitoKATP channel, mitochondrial ATP-regulated potassium channel; 5-HD, 5-hydroxydecanoic acid; SMP, submitochondrial particles
the mitochondrial inner membrane is capable of transporting K+ with characteristics similar to those of the mitoKATP channel [16,17]. Similar to the plasma membrane KATP channel, mitoKATP is probably composed of sulfonylurea receptor (mitoSUR). The usage of 125I-glibenclamide lead to labeling of 28 kDa protein in bovine heart mitochondria [18,19]. Fluorescent probe BODIPY-glibenclamide labeled a 64 kDa protein in brain mitochondria [7]. The primary function of the mitoKATP channel is to allow K+ transport into the mitochondrial matrix. This phenomenon could be involved in mitochondrial volume homeostasis [20]. The mitoKATP channel activity is probably coupled to the cellular energetic state via its inhibition by ATP and ADP [21]. Additionally, the channel is blocked by palmitoyl- or oleylCoA [37] and activated by GTP and GDP [37]. The cardiac mitoKATP channel plays an important role in cardioprotection (for reviews, see [11,20,22–24]). Cytoprotective action of potassium channel openers acting on mitochondria was also observed in a neuronal system [25]. The majority of observations concerning regulation of the mitoKATP channel is based on modulation by channel inhibitors and potassium channel openers of mitochondrial function such as matrix volume, mitochondrial potential, or oxygen consumption [3,6,8]. Another approach is based on K+ flux measurements with the use of potassium specific fluorescent dyes [3,7,13] or radioactive 86Rb+ isotope, a K+ analogue [26]. Recently, reconstitution of the mitoKATP channel allowing single channel recordings was applied successfully to study new properties of the mitoKATP channel [26–28]. Addition of xanthine/xanthine oxidase, an O 2 -generating system, resulted in a marked activation of the mitoKATP channel, further abolished by 5-hydroxydecanoic acid (5-HD) or a sulfhydryl alkylating compound, N-ethylmaleimide [27]. Moreover, a direct activation of the mitoKATP channel by the anesthetic isoflurane was observed [28]. Recently, inhibition of the cardiac mitoKATP channel by quinine was described [26]. Magnesium ions have been known for many years to affect cation transporting pathways in mitochondria [29]. These include both K+ and Na+ uniporters [29] and K+/H+ exchanger [30]. Mitochondrial potassium transport plays an essential role in maintaining mitochondrial matrix volume, due to matrix swelling or contraction, and mitochondrial cell signaling by means of reactive oxygen species or cytochrome c release [30]. In our experiments, we used purified mitochondrial inner membrane reconstituted into a planar lipid bilayer in order to study the effects of Mg2+ of the cardiac mitoKATP channel activity. Here, we show that Mg2+ interact with the mitoKATP channel affecting both the channel conductance and the open
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.01.077
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probability. Our data strongly suggest that the cardiac mitoKATP channel is regulated by magnesium from the matrix side.
2. Materials and methods 2.1. Materials L -a-Phosphatidyl-choline (asolectin), n-decane and protease (Subtilisin A, P5380) from Bacillus licheniformis were from Sigma–Aldrich, Germany. All other chemicals were of the highest purity available commercially. 2.2. Isolation of mitochondria Bovine heart mitochondria were isolated at 4 C as described earlier [27]. Briefly, a fragment of bovine heart muscle was minced in isolation buffer (200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 5 mM MOPS, 0.1% BSA, and 1 mM EGTA, pH 7.15) and homogenized with 25 U protease/gram tissue using a teflon pestle. The homogenate was then centrifuged at 8000 · g for 10 min to remove the protease. The pellet was resuspended in the isolation buffer and centrifuged again at 700 · g, for 10 min. to remove cellular debris. The supernatant was centrifuged at 8000 · g for 10 min. at 4 C to pellet the mitochondria. The mitochondria were then washed and suspended in the isolation buffer without EGTA. The suspension was loaded on top of Percoll solution (30% Percoll, 0.25 M sucrose, 1 mM EDTA, and 10 mM HEPES, pH 7.4) and centrifuged at 35 000 · g for 30 min. The mitochondrial fraction was then collected and washed twice with the isolation buffer without EGTA and resuspended at 10–20 mg of protein/ml. 2.3. Preparation of submitochondrial particles Freshly prepared mitochondria were sonicated 8 · 15 s and centrifuged at 16 000 · g for 15 min to pellet unbroken mitochondria. The supernatant was again centrifuged at 140 000 · g for 35 min and submitochondrial particles (SMP) were resuspended in the isolation buffer without EGTA at 5 mg of protein/ml. The polarity of SMP vesicles was estimated to be 40–50% outside-out (depending on the preparation), using cytochrome c oxidase activity measurements. 2.4. Black lipid membrane measurements Black lipid membranes (BLMs) were formed in a 250 lm diameter hole drilled in a Delrin cup (Warner Instrument Corp., Hamden, CT, USA), which separated two chambers (cis and trans 1 ml internal volume). The chambers contained 50/150 mM KCl (cis/trans), 20 mM Tris–HCl, pH 7.2. The outline of the aperture was coated with a lipid solution and N2 dried prior to bilayer formation to improve membrane stability. BLMs were painted using asolectin in n-decane at a final concentration of 25 mg lipid/ml. Bovine heart SMP (1–5 lg of protein) was added to the 1 ml trans compartment (Fig. 1D). Incorporation of the mitoKATP channel into the BLM was usually observed within few minutes. All measurements were carried out at air conditioned room (24 C). Formation and thinning of the bilayer was monitored by capacitance measurements and optical observations. The final accepted capacitance values ranged from 110 to 180 pF. Electrical connections were made by Ag/AgCl electrodes and agar salt bridges (3 M KCl) to minimize liquid junction potentials. Voltage was applied to the cis compartment of the chamber and the trans compartment was grounded. The current was measured using a bilayer membrane amplifier (BLM-120, BioLogic). For removing the solutions from the cis or trans compartments we used a perfusion system containing a holder with glass tip, perfusion pump (sp260p syringe pump, USA), glass syringe (FORTUNA OPTIMA Glasspritze, Aldrich) and teflon tubing (C-FLEX TUBING, Sigma). 2.5. Data analysis Signals were filtered at 500 Hz. The current were digitized at a sampling rate of 100 kHz (A/D converter PowerLab 2/20, ADInstruments) and transferred to a PC or digital tape recorder (DTR-1204, BioLogic) for off-line analysis by Chart v4.2.3 (PowerLab ADInstruments) and pCLAMP8 (Axon Laboratory). The pCLAMP8 software package was used for data processing. The channel recordings illustrated are representative of the most frequently observed conductances under
P. Bednarczyk et al. / FEBS Letters 579 (2005) 1625–1632 the given conditions. The conductance was calculated from the current–voltage relationship. The probability of channel opening (P(open)) was calculated with an automatic interval setting. The channel open (sopen) and closed (sclosed) lifetimes were calculated from logarithmic binning mode using the Marquardt-LSQ fitting method, order one, without weighting. n denotes the number of experiments, and N the number of events. sopen, sclosed, P(open) were calculated from segments of continuous recordings lasting 60 s and with N P 1000 events. Data from the experiments are reported as mean value or values ± S.E. (standard error) or S.D. (standard deviation). The permeability ratios for K+ and Cl were calculated according to the Goldman–Hodgkin–Katz voltage equation [31]: Erev ¼
RT ½Clcis þ ðP K =P Cl Þ½Ktrans ln ; zF ½Cltrans þ ðP K =P Cl Þ½Kcis
ð1Þ
where Erev is the potential at which the current is zero, R is the gas constant, T is temperature in Kelvin, z = 1 (chloride anion charge), F is FaradayÕs constant, Pion is the permeability of the ion, and [ion] is the respective concentration of the ion in the cis and trans chambers. We can rewrite Eq. (1) as P K =P Cl ¼
n½Cltrans ½Clcis ; ½Ktrans n½Clcis
ð2Þ
where zFErev n ¼ exp : RT
2.6. Protein concentration assay Protein concentration of SMP particles preparations were measured the Bio-Rad Protein Assay kit according to the instruction of the manufacturer.
3. Results 3.1. Single channel properties of the mitoKATP channel A preparation of the inner mitochondrial membrane was reconstituted into BLM and current changes characteristic for an ion channel were observed (n = 23). Fig. 1A single channel recording in a 50/150 mM KCl (cis/trans) gradient at 0 mV before and after channel incorporation (arrow) into BLM are shown. Current–time traces were recorded in the 50/150 mM KCl (cis/trans) gradient at different voltages (Fig. 1B). Fig. 1C shows current–voltage relationships for single channel opening at different voltages under gradient conditions. The reversal potential measured in the 50/150 mM KCl gradient was equal to 25 mV and this proved that the examined channel was cation-selective. The mean reversal potential calculated after curve fitting to the experimental data was equal to 25.9 ± 2.5 mV allowing calculation of the examined channel permeability PK/PCl at 19.3 (see Section 2). Substances known to modulate the mitoKATP channel activity were also used to examine the properties of the ion channel studied. Addition of 500 lM ATP/Mg2+ inhibited the channel activity (Fig. 2A). The effect was reversed by addition of a potassium channel opener – diazoxide; moreover, potassium channel inhibitors such as 5-HD and glibenclamide inhibited the channel activity (data not shown). Addition of HMR 1098, an inhibitor specific for plasma membrane KATP channels [32,33] had no influence on the channel activity (data not shown). We have recently described the pharmacological profile of the potassium channel activity under investigation [26]. The effect of ATP/Mg2+ complex and ATP on single channel activity was examined. Fig. 2A shows single chan-
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Fig. 1. Single channel recordings of bovine heart mitoKATP channel in planar lipid bilayer. (A) Single channel recordings in 50/150 mM KCl (cis/ trans) gradient before and after reconstitution of SMP vesicles (arrow) at 0 mV. – indicates the closed state. (B) Single channel recordings in 50/ 150 mM KCl (cis/trans) gradient at different voltages (n = 23). (C) Current–voltage characteristics of single channel events in 50/150 mM KCl gradient. (D) Configuration of the cis and trans compartments used in the experiments. Reconstitution of the inner mitochondrial membrane into a planar lipid bilayer was performed as described under Section 2.
nel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV before and after addition of 1 mM Mg2+ + 500 lM ATP to the cis compartment. The addition of ATP/Mg2+ inhibited the channel activity only in cis compartment within 10 min. The channel activity was not influenced by ATP/Mg2+ added to the trans side. The inhibitory effect of ATP–Mg2+was further reversed by addition of 600 lM GDP (cis). In the lower panel, the effects are also seen from the amplitude histograms fitted with Gaussian curves calculated from the same single channel recordings. Interestingly, the channel activity was not affected by application of 500 lM ATP (without the presence of magnesium ions) to the cis compartment (Fig. 2B). The experiments described above proved that the potassium channel observed after incorporation of SMP vesicles in BLM was in fact the mitoKATP channel. In further experiments the effects of Mg2+ were studied. Fig. 3A shows single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and
after addition of 1 mM Mg2+. After addition of 1 mM Mg2+ to the trans compartment were observed changes both in the current amplitude and open probability. Addition of 1 mM Mg2+ to the cis compartment was without effect on the mitoKATP channel activity (Fig. 3A). The open probability (P(open)) of the mitoKATP channel in 50/150 mM KCl (cis/ trans) gradient at 30 mV under control conditions, after addition of 1 mM Mg2+ and after perfusion in the trans compartment are shown in Fig. 3B, left panel. We observed that P(open) increased from 0.71 ± 0.14 to 0.94 ± 0.15 in the presence of 1 mM MgCl2 in the trans compartment. The righthand panel of Fig. 3B shows the current amplitude of the mitoKATP channel in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions, after addition of 1 mM Mg2+ and after perfusion in the trans compartment. The amplitude decreased from 5.4 ± 0.6 pA to 2.2 ± 0.8 pA after 1 mM MgCl2 was added to the trans compartment. Fig. 3C shows the mean open and closed time distribution of the mitoKATP channel in 50/150 mM KCl (cis/trans) gradient at
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Fig. 2. ATP, ATP/Mg2+ and GDP/Mg2+ affect the activity of the mitoKATP channel. (A) Single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and after addition of 1 mM Mg2+ + 500 lM ATP (cis) as well as in the presence of 1 mM Mg2+, 500 lM ATP (cis) and after addition of 600 lM GDP (cis) (n = 5). Shown below are amplitude histograms fitted with superimposed Gaussian curves. (B) Single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and after addition of 500 lM ATP (cis) (n = 5). Shown below are amplitude histograms fitted with superimposed Gaussian curves. – indicates the closed state of the channel. O, open state; C, closed state.
30 mV. The mean open time increased from 32 ± 2 to 172 ± 5 ms, and the mean closed time decreased from 12 ± 2 to 5 ± 2 ms upon addition of 1 mM Mg2+ to the trans compartment. Fig. 4A presents single channel recordings in 50/150 mM KCl (cis/trans) gradient at different voltages under control conditions and after addition of 1 mM Mg2+ to the trans compartment (Fig. 4A). Fig. 4B shows the current–voltage characteristics of single channel events in 50/150 mM KCl gradient under control conditions and after addition of 1 mM Mg2+ to the trans compartment. It shows that the K+ current amplitude was changed by Mg2+ addition only at negative voltages.
In the presence of 1 mM MgCl2 in the trans compartment, the mitoKATP channel conductance decreased from 110 ± 6 to 60 ± 5 pS in 50/150 mM KCl gradient only at negative voltages. Fig. 5A presents single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and after addition of 10 M, 1 mM and 10 mM MgCl2 (trans). The dose–response curve of the K+ current amplitude after MgCl2 addition to (trans) is shown in Fig. 5B. The K+ current amplitude of the mitoKATP channel decreases from 5.2 ± 0.6 pA at the control conditions to 0.0 ± 0.5 pA in the presence
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Fig. 3. Mg2+ affects the activity of the mitoKATP channel. (A) Single channel recordings in 50/150 mM KCl (cis/trans) gradient at 30 mV under control condition, after addition of 1 mM Mg2+, and after subsequent perfusion in two other experiments. MgCl2 was added only to the cis (n = 5) and or only to the trans (n = 10) compartment. – indicates the closed state. (B) Left panel shows open probability (P(open)) of mitoKATP channel in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions, after addition of 1 mM Mg2+ to the trans compartment and after perfusion. The results are presented as mean ± S.D. (n = 3). *P < 0.01 vs. control. At right panel were shown current amplitude of mitoKATP channel in gradient 50/150 mM KCl (cis/trans) solution at 30 mV under control condition, after addition of 1 mM Mg2+ to the trans compartment and after perfusion. The results are presented as mean ± S.D. (n = 3). **P < 0.001 vs. control. (C) Open and closed dwell time distribution in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions, after addition of 1 mM Mg2+ and after perfusion in trans compartment. The results are presented as means ± S.E. (n = 3). *P < 0.05 vs. control.
of 10 mM Mg2+. It was calculated that MgCl2 inhibits the K+ current amplitude of the mitoKATP channel with an EC50 value of 0.99 ± 0.02 mM.
4. Discussion Magnesium ions regulate the cation transporting systems present in the inner mitochondrial membrane [29]. Reduction of surface-bound magnesium induces Na+ uniport
[34], while depletion of intramitochondrial magnesium unmasks K+ uniport and K+/H+ antiport [30]. Regulation of K+ traffic through the inner mitochondrial membrane controls changes of mitochondrial matrix volume which, in turn, play a regulatory role in mitochondrial metabolism (for a review see [35]). There are strong indications that the mitoKATP channel may be responsible, at least partially, for the K+ uniport activity playing a role in mitochondrial volume homeostasis [36]. Hence, we have decided to study the effects of magnesium on channel activity of cardiac mitoKATP.
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Fig. 4. Single channel recordings of the mitoKATP channel activity in planar lipid bilayer at different voltages. (A) Single channel recordings in 50/150 mM KCl (cis/trans) gradient at different voltages under control and after addition of 1 mM Mg2+ in trans compartment (n = 3). (B) Current–voltage characteristics of single channel events in 50/150 mM KCl gradient under control condition (solid lines, d) and after addition of 1 mM Mg2+ in the trans compartment (solid lines, m). – indicates the closed state.
That Mg2+ interacts with the mitoKATP channel was suggested by previous observations based on flux and binding measurements [3,18,37]. First, with the use of proteoliposomes and flux assay it was shown that Mg2+ was required for ATP inhibition [3]. Also inhibition of the mitoKATP channel by long-chain acyl-CoA esters, like inhibition by ATP, exhibited an absolute requirement for Mg2+ [38]. Second, magnesium reduced the inhibitory potency of glibenclamide to block the cardiac mitoKATP channel [3]. Third, it was shown that Mg2+ caused also an increase in the total and specific binding of [3H]glibenclamide to the inner mitochondrial membrane [19]. In summary, magnesium alone had no effect in the absence of ATP on the mitoKATP channel activity measured with the use of proteoliposomes and the fluorescent, potassium selective, probe PBFI. The lack of an effect of Mg2+ on the channel activity described by Paucek et al. [3] differs from the conclusions drawn from studies in intact mitochondria showing that the K+ electrogenic uptake via K+ uniport is blocked by magnesium ions [38,39]. Measuring single channel activity with the BLM technique gave us a unique opportunity to study the regulation of mitoKATP from both sides of the membrane (cis or trans). In this paper, we have shown that Mg2+ directly affects the mitoKATP channel activity. First, it increases, in the milimolar concentra-
P. Bednarczyk et al. / FEBS Letters 579 (2005) 1625–1632
Fig. 5. Effect of Mg2+ on the activity of mitoKATP channel. (A) Single channel recording in 50/150 mM KCl (cis/trans) gradient at 30 mV under control conditions and after addition of 10 lM, 1 mM and 10 mM MgCl2 to trans compartment (n = 3). – indicates the closed state. (B) The dose–response curve for Mg2+ inhibition of K+ current amplitude. Magnesium was added to the trans compartment.
tion range, the open probability of the mitoKATP channel. Second, it decreases the K+ current amplitude so that at high doses of Mg2+, we were not able to detect any K+ current. The observed effects were reversed upon removal of Mg2+. Interestingly, the Mg2+ effects were asymmetric, i.e., they were observed only upon application of Mg2+ from the trans side. Because the effect of magnesium was observed only from the trans side of the BLM system it was likely that the magnesium regulatory site of the mitoKATP channel exhibits polarity, i.e., it faces the matrix or ‘‘cytosolic’’ side. Previously, it was shown that the inhibitory site for ATP/Mg2+ faces the ‘‘cytosolic’’ side of mitochondria [40]. Using the BLM system we have observed ATP/Mg inhibition [3] and GDP activation [37] of the mitoKATP channel only from the cis side. Hence, we assumed that the cis side represents the ‘‘cytosolic’’ side of mitochondria [40]. The regulation of the mitoKATP channel by magnesium was observed only from the side opposite to that of ATP/Mg action, i.e., from the trans side. This strongly suggests that magnesium ion regulates the mitoKATP channel from the matrix side of mitochondria (Fig. 6). This is in agreement with the observations that Mg2+ matrix (but not surface) depletion induces a K+-selective electrogenic transport in the inner mitochondrial membrane [38,39]. However, rigorous proofs of our postulate will require confirmation from studies on intact mitochondria. The above assumption on the site of Mg2+ action will also lead to the conclusion that submilimolar concentration of free Mg2+ would cause rectification of mitochondrial potassium current: the outward (out of matrix) potassium current will be lower than the inward one. Additional to the lowering of the channel conductance by Mg2+, an increase in the open probability of the mitoKATP
P. Bednarczyk et al. / FEBS Letters 579 (2005) 1625–1632
Fig. 6. Schematic representation of the regulation of the mitoKATP channel by ATP/Mg2+ and Mg2+. Activation is indicated as ¯ and inhibition as §.
channel was observed. Similar results were previously observed by measuring the activity of plasma membrane potassium channels. For example, magnesium ions increased the open probability and reduced the current amplitude of retinal glial cell Ca2+-activated K+ channels [41]. Recently, modulation of the mitoKATP channel by Ca2+ in lymphocytes was described [9]. The calcium ions alone influenced neither open probability nor the burst frequency of the mitoKATP channel. Only the presence of ATP and the initial calcium (before ATP application), modulated the behavior of the mitoKATP channel was observed [9]. This conclusion suggest that the mitoKATP channel posses calcium binding site, similar as the mitoBKCa channel described in cardiac [4] and glioma cells mitochondria [42]. Mitochondria can take up and extrude Mg2+, for example via a cAMP-dependent pathway for fast release of mitochondrial Mg2+ that might be hormonally regulated [43]. In conclusion, the results of our investigations support the concept that magnesium ions interfere with the mitoKATP channel by affecting its conductance and open probability. This poses the question regarding the role that the magnesium regulation of the mitoKATP channel plays in metabolic regulation via mitochondrial matrix volume changes. Acknowledgements: This study was supported in part by Polish State Committee for Scientific Research Grant No. 6P04A01019. This study was also supported partially by NATO collaborative Grant LST.CLG.979217 and by a grant from SGGW No. 50406020013.
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