The potassium channel opener CGS7184 activates Ca2+ release from the endoplasmic reticulum

European Journal of Pharmacology 690 (2012) 60–67

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

The potassium channel opener CGS7184 activates Ca2 þ release from the endoplasmic reticulum Antoni Wrzosek a,n, Zuzana Tomaskova b, Karol Ondrias b, Agnieszka Łukasiak c, Adam Szewczyk a a

Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warszawa, Poland Institute of Molecular Physiology and Genetics, Centre of Excellence for Cardiovascular Research, Slovak Academy of Sciences, Vlarska 5, 833 34 Bratislava, Slovakia c Department of Biophysics, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776 Warszawa, Poland b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 November 2011 Received in revised form 13 June 2012 Accepted 21 June 2012 Available online 1 July 2012

CGS7184 (ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate) is a synthetic large-conductance Ca2 þ -activated potassium (BKCa) channel opener. The existing literature suggests that potassium channels are involved in cardioprotection, particularly during ischemia-reperfusion events. However, the cellular mechanisms mediating the effects of CGS7184 remain unclear. In the present study, we investigated the effect of the BKCa channel opener CGS7184 on Ca2 þ homeostasis in H9C2 and C2C12 cell lines, Ca2 þ uptake by isolated sarcoplasmic reticulum (SR) vesicles, SR Ca2 þ -ATPase (SERCA) activity, and single-channel properties of the ryanodine receptor calcium release channel (RYR2) when incorporated into a planar lipid bilayer. The effects of CGS7184 on calcium homeostasis in C2C12 and H9C2 cell lines were measured with a Fura-2 fluorescent indicator. The BKCa channel opener CGS7184, when added to the H9C2 and C2C12 cells, caused a concentrationdependent release of calcium from internal stores. Calcium accumulation by the SR vesicles isolated from cardiac and skeletal muscle was inhibited by CGS7184 with a half-maximal inhibition value of 0.45 70.04 mM and 0.377 0.03 mM, respectively. The results of the present study indicate that the BKCa channel opener CGS7184 modulates cytosolic Ca2 þ concentration in H9C2 and C1C12 cells due to its interaction with the endoplasmic reticulum (ER). CGS7184 approximately doubled the opening probability of RYR2 channels; however, the compound seemed to most strongly affect channels with a higher control activity. These results strongly suggest that the BKCa channel opener CGS7184 affects intracellular calcium homeostasis by interacting with the sarcoplasmic reticulum RYR2 channels. & 2012 Elsevier B.V. All rights reserved.

Keywords: Potassium channel opener Ryanodine receptor Calcium channel Single-channel property Planar lipid bilayer

1. Introduction Large-conductance Ca2 þ -activated potassium (BKCa) channels are present in a variety of electrically excitable and non-excitable cells. BKCa channels are involved in cytoprotection during ischemia-reperfusion, neuronal secretion, hypertension, erectile responses, and cell metastasis (Cui et al., 2009; Eichhorn and Dobrev, 2007; Feletou et al., 2009). They link membrane excitability with intracellular Ca2 þ signalling and are important in smooth muscle contraction (Sah, 1996). BKCa channels belong to a family of K þ channels that are activated by membrane depolarisation or elevated cytosolic Ca2 þ concentrations. These channels comprise a unique class of ion channels (Cui et al., 2009; Ghatta et al., 2006; Sah, 1996). Beneficial effects of BKCa channels on neuronal survival have been attributed primarily to plasma membrane hyperpolarisation, as BKCa channels are voltage- and

n

Corresponding author. Tel.: þ48 22 589 2269; fax: þ 48 22 822 5342. E-mail address: [email protected] (A. Wrzosek).

0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.06.029

calcium-dependent potassium channels whose activation tends to reduce cellular excitability (Runden-Pran et al., 2002). BKCa channels are also present as inner mitochondrial membrane (mitoBKCa) channels. The first observed mitoBKCa channel was noted in LN229 cells using a patch-clamp technique (Siemen et al., 1999). Recently, mitoBKCa channels were discovered in inner mitochondrial membranes of various cell types (Szewczyk et al., 2009). BKCa channels are modulated by natural and synthetic compounds (Bentzen et al., 2007; Calderone et al., 2007; Candia et al., 1992; Nardi and Olesen, 2008; Sakamoto et al., 2008; Wang et al., 2008; Zhang et al., 2010). The stimulatory activities of NS004, NS1619, and NS1608 have been studied extensively and are well documented in cloned and native BKCa channels (Edwards et al., 1994; Hu and Kim, 1996; Olesen et al., 1994; Xu et al., 1994). The major limitations of this class of compounds are their weak potency and insufficient selectivity, which render them inadequate as pharmacological probes or therapeutic agents (Edwards et al., 1994; Holland et al., 1996; Hu and Kim, 1996; Xu et al., 1994). In contrast ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate

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(CGS7184), a more potent compound, has been found to produce BKCa channel-opening effect with a threshold effective concentration slightly below 0.1 mM when assessed using inside-out patches in rat and guinea pig bladder cells (Hu et al., 1997). Recently, we demonstrated that CGS7184 reduced reactive oxygen species production by respiratory chain complex I in mitochondria isolated from rat brains (Kulawiak et al., 2008). We have shown that CGS7184 affects mitochondrial function by changes in mitochondrial potential, oxygen consumption in glioma and EA.hy 926 cells, and activation of nitric oxide synthase pathways in EA.hy 926 cells (Debska-Vielhaber et al., 2009). An additional effect of CGS7184 on glioma cells is cell death, which is caused by an increase in cytosolic Ca2 þ concentration followed by the activation of calpains (Debska-Vielhaber et al., 2009). The effects triggered by CGS7184 in EA.hy 926 and glioma cells seem to be related to modulation of intracellular Ca2 þ homeostasis (Wrzosek et al., 2009). In the present study, our aim was to identify targets for the large-conductance potassium channel opener CGS7184 in the context of Ca2 þ homeostasis. A direct effect of CGS7184 on SR, especially the ryanodine receptor calcium release (RYR2) channel was observed.

2. Methods 2.1. Chemicals Dulbecco’s modified Eagle’s medium and phosphate-buffered saline were purchased from the Institute of Immunology and Experimental Therapy (Wroclaw, Poland). Foetal bovine serum, L-glutamine, and penicillin-streptomycin were obtained from GIBCO (Paisley, Scotland). Fura-2 acetoxymethyl-ester (Fura-2 AM) was purchased from molecular probes (Eugene, Oregon) and dissolved in dimethyl sulfoxide (DMSO) before use. Because some compounds of interest were dissolved in DMSO, we also used this solvent for control experiments. CGS7184 was a kind gift from Novartis (Basel, Switzerland). All other chemicals used were of high grade and obtained from Sigma unless otherwise stated. Our investigations involving animals conform to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and the experimental procedures used in the present study were approved by the local animal research committee of the Nencki Institute of Experimental Biology. 2.2. Isolation of sarcoplasmic reticulum (SR) vesicles from skeletal muscle The SR vesicles from skeletal muscle were prepared as described previously (Chu et al., 1988) with some modifications (Suko and Hellmann, 1998). Briefly, Wistar rats weighing approximately 250 g were sacrificed. All remaining procedures were performed at 0–4 1C. The white back muscles and the leg muscles (fast twitch muscle) were quickly excised; rinsed in homogenising medium; trimmed of fat, connective tissue, and red muscle; and minced. A 50 g portion of ground muscle was homogenised with 500 ml of homogenizing medium (consisting of 5 mM imidazole pH 7.4, 100 mM NaCl, and 0.1 mM PMSF adjusted with HCl at room temperature) in a Waring blender for 1.5 min at maximal speed. The homogenates were centrifuged in a Sorvall RC 6 Plus centrifuge with a SLA-1500 rotor for 20 min at 4000  g. The supernatant was filtered through layers of cheesecloth and centrifuged in a Sorvall RC 6 Plus centrifuge with a SLA-1500 rotor for 20 min at 5465  g. The supernatant was filtered through layers of cheesecloth and centrifuged in a Sorvall centrifuge with

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a Type 45 Ti rotor for 60 min at 110,000  g. The pellets were combined and resuspended in resuspension medium consisting of 5 mM imidazole-HCl (pH 7.4), 600 mM KCl, 250 mM sucrose, and 0.1 mM PMSF. Each microsomal fraction was loaded on a sucrose step-gradient constructed of 45%, 38%, 34%, 32%, and 27% sucrose (w/w) in 5 mM imidazole-HCl (pH 7.4) and 100 mM NaCl. The gradient was centrifuged in a Sorvall centrifuge with an AH-629 rotor for 3 h at 110,000  g. The 32–34% and 38–45% fractions were collected and diluted with 10 mM HEPES–Tris pH 7.4, 100 mM NaCl, and 0.1 mM PMSF and sedimented for 1 h at 125,000  g. The pellets were resuspended in storage buffer consisting of 10 mM HEPES–Tris pH 7.4, 100 mM NaCl, 250 mM sucrose, and 0.1 mM PMSF to give a protein concentration of approximately 10 mg/ml, divided into appropriate aliquots, snapfrozen in liquid nitrogen, and stored at  70 1C until use. 2.3. Isolation of sarcoplasmic reticulum vesicles from rat cardiac muscle Cardiac muscle SR vesicles were isolated from Wistar rat ventricular tissue according to a previously described method (Buck et al., 1999) with a few modifications, such as the omission of the sucrose gradient step (Chamberlain et al., 1983). Briefly, three animals weighing between 250 and 350 g were anesthetised with a sublethal dose of Nembutal (30 mg/kg) and sacrificed by cervical dislocation. The hearts were immediately removed and immersed in washing solution (0.154 M NaCl, 0.29 M sucrose) at room temperature and then transferred to ice-cold washing solution. The ventricles were trimmed of fat, atria, connective tissue, and large vessels, blotted, and weighted. The isolated ventricles were minced and homogenised in 7 volumes of homogenisation solution composed of 300 mM sucrose, 0.5 mM DTT, 0.1 mM PMSF, and 20 mM K/HEPES (pH 7.4) at 4 1C for 60 s in a Waring blender. The homogenate was centrifuged for 20 min at 9200  g in a SS-34 rotor in a Sorvall RC 6 plus centrifuge. A crude microsomal fraction was obtained from the supernatant by centrifugation for 60 min at 90,000  g in Beckman Type 60 Ti rotor. The soft pellets were resuspended for salt washing in solution (290 mM sucrose, 650 mM KCl, 0.5 mM DTT, 10 mM MOPS, pH 6.8) via manual homogenisation in a Potter tissue grinder with a Teflon pestle. After 60 min incubation on ice, the solution was centrifuged for 10 min at 4400  g in a SS-34 rotor to remove large aggregates. The supernatants were centrifuged for 60 min at 100,000  g in a Beckman Type 60 Ti rotor. The pellet was resuspended by manual homogenisation in a Potter tissue grinder to a protein concentration of 5–10 mg/ml in solution (290 mM sucrose, 200 mM KCl, 5 mM MOPS, pH 6.8). The samples were then snap-frozen in liquid nitrogen and stored at 70 1C until use. 2.4. Calcium uptake measurements The calcium uptake of the SR was measured at 37 1C based on Fura-2 pentapotassium salt fluorescence assaying (Kargacin et al., 2000; Wrzosek et al., 1992) with modifications. Briefly, fluorescence was measured using a SPEX Fluorolog fluorimeter (SPEX Instrument Inc., USA). Fura-2 free acid (1 mM) and SR vesicles were added to 3 ml of uptake buffer composed of 20 mM HEPES– Tris pH 7.0, 100 mM KCl, 100 mM sucrose, 2 mM MgCl2, 5 mM oxalate, 2 mM NaN3, 1.1 mM creatine phosphate, and 3 U/ml creatine phosphokinase in a 3-ml cuvette. Ca2 þ uptake was initiated by the addition of 1 mM ATP. The free Ca2 þ concentration, when not buffered by EGTA, was greater than 10 mM. The excitation wavelengths (340 nm and 380 nm, with emission at 510 nm) were alternated every 0.2 s, and the 340 nm/380 nm fluorescence ratio was determined at 1 s intervals.

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2.5. Cell culture conditions The rat embryonic cardiomyoblast-derived cell line H9C2 and C2C12 murine myoblasts were obtained from ECACC. The cells were cultured at 37 1C in a humidified atmosphere containing 5% CO2/95% air in Dulbecco’s Modified Eagle’s Medium supplemented with 10% foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. In the case of the C2C12 murine myoblasts, the foetal bovine serum concentration was increased to 20%. The glutamine concentration was increased to 4 mM for the H9C2 cells.

serum-free Dulbecco’s Modified Eagle’s Medium. The cells were then washed with MOPS buffer containing 120 mM NaCl, 1 mM MgCl2, 5.4 mM KCl, 0.33 mM Na2HPO4, 11 mM glucose, 30 mM MOPS, 5 mM taurine, 2 mM pyruvate, and 1.5 mM glutamine, pH 7.4. Next, the cover slips were placed in cuvettes in the same buffer and analysed using a Shimadzu RF-5301PC spectrofluorophotometer (Tokyo, Japan). The samples were excited at 340 nm and 380 nm, and the emission fluorescence was monitored at 510 nm. All Fura-2 measurements were performed at room temperature.

2.7. Biomol green phosphate assay 2.6. Measurement of cytosolic Ca2 þ concentration Ca2 þ concentration changes were monitored by observing the fluorescence of the hydrolysed form of Fura-2 AM. Cells grown on coverslips were incubated with 2 mM Fura-2 AM and 0.02% Pluronic F-127 (Sigma) for 45 min at 37 1C and 5% CO2 in air in

Ca2 þ -ATPase activities were measured using an inorganic phosphate (Pi) release assay with the Biomol Green phosphate assay reagent obtained from Enzo Life Sciences (Loerrach, Germany). The levels of Pi released during the calcium-uptake assay were determined by mixing 100 ml of sample with the

Fig. 1. Changes in cytosolic Ca2 þ concentration in the H9C2 and C2C12 cell lines following the addition of CGS7184. (A), (C) changes in the Fura-2 fluorescence ratio upon addition of CGS7184. CGS7184 was added in the absence of Ca2 þ in the extracellular medium. Calcium ions were added to achieve a final concentration of 1.5 mM, as indicated in (A) and (C). (B), (D) concentration–response curve of changes in fluorescence ratio in response to CGS7184 addition in the absence of Ca2 þ in the cell incubation medium. The Fura-2 fluorescence was measured at 510 nm with two excitation wavelengths: 340 nm and 380 nm (see Methods 2.6.). Error bars represent the S.E.M. (n ¼3–5).

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Biomol reagent, and, after a 30 min incubation at 25 1C, the reading absorbance at 620 nm. 2.8. Isolation of membrane vesicles and planar lipid bilayer measurements Cardiac sarcoplasmic reticulum vesicles were isolated from Wistar rat hearts according to the method described by Buck et al. (1999) with a few modifications (Buck et al., 1999; Gaburjakova and Gaburjakova, 2006). The formation of the lipid bilayer, fusion of membrane vesicles, and measurement of single-channel currents were performed as described in our previous studies (Gaburjakova and Gaburjakova, 2006; Marx et al., 2001). The cis chamber was filled with 1 ml of 250 mM HEPES, 125 mM Tris, 1 mM KCl, 1 mM EGTA, and 0.5 mM CaCl2 (pH 7.35). The trans chamber (corresponding to the lumen) was filled with 1 ml of 53 mM Ca(OH)2, 1 mM KCl, and 250 mM HEPES (pH 7.35). The free Ca2 þ concentration was calculated using WinMaxc32 version 2.50 (http://www.stanford.edu/_cpat ton/maxc.html). CGS7184 was applied to the cis or trans side of the lipid bilayer. Single-channel currents were filtered by a low-pass Bessel filter at a corner frequency of 1 kHz and digitised at a sampling rate of 4 kHz using a DigiData 1200 digitiser (Axon Instruments, Foster City, CA, USA). Data were then stored in an IBM-compatible computer using pClamp5 software (Axon Instruments), which was also used for processing the data. Data are presented as the median open probability and corresponding interquartile range (IQR, Q1–Q3). The current and conductance are presented as the mean7S.D. from N experiments. A non-parametric Wilcoxon signed rank test was used to determine the significance of the effect of CGS7184 on ion channel activity. A probability value (P) less than 0.05 was considered statistically significant.

3. Results 3.1. Effects of the BKCa channel opener CGS7184 on calcium levels in H9C2 and C2C12 cells A rat embryonic heart cell line (H9C2) and mouse myoblasts (C2C12) were used to study the Ca2 þ homeostasis changes caused

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by the addition of CGS7184. CGS7184, at a concentration of 5 mM, increased cytosolic Ca2 þ concentration in H9C2 (Fig. 1A) and C2C12 (Fig. 1C) cell lines cultured on glass cover slips. In a calcium-free bath medium, CGS7184 caused a substantial increase in the Fura-2 fluorescence ratio in both cell lines due to the release of intracellular Ca2 þ ([Ca2 þ ]i) from intracellular stores (Fig. 1A and C). Titration with increasing concentrations of CGS7184 in both types of cells caused a concentration-dependent elevation in intracellular Ca2 þ concentration (Fig. 1B and D), with an EC50 of approximately 0.6 mM and a maximal response at 5 mM. In the H9C2 cells, 10 mM CGS7184 caused an increase in [Ca2 þ ]i, assessed using the Fura-2 fluorescence ratio, of 307 741% when compared to the control condition (Fig. 1B). In C2C12 cells, the Fura-2 fluorescence ratio increased by 327 761% when compared to the control condition (Fig. 1D). The addition of CaCl2 (1.5 mM) before CGS7184 resulted in a insignificant increase in [Ca2 þ ]i at the 0.05 level of significance. The Fura-2 fluorescence ratio in the absence of Ca2 þ in H9C2 cells was 0.80070.167 and increased to 0.97270.263 after the addition of 1.5 mM CaCl2. In the case of the C2C12 cells, the Fura-2 fluorescence ratio changed from 0.7107 0.139 and 0.76670.198, respectively, in the absence and presence of Ca2 þ in the incubation medium. The addition of CaCl2 (1.5 mM) following the application of CGS7184 led to an immediate increase in [Ca2 þ ]i (Fig. 1A and C). 3.2. Effects of CGS7184 on calcium uptake and Ca2 þ -ATPase activity in SR vesicles To determine whether CGS7184 directly inhibits the SR Ca2 þ -ATPase, colourimetric measurements of Pi release from ATP by SERCA were made using the Biomol Green compound (see Methods 2.7). The samples analysed for Pi release were taken directly from the calcium-uptake measurement reaction medium (see Fig. 2B). The uptake of Ca2 þ into SR vesicles derived from fast-twitch rat skeletal muscle was inhibited by CGS7184 in a concentration-dependent manner (Fig. 2B), but the SERCA pump hydrolytic activity increased (Fig. 2A) similarly to that obtained in the presence of ionomycin. These results suggest that the process of Ca2 þ -uptake inhibition is related to the release of Ca2 þ from SR vesicles.

Fig. 2. Effect of CGS7184 on Ca2 þ -ATPase activity in skeletal muscle sarcoplasmic reticulum (SR) (A), and calcium uptake measured in SR isolated from rat skeletal muscle (B). (A) changes in Ca2 þ -ATPase activity, as indicated by changes in Biomol Green absorbance (see Methods 2.7.) in skeletal muscle SR under control conditions (-’-) and after treatment with 0.1 mM (-K-), 0.3 mM (-m-), and 1 mM (-.-) CGS7184, respectively. For Ca2 þ -ATPase activity measurements, the samples were taken at the time indicated in panel B. (B) Representative traces of calcium uptake, as indicated by changes in the fluorescence ratio of Fura-2, in SR vesicles under the same conditions as in panel A. Symbols are the same for both panels A and B. Error bars represent the S.D. (n¼3).

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Fig. 3. Changes in rate of Ca2 þ -uptake by SR vesicles isolated from rat cardiac (A) and skeletal muscle (B). The maximal rate of Ca2 þ -uptake was calculated from the straight part of curve of changes in fluorescence ratio of Fura-2 for each concentration of CGS7184. The insets represent calcium release from loaded SR vesicles after the addition of a submaximal concentration of CGS7184 (5 mM). Error bars represent the S.D. (n ¼3).

3.3. Effect of CGS7184 on Ca2 þ handling by SR vesicles The effect of CGS7184 on the rate of Ca2þ accumulation by SR vesicles isolated from rat cardiac and skeletal muscles in the presence of oxalate were measured (Fig. 3). The rate of Ca2 þ -uptake was evaluated as the maximal speed of change in the Fura-2 fluorescence ratio as a function of Ca2þ concentration. This method is very sensitive for detecting the equilibrium of Ca2 þ -uptake and release by SR vesicles suspended in buffer solution (Kargacin et al., 2000). As calculated from the straight part of the Fura-2 fluorescence ratio decay curve the half-inhibition values by CGS7184 for Ca2 þ uptake by SR vesicles from rat cardiac and skeletal muscle were 0.4570.04 mM and 0.3770.03 mM, respectively. The process of Ca2 þ -uptake into SR vesicles continued until steady-state conditions were reached (Fig. 3. A and B inset). The addition of CGS7184 (5 mM) evoked the release of accumulated Ca2 þ from SR vesicles, as detected by an increase in the fluorescence ratio of Fura-2 in suspensions of SR preparations obtained from rat cardiac and skeletal muscles (Fig. 3. A and B see insets). The increase in the fluorescence ratio of Fura-2 in the presence of CGS7184 was comparable to that obtained for ionomycin or thapsigargin (1 mM each). It is well known that compounds that influence Ca2 þ handling by SR vesicles can act on calcium release (RYR and IP3-receptor) channels, SR membrane permeability to Ca2 þ or inhibition of SERCA pump activity. We have used ruthenium red (1 mM), a potent inhibitor of RYR channels activity and heparin (1 mM), an inhibitor of the IP3 receptor, to study the effect of CGS7184 on Ca2 þ -uptake by SR vesicles isolated from rat skeletal muscle (Fig. 4). The addition of ruthenium red and heparin to the incubation medium of the SR vesicles isolated from rat skeletal muscle increased the CGS7184 inhibitory concentration (Fig. 4). The activation value for CGS7184 changes from 0.54370.009 to 0.50870.002 at the 0.05 level of significance in the absence and in the presence of ruthenium red, respectively. Using the planar lipid bilayer technique, we demonstrated that CGS7184 did not significantly change the conductance of preformed lipid bilayers at concentrations of up to 5 mM. To test the hypothesis that the molecular target for CGS7184 is a Ca2 þ release RYR channel, SR vesicle preparations from rat cardiac muscle were fused with the lipid bilayer, and single RYR2 channel activity was measured.

Fig. 4. Changes in Ca2 þ -uptake caused by different CGS7184 concentration in the absence and presence of ruthenium red and heparin (1 mM each). Changes in the rate of Ca2 þ -uptake in control conditions (-’-) and after addition of 1 mM Ruthenium red and heparin (-K-). Error bars represent the S.D. (n¼ 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Effect of CGS7184 on RYR2 channel opening probability After incorporating the SR membrane vesicles isolated from rat cardiac muscle into a bilayer lipid membrane (BLM), we examined the activity of the Ca2 þ -permeable channels. The observed channels (n ¼22) had properties typical of RYR2 channels, with a conductance of 107 712 pS and calcium current at 0 mV of 3.370.3 pA. They were activated by caffeine, and ryanodine

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locked the channels at approximately 50% of the maximum conductance (data not shown). High Ca2 þ concentrations on the cytosolic side of RYR2 inhibit the channel (Laver et al., 1995). Therefore, in our experiments, the measured RYR2 channels faced the cis solution with the cytosolic side. The channels with cytosolic side facing the trans solution were inhibited by the high calcium concentration present in the solution. This experimental design ensured the consistent orientation of all of the measured channels. The cytosolic side of all the measured channels faced the cis solution, and their luminal side faced the trans solution. To study the effect of CGS7184 on RYR2 channels, 5 mM of CGS7184 was added on either the cis or trans side of the channel. The channel activity was quantified by the median open probability, Po. When the compound was applied to the cis side (N ¼ 4), the Po increased from 0.0075 (0.0056–0.0444) to 0.0142 (0.0087– 0.3515). Application to the trans side (N ¼4) shifted Po from 0.0029 (0.0019–0.0201) to 0.0191 (0.0088–0.2480). Thus, the presence of CGS7184 approximately doubled Po when applied from the cis side, and increased Po approximately 6-fold from the trans side (Fig. 5A). This effect was, however, not significant (P¼0.2500 for the cis- and P¼0.1250 for the trans-side effect). The heterogeneity of the channel activity after CGS7184 addition, as seen from the broad interquartile range, was responsible for the lack of significance. The effect seemed more pronounced when the RYR2 channel was more active.

Fig. 5. The effect of 5 mM CGS7184 on the RYR2 channels from both the cytosolic (cis) and luminal (trans) sides. (A) The control conditions corresponded to 90 nM free Ca2 þ on the cytosolic side. Due to the heterogeneity of the channel activity, there was no significant increase of RYR2 median open probability. However, when 3 mM caffeine was added to the cytosolic side (B), the effect was more homogenous and the activation was statistically significant (indicated by an asterisk) when CGS7184 was applied from the cis or trans side of the RYR2 channel. All data data are presented as the median (horizontal line), interquartile range (box), and minimum and maximum (whiskers).

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To increase the Po of the RYR2 channels, 3 mM caffeine was applied to the cytosolic side of the channels and set as a control. The subsequent addition of 5 mM CGS7184 to the cis compartment (N¼ 7) further enhanced the channel activity and the median Po increased 2.1-fold compared to the control. The Po value changed from 0.0888 (0.0422–0.1090) to 0.1909 (0.0922– 0.3709). The effect from the cytosolic side was statistically significant, with P¼ 0.0156, as was the effect from the luminal side (P¼0.0234, Fig. 5B). As in the previous set of experiments, the effect was more evident from the luminal side (2.6-fold increase, N ¼7). Although the absolute values of Po were lower, Po increased from 0.0253 (0.0112–0.3833) to 0.0661 (0.0215– 0.4817) following luminal-side addition. The reversibility of the CGS7184 effect was studied only from the cytosolic side, as only the cis compartment could be perfused. The effect was fully reversible (N ¼4). A representative current trace is shown in Fig. 6. Two RYR2 channels were incorporated into the BLMs in presence of 90 nM free cytosolic Ca2 þ after the addition of 3 mM caffeine and the subsequent application of 5 mM CGS7184 to the cytosolic (cis) side of the RYR2 channels. The two lowest traces

Fig. 6. Representative current traces of ryanodine receptors incorporated into the planar lipid bilayer. Two channels were present in the lipid membrane. (A) Current trace at 90 nM free cytosolic Ca2 þ . (B) The channel was activated by 3 mM caffeine from the cytosolic (cis) side. (C) The channel was further activated by the addition of 5 mM CGS7184 to the cis side. (D) The activation effect was reversible. The channel activity was reduced after reperfusions of the cis compartment with a solution containing 90 nM free cytosolic Ca2 þ as in trace (A). (E) The channel was still sensitive to caffeine. Black solid lines at the left side of the traces indicate the closed level. The dotted lines indicate the open levels for one channel and two channels. The channels open upwards.

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indicate the reversibility of CGS7184 effect and the response to caffeine after CGS7184 wash-out.

4. Discussion Our results indicate that CGS7184 directly affects the RyR2 channels isolated from rat cardiac muscle. When CGS7184 is added to a suspension of SR vesicles from rat cardiac and skeletal muscles, the ryanodine receptor is significantly activated, which increases leakage from SR vesicles. Our results suggest that the CGS7184 direct effect on RYR channels may help explain the depletion of intracellular stores in H9C2 and C2C12 cells. In addition to their role in regulating K þ homeostasis, potassium channel openers are able to affect the passage of other ions across biological membranes. The activation of BKCa channels in the plasma membrane leads to its hyperpolarisation. Cell membrane hyperpolarisation in non-excitable cells causes an increase in Ca2þ influx, whereas hyperpolarisation in excitable cells decreases in Ca2þ influx because of the inhibition of the voltage-dependent calcium channels (Feletou et al., 2009). Previous studies have shown that the potassium channel opener CGS7184 induced an elevation of cytosolic Ca2 þ concentration in glioma and endothelial EA.hy 926 cells, and it was strongly suggested that this process is, at the least, related to the endoplasmic reticulum (Debska-Vielhaber et al., 2009; Wrzosek et al., 2009). Heart-derived H9C2 myoblasts that do not express BKCa channels (Wang et al., 2006) and C2C12 cells expressing these channels are also influenced by CGS7184 (Fig. 1) and demonstrate entry of extracellular calcium ions entry after the emptying of the Ca2 þ stores (as observed in glioma and endothelial cells) (DebskaVielhaber et al., 2009; Wrzosek et al., 2009). In our experiments, the L-type calcium channel should be in a closed state because the activation of BKCa channels in the plasma membrane leads to its hyperpolarisation. In this case, the calcium influx should be related to the store-operated Ca2þ entry (Dirksen, 2009; Huang et al., 2006). It is possible that CGS7184 can interact directly with L-type channels, but our experiments with nifedipine did not support this hypothesis (data not shown). Recently, the RYR channels were examined in heart mitoplasts with its biophysical and pharmacological properties, and the channel can also affect Ca2 þ homeostasis in studied cell lines (Ryu et al., 2011). But, it is not a case, because the all CGS7184 effects were observed in the isolated SR vesicles with similar potency (Fig. 3). The uptake of Ca2 þ into the SR continues until active transport of Ca2 þ is balanced by the passive leak of calcium ions from the SR (Brini and Carafoli, 2009). The SERCAs pumps are regulated in vivo by the cytosolic concentration of Ca2 þ , and in cardiac muscle, the pump is further regulated by the phosphorylation of the SR regulatory protein phospholamban (Traaseth et al., 2008). The release of Ca2 þ from the SR, as mentioned above, can be caused by inhibition of the SERCA pump, increases in permeability to calcium ions caused by CGS7184, or activation of RYR or IP3 channels (Endo, 2009; Wray and Burdyga, 2010; Wrzosek et al., 1992). The possibility that CGS7184 affects the SERCA pump, thus causing the inhibition of Ca2þ uptake into SR vesicles (Fig. 2), is contradicted by the CGS7184-stimulated increase in ATP hydrolytic activity, which was similar to that caused by ionomycin, which promotes constant SERCA hydrolytic activity due to Ca2 þ leakage through the SR membrane. The measurements of SERCA pump hydrolytic activity via the monitoring of Pi release with Biomol Green reagent should consider that inorganic phosphate can be released by phosphatase enzyme. In our case the basal release of Pi from SR vesicle preparations after treatment with thapsigargin was lower than a few percent. Another possibility is that CGS7184 acts to uncouples Ca2 þ transport from SERCA ATPase activity (Nigro et al., 2009). However, it is unlikely that this heat-producing state actually occurs because the same effect was observed in SR isolated from rat cardiac muscle (Fig. 3). The increased permeability of the SR

membrane is also unable to account for the results because the concentration of CGS7184 used in the experiments did not affect the permeability of the planar lipid bilayer (see Methods 3.4). The strongest evidence that the targets for CGS7184 are the RYR channels is the inhibition caused by ruthenium red and heparin (Fig. 4). Ruthenium red is a potent inhibitor of RYR channel activity and induces subconductance by binding to the cytosolic and luminal sites of the skeletal and cardiac ryanodine channels (Neumann et al., 2011; Xu et al., 1999). The literature contains reports that state that the IP3receptor is expressed in skeletal muscle, and this receptor is present in SR membranes (Cardenas et al., 2010). Thus, the SR preparations could contain the IP3-receptor, resulting in the release of calcium ions from the SR vesicles independently from the RYR channels but no changes in calcium uptake by the SR vesicles in the presence or absence of 1 mM heparin was observed (data not shown). Even if CGS7184 does not interact directly with the IP3-receptor, calcium released from the SR could activate the IP3 receptor via indirect activation by Ca2 þ released from the SR vesicles via RyR channel. CGS7184 had a weak activation effect on the RYR2 channels, but the effect was not seen for channels with very low activity (Po o0.01). Interestingly, the effect was similar on both sides of the RYR2 channel, though the effect on the luminal side was more pronounced. This finding may indicate that the mechanism of activation is not connected to some specific binding site and that the compound affects the conformation of the channel protein when the channel is open for relatively long time. The results suggest that the open conformation of the RYR2 channel is slightly energetically favoured. The activation of the RYR2 channel by CGS7184 influences excitationcontraction coupling, which, in turn, influences the function of the heart. Few exogenous compounds can activate the RYR2 channel, including caffeine and ryanodine; therefore, CGS7184 may be a good candidate for further studies of the effect of the RYR2 channel on heart function. To keep the SR space electrically neutral, the movement of positively charged Ca2 þ ions is likely to be accompanied by the efflux of Cl  anions or influx of K þ cations (Kargacin et al., 2000; Nigro et al., 2009). RYR channels exist in the mitochondrial inner membrane (mitoRYR); therefore, the action of CGS7184 on mitochondria could also be related to such channels (Feissner et al., 2009). However, the localisation of the RYR channel in the mitochondrial inner membrane in rat ventricular myocytes has been questioned (Salnikov et al., 2009). There are no available compounds with specificity for only the plasmalemmal or mitochondrial BKCa channels, which suggests that BKCa-channel modulators have many sites of action (Park et al., 2007). We conclude that the potassium channel opener CGS7184 not only acts on BKCa channels but also directly activates RYR2 channels in rat cardiac muscle. CGS7184 is a potent compound that evokes the release Ca2 þ from intracellular stores in H9C2 and C2C12 cells. We have demonstrated that one of the targets is the SR network. We also have demonstrated that the BKCa channel opener CGS7184 has no inhibitory effect on the activity of SERCA pumps isolated from rat skeletal and cardiac muscle. Acknowledgments This study was supported by the European Union with resources from the European Regional Development Fund under the Innovative Economy Programme (POIG.01.01.02-00-069/09). We would like to thank Dr. Michele Chiesi of Novartis Pharma (Basel) for the CGS7184 compound. References Bentzen, B.H., Nardi, A., Calloe, K., Madsen, L.S., Olesen, S.r.-P., Grunnet, M., 2007. The small molecule NS11021 is a potent and specific activator of Ca2 þ -activated big-conductance K þ channels. Mol. Pharmacol. 72, 1033–1044.

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