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• W2133173611 abstract "The cardiac acetylcholine-activated K+ channel (IK,Ach) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K+ (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K+ efflux through the GIRK1/4 channel (carbachol vs. control solution, Z′ factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl2 and enhanced by increasing the driving force for K+ across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na+ and K+ channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF. The cardiac acetylcholine-activated K+ channel (IK,Ach) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K+ (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K+ efflux through the GIRK1/4 channel (carbachol vs. control solution, Z′ factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl2 and enhanced by increasing the driving force for K+ across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na+ and K+ channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF. The acetylcholine-activated K+ channel (IK,Ach) is a member of the superfamily of proteins known as the G-protein-coupled inward rectifier K+ (GIRK) channels.1Dascal N Signalling via the G protein-activated K+ channels.Cell Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar,2Hibino H Inanobe A Furutani K Murakami S Findlay I Kurachi Y Inward rectifying potassium channels: their structure, function and physiological roles.Physiol Rev. 2010; 90: 291-366Crossref PubMed Scopus (1042) Google Scholar GIRK channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in cardiac muscle, skeletal muscle, neurons, and endocrine tissue.1Dascal N Signalling via the G protein-activated K+ channels.Cell Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar,2Hibino H Inanobe A Furutani K Murakami S Findlay I Kurachi Y Inward rectifying potassium channels: their structure, function and physiological roles.Physiol Rev. 2010; 90: 291-366Crossref PubMed Scopus (1042) Google Scholar The cardiac IK,Ach is composed of the inward rectifier K+ (Kir) channel subunits Kir3.1 and Kir3.4 (GIRK1/4), which are arranged in a tetramer.1Dascal N Signalling via the G protein-activated K+ channels.Cell Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar,2Hibino H Inanobe A Furutani K Murakami S Findlay I Kurachi Y Inward rectifying potassium channels: their structure, function and physiological roles.Physiol Rev. 2010; 90: 291-366Crossref PubMed Scopus (1042) Google Scholar In the heart, binding of muscarinic agents such as acetylcholine or carbachol to the muscarinic (M2) receptor causes a dissociation of the βγ subunits of the G inhibitory protein (Gi), which subsequently bind to and activate IK,Ach.1Dascal N Signalling via the G protein-activated K+ channels.Cell Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar,2Hibino H Inanobe A Furutani K Murakami S Findlay I Kurachi Y Inward rectifying potassium channels: their structure, function and physiological roles.Physiol Rev. 2010; 90: 291-366Crossref PubMed Scopus (1042) Google Scholar Once opened, IK,Ach allows the movement of K+ out of the cardiac myocyte, causing the cell membrane potential to become more negative. Cardiac arrhythmias are defined as abnormalities in the generation or conduction of electrical impulses in the heart. Atrial fibrillation (AF) is a rapid and irregular atrial muscle arrhythmia that results in erratic and incomplete cardiac contractions. AF is the most prevalent arrhythmia in the United States, affecting more than 2 million adult patients.3Tsikouris JP Cox CD A review of class III anti-arrhythmic agents for atrial fibrillation: maintenance of normal sinus rhythm.Pharmacotherapy. 2001; 21: 1514-1529Crossref PubMed Scopus (25) Google Scholar,4Savelieva I Camm J Anti-arrhythmic drug therapy for atrial fibrillation: current anti-arrhythmic drugs, investigational agents and innovative approaches.Europace. 2008; 10: 647-665Crossref PubMed Scopus (119) Google Scholar The frequency of AF increases with age, occurring in 3% to 5% of those older than 65 years of age and 9% of people older than 80 years.3Tsikouris JP Cox CD A review of class III anti-arrhythmic agents for atrial fibrillation: maintenance of normal sinus rhythm.Pharmacotherapy. 2001; 21: 1514-1529Crossref PubMed Scopus (25) Google Scholar,4Savelieva I Camm J Anti-arrhythmic drug therapy for atrial fibrillation: current anti-arrhythmic drugs, investigational agents and innovative approaches.Europace. 2008; 10: 647-665Crossref PubMed Scopus (119) Google Scholar Regardless of the underlying cardiac disease, chronic AF is associated with increased morbidity and mortality. Current antiarrhythmic drugs used in the treatment of AF are limited by suboptimal efficacy and a high incidence of toxicity.3Tsikouris JP Cox CD A review of class III anti-arrhythmic agents for atrial fibrillation: maintenance of normal sinus rhythm.Pharmacotherapy. 2001; 21: 1514-1529Crossref PubMed Scopus (25) Google Scholar,4Savelieva I Camm J Anti-arrhythmic drug therapy for atrial fibrillation: current anti-arrhythmic drugs, investigational agents and innovative approaches.Europace. 2008; 10: 647-665Crossref PubMed Scopus (119) Google Scholar One novel target for AF drug therapy is the GIRK1/4 channel.4Savelieva I Camm J Anti-arrhythmic drug therapy for atrial fibrillation: current anti-arrhythmic drugs, investigational agents and innovative approaches.Europace. 2008; 10: 647-665Crossref PubMed Scopus (119) Google Scholar, 5Ehrlich JR Inward rectifier potassium currents as a target for atrial fibrillation therapy.J Cardiovasc Pharmacol. 2008; 52: 129-135Crossref PubMed Scopus (70) Google Scholar, 6Li D Sun H Levesque P Anti-arrhythmic drug therapy for atrial fibrillation: focus on atrial selectivity and safety.Cardiovasc Hematol Agents Med Chem. 2009; 7: 64-75Crossref PubMed Scopus (27) Google Scholar Recent studies indicate that IK,Ach is constitutively active in patients with AF.7Dobrev D Friedrich A Voigt N Jost N Wettwer E Christ T et al.The G protein-gated potassium current IK,Ach is constitutively active in patients with chronic atrial fibrillation.Circulation. 2005; 112: 3697-3706Crossref PubMed Scopus (364) Google Scholar This constitutively active channel causes the atrial action potential duration to shorten with a resulting increase in cell excitability.5Ehrlich JR Inward rectifier potassium currents as a target for atrial fibrillation therapy.J Cardiovasc Pharmacol. 2008; 52: 129-135Crossref PubMed Scopus (70) Google Scholar,7Dobrev D Friedrich A Voigt N Jost N Wettwer E Christ T et al.The G protein-gated potassium current IK,Ach is constitutively active in patients with chronic atrial fibrillation.Circulation. 2005; 112: 3697-3706Crossref PubMed Scopus (364) Google Scholar Therefore, GIRK1/4 channel blockers, by decreasing atrial excitability, should reduce the incidence of AF. However, discovery of new drugs that bind to and block the cardiac IK,Ach has been hampered by the absence of a cell-based screening assay that uses the GIRK1/4 channel. In the present study, immortalized cardiac atrial cells, expressing the GIRK1/4 channel, were analyzed using patch-clamp recording procedures and fluorescent imaging plate reader (FLIPR) measurements. Application of carbachol activated a whole-cell IK,Ach current that was blocked by antiarrhythmic agents and the Kir toxin tertiapin. In cells loaded with the membrane potential–sensitive fluorescent dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)), carbachol caused a rapid, time-dependent decrease in the fluorescent signal. This carbachol-induced decrease in fluorescence was blocked by BaCl2 and enhanced by increasing the driving force for K+ across the cell membrane. The GIRK1/4 channel/DiBAC4(3) assay was screened against a small-compound library of Na+ and K+ channel modulators. Analogues of propafenone and amiloride were identified as GIRK channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay will be useful for expanding the limited pharmacology of IK,Ach blockers and for developing new and selective agents for treating AF. The immortalized cardiac HL-1 cell line was generously supplied by Dr. William Claycomb (LSU Medical Center, New Orleans, LA). Cells were maintained in Claycomb/Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and L-glutamine (Sigma-Aldrich, St. Louis, MO). Myocytes were platted on gelatin-coated glass coverslips (5000 cells per coverslip; patch-clamp recording) and in black 96-well plates (25,000 cells per well; FLIPR measurements). Cells were stored in an incubator at 37°C (5% O2/95% CO2) and used on days 2 to 4 after plating. The patch-clamp method8Hamill OP Marty A Neher E Sakmann B Sigworth J Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches.Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15138) Google Scholar was used to record the whole-cell IK,Ach using L/M EPC-7 (Adams & List Associates, Great Neck, NY) and Axopatch 200 (Axon Instruments, Sunnyvale, CA) amplifiers. Our procedures for measuring cardiac K+ channels have been described.9Walsh KB Zhang J Neonatal rat cardiac fibroblasts express three types of voltage-gated K+ channels: regulation of a transient outward current by protein kinase C.Am J Physiol Heart Circ Physiol. 2008; 294: H1010-H1017Crossref PubMed Scopus (39) Google Scholar Pipettes were made from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) and had resistances of 1 to 2 Mohms when filled with internal solution. All experiments were conducted on isolated, noncoupled HL-1 cells at room temperature (22-24°C). For the measurement of IK,Ach, cells were placed in a normal Tyrode’s solution consisting of 132 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM dextrose, and 5 mM HEPES (pH 7.4; with NaOH) (280 mOsm). INa was blocked with tetrodotoxin (10 µM) and the Na+ channels inactivated by maintaining the myocytes at a holding potential of –40 mV. L-type Ca2+ channels were blocked with 500 nM nisoldipine. The internal solution consisted of 50 mM KCl, 60 mM K+-glutamate, 2 mM MgCl2, 1 mM CaCl2, 11 mM EGTA, 3 mM adenosine triphosphate (ATP), and 10 mM HEPES (pH 7.3; with KOH) (280 mOsm). Membrane currents were recorded with 12-bit analog/digital converters (Axon Instruments). Data were sampled at 5 KHz and filtered at 1 KHz with a low-pass Bessel filter (Frequency Devices, Ottawa, IL). Series resistance was compensated to give the fastest possible capacity transient without producing oscillations. With this procedure, more than 70% of the series resistance could be compensated. Following the measurement of the HL-1 cell background current, IK,Ach was activated by the addition 10 µM carbachol using a rapid perfusion system. IK,Ach was defined as the BaCl2-sensitive current (see Fig. 1). IK,Ach blockers were added and inhibition determined immediately after carbachol-induced activation. This limited errors in quantifying drug block due to GIRK1/4 channel desensitization and rundown. Averaged current values presented are means ± SE. Cell lysates for Western blot analysis were prepared by placing the HL-1 cell cultures into a lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 50 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethysulfonyl fluoride, 1 mM EGTA, 0.25% sodium deoxycholate, 2 µg/mL aprotinin, and protease inhibitor cocktail [Roche, Basel, Switzerland]). The protein content of the cell preparations was determined using a protein assay kit (Pierce, Rockford, IL). For Western blot analysis of the GIRK1 channel subunit, proteins were separated by electrophoresis on 8% sodium dodecyl sulfate (SDS) polyacrylamide gels using a mini PROTEAN cell (Bio-Rad, Hercules, CA). The running buffer contained 25 mM Tris, 193 mM glycine (pH 8.3), and 0.1% SDS. Proteins were transferred to polyvinylidene difluoride membranes using a Trans-Blot apparatus (Bio-Rad). The transfer buffer contained 25 mM Tris, 192 mM glycine (pH 8.5), and 20% methanol. For immunodetection, membranes were blocked in Tris-buffered saline (TBS) containing 0.1% Tween-20, bovine serum albumin, and 0.025% Na-azide for 60 min at room temperature. Antibodies to the Kir3.1 (GIRK1) channel (1:200) (Alomone, Jerusalem, Israel) were incubated with the membranes overnight at 4°C. After primary antibody treatment, the membranes were washed with TBS–0.1% Tween-20 and incubated with a secondary antibody (horseradish peroxidase [HRP]–conjugated rabbit IgG; Cell Signaling Technology, Danvers, MA). Immunoreactive bands were visualized on X-ray film (Kodak) using the enhanced chemiluminescence method (Pierce). HL-1 cells were incubated for 1 h in Tyrode’s solution containing 5 µM DiBAC4(3) (AnaSpec, Fremint, CA) and fluorescent signals recorded using a Synergy2 microplate reader (BioTek, Winosski, VT) at 28°C. Test compounds were dissolved in DMSO at a stock concentration of 10 mM, diluted to various concentrations, and applied to the cells for 5 min prior to the fluorescent measurements. DiBAC4(3) was present in all experimental solutions. DMSO, up to 1% in Tyrode’s solution, had no adverse effects on the cell response. Carbachol or control solution (20 µL) was added to each well (total volume = 220 µL) at time zero using the Synergy2 injector system. Data points were collected at 10-s intervals over a 300-s sampling period at excitation and emission wavelengths of 480 and 520 nm, respectively. Positive (carbachol or carbachol + 1 mM BaCl2) and negative (water or DMSO) control experiments were performed to calculate Z′ factors.10Zhang J-H Chung TDY Oldenburg KR A simple statistical parameter for use in evaluation and validation of high throughput screening assays.J Biomol Screen. 1999; 4: 67-73Crossref PubMed Scopus (5325) Google Scholar The Z′ factor is defined as Z′ = 1 – (3σP + 3σN)/|µP – µN|, where µP and µN are the means of the positive control and negative control signals, and σP and σN are the standard deviations of the positive control and negative control signals, respectively.10Zhang J-H Chung TDY Oldenburg KR A simple statistical parameter for use in evaluation and validation of high throughput screening assays.J Biomol Screen. 1999; 4: 67-73Crossref PubMed Scopus (5325) Google Scholar Plates used for Z′ factor determination contained 2 rows each of positive and negative controls. Dose-response curves for selected compounds were obtained and drug potencies determined by fitting the data with the following curve: Max/(1 + ([drug]/IC50)k), where the IC50 is the concentration of the compound producing a 50% decrease in the maximal carbachol response (Max), and k is the slope factor. Interference of the test compounds with the DiBAC4(3) fluorescence was determined in the absence of cells by adding various concentrations of the compounds to Tyrode’s solution containing the dye. Carbachol, KR-32568, antiarrhythmic agents, and an Na+, K+ channel modulator kit, containing 66 compounds, were purchased from Sigma-Aldrich. The propafenone analogues 1-{2-[3-(tert-butylamino)-2-hydroxypropoxy]phenyl}-3- phenyl-1-propanone, 1-{2-[2-(diethylamino)ethoxy]phenyl}-3- phenyl-1-propanone, 1-{2-[3-(diethylamino)-2-hydroxypropoxy]phenyl}-3-phenyl-1-propanone, 5-hydroxy propafenone, 1-{2- [2-hydroxy-3-(1-piperidinyl)propoxy]phenyl}-3-phenyl-1- propanone, 1-{2-[2-hydroxy-3-(4-morpholinyl)propoxy]phenyl}- 3-phenyl-1-propanone, 1-{2-[3-(ethylamino)-2-hydroxypropoxy]phenyl}ethanone, and 1-(1-piperidinyl)-3-(4-propylphenoxy)-2-propanol were obtained from ChemBridge Corp. (San Diego, CA). IK,Ach is highly expressed in cardiac sinoatrial nodal, atrioventricular nodal, and atrial tissues.1Dascal N Signalling via the G protein-activated K+ channels.Cell Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar,2Hibino H Inanobe A Furutani K Murakami S Findlay I Kurachi Y Inward rectifying potassium channels: their structure, function and physiological roles.Physiol Rev. 2010; 90: 291-366Crossref PubMed Scopus (1042) Google Scholar HL-1 cells are an immortalized atrial cell line that displays an adult-like cardiac genotype and contracts spontaneously in cell culture.11Whit SM Constantin PE Claycomb WC Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function.Am J Physiol Heart Circ Physiol. 2004; 286: H823-H829Crossref PubMed Scopus (332) Google Scholar,12Claycomb WC Lanson Jr, NA Stallworth BS Egeland DB Delcarpio JB Bahinski A et al.HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.Proc Natl Acad Sci USA. 1998; 95: 2979-2984Crossref PubMed Scopus (1235) Google Scholar Figure 1 displays IK,Ach currents measured in the HL-1 cells using the whole-cell arrangement of the patch-clamp technique. Cells were bathed in normal Tyrode’s solution (5 mM K+), and the pipette contained a KCl/K+-glutamate (140 mM K+) solution. Application of carbachol to the recording chamber resulted in the activation of an inward rectifying current (Fig. 1). As expected for an inward rectifier K+ channel, the carbachol-activated current was completely blocked by BaCl2 (Fig. 1). The reversal potential (Erev) for the current (–72 mV) was within range of the calculated Nernst equilibrium potential (EK) (–85 mV) for a K+-selective channel under these conditions. The cardiac IK,Ach is composed of the inward rectifier Kir3.1 and Kir3.4 (GIRK1 and GIRK4) subunits.1Dascal N Signalling via the G protein-activated K+ channels.Cell Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar,2Hibino H Inanobe A Furutani K Murakami S Findlay I Kurachi Y Inward rectifying potassium channels: their structure, function and physiological roles.Physiol Rev. 2010; 90: 291-366Crossref PubMed Scopus (1042) Google Scholar As shown in Figure 1, an immunoreactive doublet protein corresponding to the approximately 50- to 55-kDa GIRK1 subunit was identified in HL-1 cells but not in ventricular myocytes using an anti-GIRK1 antibody. The pharmacological properties of the HL-1 cell IK,Ach current were also examined. A number of agents, including the Kir toxin tertiapin (100 nM) and the antiarrhythmic drugs flecainide (100 µM) and propafenone (10 µM), block IK,Ach in primary cultures of adult atrial myocytes.13Inomata N Ohno T Ishihara K Akaike N Antiarrhythmic agents act differently on the activation phase of the Ach-response in guinea pig atrial myocytes.Br J Pharmacol. 1993; 108: 111-115Crossref PubMed Scopus (35) Google Scholar, 14Watanabe Y Hara Y Tamagawa M Nakaya H Inhibitory effect of amiodarone on the muscarinic acetylcholine receptor-operated potassium current in guinea pig atrial cells.J Pharmacol Exp Ther. 1996; 279: 617-624PubMed Google Scholar, 15Hashimoto N Yamashita T Tsuruzoe N Tertiapin, a selective IK,Ach blocker, terminates atrial fibrillation with selective atrial effective refractory period prolongation.Pharmacol Res. 2006; 54: 136-141Crossref PubMed Scopus (96) Google Scholar All 3 of these agents strongly blocked IK,Ach at the concentrations tested (Fig. 2). In contrast, addition of control solution (containing drug vehicle) produced no significant change in the current (Fig. 2). The full dose-response curve for the propafenone block of IK,Ach is included in Figure 2. The IC50 for the propafenone block was 2 µM. Thus, the inward rectifying current versus voltage relationship, the Ba2+-sensitivity and pharmacology of the whole-cell IK,Ach current, and the presence of Kir3.1 subunits are all consistent with the expression of GIRK1/4 channels in the HL-1 cells. Having demonstrated that the IK,Ach current–GIRK1/4 channel is expressed in the HL-1 cell line, it was determined if these cells could be used for FLIPR measurements of the channel. The experimental design of the screening assay is outlined in Figure 3. HL-1 cells were cultured in 96-well plates and loaded with the fluorescent membrane potential–sensitive dye DiBAC4(3). Membrane potential–sensitive dyes such as DiBAC4(3) distribute across the plasma membrane when cells are in the rested state and reach equilibrium (Fig. 3, top panel). The DiBAC4(3) molecules inside the cells become strongly fluorescent upon binding to intracellular proteins and other cytoplasmic components. Treatment of the cells with carbachol activates Gi, stimulating the GIRK1/4 channels to open (bottom panel). The resulting efflux of K+ out of the cell should therefore cause the resting membrane potential to become more negative and the DiBAC4(3) molecules to redistribute to the outside of the cell. As a result, the cell fluorescent signal should decrease. The applicability of the GIRK1/4 channel fluorescent assay for drug testing is evaluated in Figure 4. Each figure plots the DiBAC4(3) fluorescence signal measured over time in the 96-well plates using a Synergy2 microplate reader (BioTek). As predicted by the experimental model, addition of carbachol (10 µM) to the cardiac cells caused a rapid, time-dependent decrease in the DiBAC4(3) fluorescent signal (Fig. 4, top left panel). In contrast, addition of control solution produced only a small instantaneous rise in the fluorescence (Fig. 4). Qualitatively similar results were obtained using the oxonol dye HLB 021-152 (AnaSpec; results not shown). Comparison of peak fluorescent changes in wells injected with control and carbachol solutions gave Z′ factors ranging from 0.50 to 0.64 (4 plates from 2 experimental days). The peak carbachol-induced fluorescent signal obtained from these plates was 0.956 ± 0.008 (mean ± SD). To quantify the GIRK1/4 channel fluorescent signal, we subtracted the averaged control measurement, obtained in each 96-well plate, from the records measured in the presence of carbachol (Carb – Con; Fig. 4). This control-subtracted fluorescent signal (i.e., the carbachol- sensitive component) was subsequently obtained and analyzed throughout the rest of the study. The pharmacological and biophysical properties of the carbachol-sensitive fluorescent signal are summarized in Figure 4. Carbachol activated the GIRK1/4 channel in a concentration-dependent manner with a half-maximal effective concentration (EC50) of 110 nM (Fig. 4, top right panel). The carbachol signal was blocked by 1 mM BaCl2 and inhibited during pretreatment with the muscarinic antagonists scopolamine (1 µM; Fig. 4, bottom left panel) and atropine (1 µM; results not shown). The effect of altering the driving force for K+ across the plasma membrane on the GIRK1/4 channel signal was also determined with the HL-1 cells. Carbachol-induced changes in the DiBAC4(3) signal were measured in Tyrode’s solutions containing 1, 5, and 20 mM KCl. Under these conditions, the reversal potential (Erev) for the GIRK1/4 channel should follow EK and approach –120 mV and –50 mV in 1-mM and 20-mM KCl solutions, respectively. As shown in Figure 4 (bottom right panel), reducing the extracellular K+ concentration ([K]o) from 5 mM to 1 mM produced a 2-fold increase in the carbachol-sensitive signal (mean = 2.0 ± 0.1, 3 plates). In contrast, increasing [K]o to 20 mM, which should shift EK close to the resting potential of the HL-1 cell (–50 to –65 mV),16Xiao YF TenBroek EM Wilhelm JJ Iaizzo PA Sigg DC Electrophysiological characterization of murine HL-5 atrial cardiomyocytes.Am J Physiol Cell Physiol. 2006; 291: C407-C416Crossref PubMed Scopus (4) Google Scholar completely eliminated the carbachol-mediated decrease in the fluorescent signal. IK,Ach is blocked by several antiarrhythmic drugs, including propafenone, flecainide, and amiodarone.13Inomata N Ohno T Ishihara K Akaike N Antiarrhythmic agents act differently on the activation phase of the Ach-response in guinea pig atrial myocytes.Br J Pharmacol. 1993; 108: 111-115Crossref PubMed Scopus (35) Google Scholar, 14Watanabe Y Hara Y Tamagawa M Nakaya H Inhibitory effect of amiodarone on the muscarinic acetylcholine receptor-operated potassium current in guinea pig atrial cells.J Pharmacol Exp Ther. 1996; 279: 617-624PubMed Google Scholar, 15Hashimoto N Yamashita T Tsuruzoe N Tertiapin, a selective IK,Ach blocker, terminates atrial fibrillation with selective atrial effective refractory period prolongation.Pharmacol Res. 2006; 54: 136-141Crossref PubMed Scopus (96) Google Scholar Therefore, the GIRK1/4 channel assay was screened against a group of antiarrhythmic agents. Propafenone was the most potent of the drugs tested, with an IC50 of 3 µM (Fig. 5). This inhibitory potency was similar to that measured for the propafenone block of the IK,Ach current (IC50 = 2 µM; Fig. 2). Several structural analogues of propafenone were obtained from ChemBridge (see Materials and Methods) and tested for their inhibitory action. Of these compounds, only 1-{2-[3-(tert-butylamino)-2-hydroxypropoxy] phenyl}-3-phenyl-1-propanone (TBPP; IC50 = 2 µM) was more potent than propafenone (Fig. 5). Other antiarrhythmic agents, including ibutilide (IC50 = 16 µM), quinidine (IC50 = 24 µM), and flecainide (IC50 = 56 µM), were also effective in inhibiting the carbachol-sensitive fluorescence signal. In contrast, drugs such as sotalol (blocker of the rapid component of the cardiac-delayed rectifier K+ channel), glybenclamide (ATP-sensitive K+ channel blocker), and procainamide (voltage-gated Na+ channel blocker) caused no block at the concentrations tested (Fig. 5). Amiodarone, another antiarrhythmic agent that blocks K+ channels,14Watanabe Y Hara Y Tamagawa M Nakaya H Inhibitory effect of amiodarone on the muscarinic acetylcholine receptor-operated potassium current in guinea pig atrial cells.J Pharmacol Exp Ther. 1996; 279: 617-624PubMed Google Scholar was not evaluated since it interfered with the DiBAC4(3) fluorescence at concentrations equal to or greater than 1 µM. A small-compound library, consisting of Na+ and K+ channel modulators (Sigma-Aldrich), was also tested using the GIRK1/4 channel assay. Several analogues of the epithelial Na+ channel blocker amiloride were identified as inhibitors of the carbachol-sensitive signal when the modulators were screened at 1 µM. The full dose-response curves for these drugs are plotted in Figure 6. Both 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; IC50 = 0.8 µM) and 5-(N, N-hexamethylene)-amiloride (HMA; IC50 = 0.9 µM) were effective at concentrations below 1 µM. In contrast, amiloride caused only a partial block at concentrations up to 100 µM (Fig. 6). Since EIPA and HMA inhibit the Na+/H+ exchanger (NHE),17Levitsky J Gurell D Frishman WH Sodium ion/hydrogen ion exchange inhibition: a new pharmacologic approach to myocardial ischemia and reperfusion injury.J Clin Pharmacol. 1998; 38: 887-897Crossref PubMed Scopus (77) Google Scholar the effect of the selective NHE inhibitor KR-32568 ([5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl] guanidine) was determined in the assay. However, at drug concentrations up to 1 µM, KR-32568 caused no significant change in the carbachol-sensitive fluorescence (Fig. 6). The present study demonstrates that immortalized cardiac HL-1 cells can be used for identifying new GIRK1/4 channel blockers. Consistent with the presence of the GIRK1/4 channel, HL-1 cells displayed a carbachol-activated Kir current and expressed the Kir3.1 channel subunit. Application of carbachol to cells loaded with a membrane potential–sensitive dye caused a rapid, time-dependent decrease in the fluorescent signal that was inhibited by BaCl2 and muscarinic receptor antagonists. Finally, structural analogues of propafenone and amiloride were identified as GIRK1/4 channel blockers at concentrations less than 1 µM. The traditional approach to ion channel drug discovery has involved the expression of recombinant channels in heterologous cell lines such HEK-293 and Chinese hamster ovary (CHO) cells. However," @default.
• W2133173611 created "2016-06-24" @default.
• W2133173611 creator A5069088638 @default.
• W2133173611 date "2010-12-01" @default.
• W2133173611 modified "2023-10-06" @default.
• W2133173611 title "A Real-Time Screening Assay for GIRK1/4 Channel Blockers" @default.
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