Tetraethylammonium (TEA, Sigma), NMDG (Sigma), 4-aminopyridine (≥99%; Sigma and Tocris Bioscience, Bristol, United Kingdom), 2-aminopyridine (2-AP; Sigma), 3-aminopyridine (3-AP; Sigma), 3,4-diaminopyridine (3,4-DAP; Sigma), 1,3-Bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS 1643, Tocris), 10,10-bis(4-Pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991, Tocris), and N-[4-[[1- [2-(6-Methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl] methanesulfonamide dihydrochloride (E-4031, Tocris) were diluted in extracellular solution to concentrations indicated in “Results.” All substances were applied locally via a glass capillary through a custom-made application system.
E 4031
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
E-4031 is a small molecule compound that inhibits the hERG potassium channel. It is commonly used as a research tool to study the role of the hERG channel in various biological processes.
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Lab products found in correlation
Verapamil, by Merck Group (2 mentions)
Chromanol 293b, by Bio-Techne (2 mentions)
4 aminopyridine 4 ap, by Merck Group (2 mentions)
Tetraethyl ammonium tea, by Merck Group (1 mentions)
4 aminopyridine, by Merck Group (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Dofetilide, by Merck Group (1 mentions)
Ranolazine, by Merck Group (1 mentions)
Haloperidol, by Merck Group (1 mentions)
Thioridazine, by Merck Group (1 mentions)
Moxifloxacin, by Merck Group (1 mentions)
Tetrodotoxin ttx, by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Gs 967, by Gilead Sciences (1 mentions)
Bekm 1, by Alomone (1 mentions)
Na hepes, by Thermo Fisher Scientific (1 mentions)
K4bapta, by Santa Cruz Biotechnology (1 mentions)
Nifedipine, by Merck Group (1 mentions)
Apamin, by Merck Group (1 mentions)
16 protocols using e 4031
1
Pharmacological Modulation of Ion Channels
2
Effects of KCNH2 Inhibitor E-4031 on Cardiomyocyte Beating
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To determine the effect of E-4031 (an inhibitor of the KCNH2 channel) on beating frequency, videos were taken at the baseline (drug-free culture medium), with the drug, and following washout of the drug. Baseline videos were first acquired for EBs from all groups, in fresh CM Media after equilibration. After baseline videos were acquired, 5 μM of E-4031 (Tocris) were supplemented to fresh CM Media and the EBs were allowed to equilibrate for 20 min before taking videos. EBs were then washed with CM media three times, allowed to equilibrate for 20 min, and videos of the same EBs were taken the third time to determine the frequency of beating after the drug washout.
To determine the effect of constitutive culture with an inhibitor of KCNH2, 5 μM E-4031 was added to the media at the start of the stimulation period. Videos were taken on days 1, 3 and 7 following 20 min of equilibration to fresh media containing E-4031. At the end of the culture period, the cardiomyocytes were washed with fresh CM Media without E-4031 three times, allowed to equilibrate for 20 min and re-evaluated for frequency.
To examine the effects of isoproterenol on cardiomyocytes, videos were first taken at the baseline in fresh CM media after equilibration, and then in CM media containing 1 μM isoproterenol (Sigma).
To determine the effect of constitutive culture with an inhibitor of KCNH2, 5 μM E-4031 was added to the media at the start of the stimulation period. Videos were taken on days 1, 3 and 7 following 20 min of equilibration to fresh media containing E-4031. At the end of the culture period, the cardiomyocytes were washed with fresh CM Media without E-4031 three times, allowed to equilibrate for 20 min and re-evaluated for frequency.
To examine the effects of isoproterenol on cardiomyocytes, videos were first taken at the baseline in fresh CM media after equilibration, and then in CM media containing 1 μM isoproterenol (Sigma).
3
Comprehensive Pharmacological Screening Protocol
4
Animal Study Protocols for Cardiac Research
5
Characterization of Cardiac Ion Channels
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Krebs buffer contained: 146.9 mM NaCl, 4.7 mM KCl, 2 mM CaCl2·2H2O, 1.2 mM MgSO4, 1.2 mM NaH2PO4·H2O, 3 mM NaHCO3, 1.5 mM Na-HEPES, and 5 mM D-glucose (pH 7.4). An identical buffer (“Krebs-BSA”) also contained 0.5% bovine serum albumin. Krebs-BSA buffer was present both luminally and abluminally during cannulation, but the abluminal solution was constantly exchanged with plain Krebs during the experimental protocol. For Ca2+-free Krebs, 3 mM EGTA replaced CaCl2·2H2O. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) except BSA (US Biochemicals; Cleveland, OH), MgSO4 and Na-HEPES (ThermoFisher Scientific). E-4031 was obtained from Tocris (#1808) and dissolved in water to make a 1 mM stock solution. BeKm-1 was obtained from Alomone Labs (#STB-470) and dissolved in water to make a 100 mM stock solution. RPR-260243, type 1 ERG channel agonist, was obtained from MedChemExpress (#HY-16915) and dissolved in DMSO to make a 10 mM stock solution. ICA-105574, a type 2 ERG channel agonist, was obtained from Abious (#AOB2681) and dissolved in DMSO to make a 10 mM stock solution.
6
Preparation of Agonist Stock Solutions
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Ado and ACh (Sigma-Aldrich, St Louis, MO, USA) were dissolved in deionized water to make 10 mM stock solutions and stored at -20°C. Working agonist concentrations were freshly prepared in the recording extracellular solution. E-4031 and chromanol 293B were obtained from Tocris Bioscience (Ellisville, MO, USA), K4BAPTA was acquired from Santa Cruz Technology (Dallas, TX, USA), and all other reagents were from Sigma-Aldrich.
7
Evaluation of Ion Channel Blockers on Atrial Tissue
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Various general ion channel blockers with well-known effects on human atrial tissue and/or cells were used to test their ability to modify AP characteristics when applying current injection and to validate if potential drug effects depend on the IK1 or constant current injection approach used. Each drug was tested in myocytes isolated from two patients. 4-Aminopyridine (4-AP; Merck) was freshly prepared as 10 mM stock solution in Tyrode’s solution and pH was readjusted to 7.4 (HCl). Nifedipine (Sigma) was prepared as 5 mM stock solution in ethanol, and stored in the dark. E-4031 (Tocris), apamin (Sigma), and BaCl2 (Sigma) were prepared as 5, 0.1, and 1000 mM stock solutions, respectively, in distilled water. All stock solutions and lidocaine (10 mg/ml; Braun) were diluted appropriately before use.
8
Lipid Compounds and Their Preparation
9
Preparation and Handling of Stock Solutions for Cell Experiments
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All drugs were obtained from Sigma-Aldrich (MO, USA), except for E-4031, PK-THPP, ML-365 (Tocris, MN, USA), Sub-P (Enzo Life Sciences, NY, USA), and TTX (Abcam Biomedicals, Cambridge, UK).
DIDS was freshly prepared as a 0.5 M and chromanol 293B as a 0.1 M stock solution in DMSO. Nifedipine was prepared as a 5 mM stock solution in ethanol. E-4031 and TTX were prepared as a 5 and 30 mM stock solution in distilled water. Senktide was prepared as a 1 mM stock solution in ethanol. PK-THPP and ML-365 were prepared as a 1 mM stock solutions in DMSO. Spadin was prepared as a 1 mM Stock solution in distilled water. ZnCl2 was prepared as a 100 mM stock solution in distilled water. All stock solutions were diluted appropriately before use. Sub-P was freshly dissolved at its final concentration. DIDS and nifedipine were stored in the dark.
DIDS was freshly prepared as a 0.5 M and chromanol 293B as a 0.1 M stock solution in DMSO. Nifedipine was prepared as a 5 mM stock solution in ethanol. E-4031 and TTX were prepared as a 5 and 30 mM stock solution in distilled water. Senktide was prepared as a 1 mM stock solution in ethanol. PK-THPP and ML-365 were prepared as a 1 mM stock solutions in DMSO. Spadin was prepared as a 1 mM Stock solution in distilled water. ZnCl2 was prepared as a 100 mM stock solution in distilled water. All stock solutions were diluted appropriately before use. Sub-P was freshly dissolved at its final concentration. DIDS and nifedipine were stored in the dark.
10
Optogenetic Imaging of Ion Channel Modulators
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Dried powders of E-4031 and JNJ-303 (Tocris) were dissolved in DMSO to make a stock concentration of 10 mM. Compounds were solubilized by vortexing the stock solution at RT and stock solutions were stored at −20°C until use. The drug dilutions were prepared fresh at the day of the experiment from stocks in imaging buffer and kept at 37°C in 5% CO2. For optogenetic imaging, the addition of drugs started from a blank (fresh imaging buffer) to check proper beating of the monolayer, followed by vehicle and drug doses. The entire volume (2 ml) in the dish was exchanged at each drug dose, as we noted that the cells needed fresh buffer at regular intervals for proper beating. A delay of ~1 min before imaging was allowed in order for the drug to take effect. The vehicle DMSO concentrations were 0.001% (v/v) for E-4031 series and 0.03% (v/v) for JNJ-303 series. The final concentrations for E-4031 were 3, 10, 30, and 100 nM and the final concentrations for JNJ-303 were 0.03, 0.1, 0.3, 1, 3, and 10 μM. For E-4031 patch clamp measurements drugs were diluted in bathing solution and administered through perfusion.
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