Cell-based hERG Channel Inhibition Assay in High-throughput Format

Abstract

Human ether-a-go-go-related gene (hERG) channel plays an essential role in the repolarization of the cardiac action potential. Genetic mutations and some chemicals/drugs interfere with hERG channel activity, which may prolong the QT interval and potentially cause long QT syndrome. The FluxOR™ thallium flux assay performed in two cell lines, U2OS and HEK293, with stable hERG expression can be used to identify compounds that inhibit hERG channel activity. This chapter describes a cell-based hERG channel inhibition assay that has been optimized and performed in a 1536-well plate format. The homogeneous and robust assay can be used to identify compounds that inhibit hERG channel activity.

1. Introduction

Human ether-a-go-go-related gene (hERG) channel (KCNH2a, Kv11.1), a member in a voltage-gated potassium (K⁺) channel (Kv) family [1], functions by conducting the rapid delayed rectifier K⁺ current [2]. hERG channel plays an important role in the proper repolarization of the action potential in a normal heart as well as the prevention of arrhythmias induced by ectopic depolarizations [1]. Inhibition of hERG channel activity by some drugs or genetic mutations in the hERG channel leads to potentially fatal Long QT Syndrome (LQTS). Several FDA approved drugs, such as terfenadine and cisapride, have been removed from the market due to their hERG blocking activity [3,4].

Several hERG inhibition assays including radioligand binding [5,6], automated planar patch clamp [6], rubidium ion efflux [7], and thallium flux [8] are currently available to assess the compound inhibitory effect on hERG channel. Previously we have optimized a thallium flux assay in U2OS cells transduced with hERG channel and validated this assay by screening LOPAC and NTP compound collections [8,9]. In this chapter, we evaluated two cell lines, U2OS and HEK293, stably expressing hERG for their ability to inhibit hERG channel in thallium flux assay using FDSS-7000 kinetic plate reader (Hamamatsu Corp., Hamamatsu City, Japan) [8]. This assay measures hERG channel activity using thallium ions as surrogate ions. Prior to the experiment, hERG-U2OS or hERG-HEK293 stable cell lines expressing hERG channel are first loaded with FluxOR dye containing aminomethyl (AM) ester groups that can freely enter cell membrane from extracellular medium. When FluxOR dye presents in the cytosol, its AM ester groups are cleaved by intracellular esterase, becoming charged and trapped inside of the cell. Upon stimulation by KCl, thallium ions enter the cells through open hERG channels and bind to the dye, producing green fluorescence (480 nm excitation and 530 nm emission) upon excitation (Fig 1). The fluorescence signal is proportional to the number of open hERG channel. In the presence of a hERG inhibitor, the intracellular fluorescence signal will be decreased.

Fig 1.

Principle of the thallium flux assay. At resting state when hERG channel is closed, hERG expressing cells are loaded with FluxOR dye. Upon stimulation, thallium (TI⁺) ions enter the cells through open hERG channels and bind to the dye, yielding green fluorescence (This figure was created with BioRender.com).

2. Materials

2.1. Cell lines and cell culture condition

1. Cell lines: hERG-U2OS and hERG-HEK293 stable cell lines (Codex BioSolutions Inc., Gaithersburg, MD).

2. Culture media: Dulbecco’s Modified Eagle Medium (DMEM) with glutamax, fetal bovine serum (FBS), Non-Essential Amino Acid (NEAA), penicillin-streptomycin, and puromycin.

3. Thaw media: same as culture medium but without puromycin.

4. Assay media: same as culture medium.

5. Freezing Medium: Recovery™ Cell Culture Freezing Medium (Life Technologies, Carlsbad, CA).

6. 0.05% Trypsin/EDTA.

7. Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium.

2.2. Assay Reagents and Chemicals

1. Reagents for hERG channel assay: FluxOR II Potassium Ion Channel Assay kit (Life Technologies, Carlsbad, CA).

2. Dimethyl sulfoxide (DMSO).

3. Astemizole (Sigma–Aldrich, St. Louis, MO).

2.3. Supplies and Equipment

1. T75 and T225 cell culture flasks (Corning, Corning, NY).

2. 1536-well polystyrene assay plates: a black clear bottom, cell culture treated plate (Greiner Bio One North America, Monroe, NC).

3. 1536-well cycloolefin compound storage plates (Greiner Bio One North America, Monroe, NC).

4. Cell strainer: a receptacle with a 40 μm nylon filter that is used to remove clumped cells from cell suspensions (Corning, Corning, NY).

5. Lids for assay and compound plates: these reusable lids are made from stainless steel and contain a rubber gasket that sits around the top outer edge. The cellular assay lid contains small evenly placed holes that allow air exchange necessary for cellular assays. The weight of the lid allows the gasket to form a strong barrier around the plate, virtually eliminating edge effects.

6. BioRaptr Flying Reagent Dispenser (Beckman Coulter, Brea, CA): a liquid handling system that can transfer of 0.2–10 μL of up to four different reagents or cells simultaneously into a 1536 well plate.

7. Multidrop Combi Dispenser (Thermo Fisher, Waltham, MA): a high-speed dispenser capable of one reagent or cells using eight-channel detachable dispensing cassettes.

8. CyBi-well Vario Pipettor (Analytik Jena, Upland, CA), a 96-, 384- and 1536-channel simultaneous pipettor: It requires the use of disposable tips, which is more suitable for the preparation of positive control plate in 1536-well plate format.

9. Cellometer Auto T4 Cell Viability Counter (Nexcelom Bioscience, Lawrence, MA) is used to count viable cells.

10. Functional Drug Screening System (FDSS) 7000EX kinetic plate reader (Hamamatsu, Japan). FDSS is a kinetic plate reader with an integrated dispensing head and imaging-based detector. Simultaneous dispensing into the entire 1536-well plate and simultaneous detection of the kinetics of the fluorescence intensity allow quick measurements with no time lag for the 1536-well format.

3. Methods

3.1. Cell Culture

• 1.Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath with gentle agitation for 1–2 min.
• 2.Place the vial into a laminar flow hood. Before opening the vial, wipe the outside of the vial with 70 % ethanol.
• 3.Transfer the thawed cells into a 50 mL conical sterile centrifuge tube with 30 mL prewarm thaw medium.
• 4.Centrifuge the cell supernatant for 4 min at 200 x g at room temperature.
• 5.Carefully aspirate supernatant without disturbing the cell pellet.
• 6.Gently re-suspend cell pellet with thaw medium (Table 1).
• 7.Transfer the desired amount of the cells to a T225 tissue culture flask.
• 8.Place the flask in an incubator at 37°C under a humidified atmosphere and 5% CO_2. Maintain the cells at 30% to 90% confluence prior to passage (see Note 1 & 2). During the first passage, switch to culture medium (Table 1).
• 8.After 48 −72 h culture or cells with 80 – 90% confluence, aspirate medium and rinse once with 10 mL DPBS, followed by the addition of 10 mL of 0.25% Trypsin/EDTA and swirl to coat the cells evenly around flask.
• 9.Place the flask in incubator at 37°C for 2–3 min or until the cells detach.
• 10.Add 10 mL of culture medium to deactivate Trypsin.
• 11.Transfer the cells to a 50 mL conical tube and centrifuge at 200 × g for 4 min at room temperature.
• 12.Carefully aspirate supernatant and re-suspend cell pellet in the culture medium.
• 13.Count cells using a Cellometer auto cell counter or manually account.
• 14.Transfer the cell suspension to a T225 tissue culture flask.
• 15.Incubate cells at 37 °C, 5 % CO 2 and 95 % humidity until next passage or assay.

Table 1.

Components of cell culture and assay medium

Component	Culture Medium	Assay Medium	Thaw Medium
DMEM with Glutamax	90%	90%	90%
FBS	10%	10%	10%
NEAA	0.1 mM	0.1 mM	0.1 mM
Penicillin	50 U/mL	50 U/mL	50 U/mL
Streptomycin	50 μg/mL	50 μg/mL	50 μg/mL
Puromycin	1 μg/ml	1 μg/ml

3.2. Thallium flux assay (see Table 2 for simplified protocol)

Table 2.

Protocol for U2OS-hERG inhibition assay in a 1536-well plate format

Sequence	Parameter	Value	Description
1	Cells	1000 cells/well/3uL	In 1536-well black, clear bottom plate
2	Incubation	18 h	At 37°C and 5%CO2
3	Reagent #1	3 μL	Loading Buffer
4	Incubation	1 h	At room temperature in the dark
5	Compounds	23 nL	Compounds or control (astemizole)
6	Incubation	10 min	At room temperature
7	Reagent #2	1 μl	Stimulation Buffer
8	Detection	2 min	FDSS 7000EX kinetic plate reader with a filter set of Ex/Em=480/540 for 2 min at 1 sec interval

1. Harvest the hERG-U2OS or hERG-HEK293 cells and resuspend the cell pellet in culture medium.

2. Place the cells on a cell strainer to remove clumped cells before counting cells.

3. Count cell number and determine cell viability (see Note 3). Cell viability of 95% or greater will have a better window of signal to basal level.

4. Prepare cell stock in assay medium at density of 0.33 × 10⁶ cells/mL.

5. Dispense 3 μL (1000cells/well/3μL) of cells prepared at step 4 into each well of a 1536-well black/clear-bottom, tissue culture-treated assay plates using a Multidrop Combi dispenser (see Note 4).

6. Place a pre-cleaned plate lid over the plate (see Note 5) and incubate assay plates at 37°C under a humidified atmosphere and 5% CO₂ for 18 h to allow cells to attach.

7. Dispense 3 μL of Loading Buffer to each well using a BioRaptr dispenser (see Note 6).

8. Incubate the assay plates at room temperature in the dark for 1 h.

9. Treat cells with 23 nL of test compounds and controls using FDSS PinTool. The test compounds were transferred to columns 5–48, the astemizole (positive control) was transferred to columns 1–3 compound and DMSO (negative control) was transferred to column 4 (Fig. 2).

10. Incubate the plates in the dark at room temperature for 10 min.

11. Add 1 μL of Stimulation Buffer containing thallium (see Note 7) into each assay well.

12. Continuously measure fluorescence intensity at 480 nm excitation and 540 nm emission for 2 min at 1 sec interval using a Functional Drug Screening System (FDSS) 7000EX kinetic plate reader (see Note 8).

13. Astemizole inhibits hERG channel activity in a concentration–dependent manner (Fig. 3). The concentration-response curves of astemizole inhibition were analyzed using Prism software (GraphPad, San Diego, CA).

Fig 2.

Control plate map in a 1536-well plate format. Dose titration of astemizole ranging from 76 μM to 2.3 nM in column 1; 9.6 μM and 4.8 μM astemizole in column 2 and 3, respectively, and DMSO only in column 4.

Fig 3.

Astemizole inhibited hERG channel activity in hERG-U2OS (a) and hERG-HEK293 (b) cells in a 1536-well plate format. Each value represents the slope of fluorescence intensities vs the time of first 30 sec after addition of the stimulation buffer calculated from the kinetic readouts.

4. Notes

1. The cells should pass at least one passage after thawing for screening.

2. It is very important that the cells do not reach >90% confluence. For the best value of signal to base ratio, split cells before they reach 90% confluence.

3. It is recommended to count cells three times and take the average cell density to ensure accurate cell counts for plating.

4. The fluorescence signals of hERG assay are read from the bottom of assay plates. Be sure the bottoms of assay plates are untouched, which otherwise might affect fluorescence assay readouts.

5. Be sure the metal lid is on the assay plate properly to prevent evaporation.

6. The Loading Buffer and Stimulus Buffer should be freshly prepared before use. Discard any excess buffer.

7. Thallium is a toxic substance. Use caution when handling thallium sulfate and all solutions prepared containing thallium sulfate. Properly dispose of any waste containing thallium in compliance with all pertaining regulations.

8. The reagents should be stored in the amber bottle to reduce photobleaching during the kinetic reading.