PrecisION^® hERG ion channel membrane preparation

32011203

NA.41

Human ERG
The human ether-a-go-go related gene (hERG) is a potassium ion channel which is essential for normal cardiac repolarization. In drug screening models, the hERG K⁺ channel has been indicated to inhibit a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias (Murphy et al. 2006). Cardiac safety relating to I_Kr K⁺ channels has become a major concern of regulatory agencies, as hERG channel inhibition has been identified as the firmest link to QT prolongation (Chiu et al. 2004). Millipore′s hERG membrane preparations are crude membrane preparations made from HEK293 stable recombinant cell lines (Millipore cat. # CYL3039), which are ideal HTS tools for screening antagonists against the hERG channel. The membrane preparations exhibit a Kd of 5.2 nM for [³H]-Astemizole. With 10 μg/well hERG Membrane Prep and 3.0 nM [³H]-Astemizole, a greater than 4-fold signal-to-background ratio was obtained.

GPCR Cell Lines

Radioligand binding assay

Target Sub-Family: Potassium
Protein Target: hERG

Table 1. Signal: background and specific binding values obtained in a competition binding assay for the hERG membrane prep.

	10 µg/well
Signal:Background	4.6
Specific Binding (cpm)	435

SPECIFICATIONS: 1 unit = 10 μg membrane preparation with [³H]-astemizole
Bmax with [³H]-astemizole: 6.7 pmol/mg
Kd for [³H]-astemizole: 5.2 nM

Table 2. Rank order comparison of various small molecule hERG inhibitors using [³H]-astemizole binding assay and E-Phys assays:

	Binding Ki (nM) with hERG membrane preps		Electrophysiology IC50 (nM) with hERG cell lines
	Millipore CYL4039M	Literature value*	Millipore CYL4039 by IonWorks PPC	Millipore CYL4039 by PatchExpress	Literature value by Patch Clamp
Astemizole	3.4 ± 0.7	3.3 ± 0.7	4.6 ± 0.003	ND	0.5
Dofetilide	5.8 ± 2.4	28 ± 6	ND	ND	15.3 ± 2.5
E-4031	6.1 ± 0.8	58 ± 5	115 ± 0.02	144 ± 44	14
Pimozide	18.8 ± 2.3	14 ± 4	ND	ND	18
Cisapride	104.9 ± 30.9	123 ± 25	75.6 ± 0.02	5 ± 1	44.5 ± 10.6
Haloperidol	241.4 ± 21.4	234 ± 20	ND	ND	63
Risperidone	>4000	4302 ± 319	ND	ND	167
Verapimil	>4000	3902 ± 529	1332 ± 120	3100 ± 400	830

*Data obtained from Chiu et al., 2004

Inucbation Conditions
RECOMMENDED ASSAY CONDITIONS: Membranes are mixed with radioactive ligand and unlabeled competitor (see Figures 1 and 2 for concentrations tested) in binding buffer in a nonbinding 96-well plate, and incubated for 1-2 h. Prior to filtration, an FB 96-well harvest plate (Millipore cat. # MAHF B1H) is coated with 0.33% polyethyleneimine for 30 min, then washed with 25 mM Tris, pH 7.4, 130 mM NaCl, 5 mM KCl, 0.8 mM CaCl₂, 0.1% BSA. Binding reaction is transferred to the filter plate, and washed 6 times (250μL per well per wash) with Wash Buffer. The plate is dried and counted.

Binding buffer: 10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl, 0.8 mM MgCl₂, 1 mM NaEGTA, 10 mM glucose, 0.1% BSA, filtered and stored at 4°C

Radioligand: [³H]-Astemizole (Perkin Elmer # NET-1140)

Wash Buffer: 25 mM Tris, pH 7.4, 130 mM NaCl, 5 mM KCl, 0.8 mM MgCl₂, 0.05 mM CaCl₂, 0.1% BSA, filtered and stored at 4°C


One package contains enough membranes for at least 200 assays (units), where a unit is the amount of membrane that will yield greater than 4-fold signal: background with ³H-labeled Astemizole.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA with no preservatives.
Packaging method: Membrane protein was adjusted to the indicated concentration in packaging buffer, rapidly frozen, and stored at -80°C.

Precision B+ is a registered trademark of Precision Scientific

100

human

expressed in HEK 293 cells

PrecisION

U04270

dry ice

Maintain frozen at –70°C up to expiration date indicated on the label. Do not freeze and thaw.

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10 - Combustible liquids

WGK 2