Download Human Kv11.1 (hERG) Channel Membrane Preparation

Human Kv11.1 (hERG) Channel Membrane Preparation
Technical Manual No. TM0516
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Version 03092011
Introduction ….…………………………………………………………………………….
Background ………..……………………………………………………………………….
Representative Data.………………………………………………………………………
Brief Competition Binding Protocol ………………………………………………………
References ……………………………………………………………………………….
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Introduction
Catalog Number: M00421
Cell Line Name: HEK293/hERG
Gene Synonyms: ERG, ERG1, HERG
Gene Expressed: GenBank Accession Number NM_000238.2; no expressed tags
Host Cell: HEK293
Quantity: 1 vial (1 ml per vial)
Protein Concentration: 1 mg/ml
Storage Buffer: 50 mM HEPES, 0.1 mM EDTA, 10 % glycerol
Application: Binding assay for human Kv11.1 (hERG) channel
Storage: Store at -80°C
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Background
hERG (human ether-a-go-go related gene) codes for a protein known as Kv11.1 potassium ion channel. This ion
channel mediates the repolarizing IKr current in the cardiac action potential. Suppression of IKr by loss of function
mutations in the hERG gene or by untoward drug block can prolong the QT interval and predispose patients to the
potentially lethal arrhythmia Torsades de Pointes (TdP). A number of clinically drugs in the market have had the
tendency to inhibit hERG, which can increase concomitant risk of sudden death as well as cause other side effects.
Therefore, hERG channel is an important antitarget to avoid during drug development.
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Representative Data
pmol/mg protein
Saturation Binding for hERG Channel
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Total binding
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Specific binding
NSB
Bmax = 2.61 pmol/mg protein
Kd = 3.57 nM
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10
20
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[3H]Dofetilide (nM)
Figure 1 10 μg of membranes prepared from 293 cells stably expressing hERG channels were incubated with
indicated concentrations of [3H]Dofetilide in the absence (total binding) or presence of 1000-fold excess unlabeled
Dofetilide (nonspecific binding, NSB). Binding was terminated by rapid filtration. Specific binding was defined by
subtracting NSB from total binding. Data were fit to one-site binding equation using a non-linear regression
method.
Competition Binding for hERG Channel
Inhibition %
100
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60
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Ki = 3.4 nM
IC50 = 7.2 nM
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Log[Astemizole] M
Figure 2 10 μg of membranes prepared from 293 cells stably expressing hERG channels were incubated with
indicated concentrations of Astemizole in the presence of 10 nM [3H]Dofetilide. Binding was terminated by rapid
filtration. Data were fit to one-site competition equation using a non-linear regression method.
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Brief Competition Binding Protocol
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Incubated 10 μg membranes with radio labeled ligand and unlabeled competitor (see Figures 1 and 2 for
concentrations tested) in binding buffer in a nonbinding 96-well plate. Incubated for 60 min at 37°C.
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Prior to filtration, coat a GF/C 96-well filter plate with 0.5% polyethyleneimine (PEI) for 30 min at 4°C, then
washed the plate with 1 ml/well 50mM HEPES, 0.5% BSA (pH 7.4).
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Transfer the binding mixtures then to the filter plate. After quick filtration, wash the plate for 3 times (3 ml per
well totally) with Wash Buffer.
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Dry the plate for 0.5 h and then add 50 μl scintillation cocktail (Microscint20). Stay for 1min and count on
TopCount NXT for 1 min/well.
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Binding buffer: 10 mM HEPES, pH7.4, 130 mM NaCl, 60 mM KCl, 0.8 mM MgCl2, 1 mM NaEGTA, 10 mM
glucose, 0.1% BSA, filtered and stored at 4°C
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Wash Buffer: 10 mM Tris-HCl, pH 7.4, filtered and stored at 4°C.
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References
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Diaz G. et. al. (2004) The [3H]dofetilide binding assay is a predictive screening tool for hERG blockade and
proarrhythmia: Comparison of intact cell and membrane preparations and effects of altering [K+]o. J.
Pharmacol. Toxicol. Methods.50(3):187-99.
2.
Chen, M. et al. (2007). Improved functional expression of recombinant human ether-a-go-go (hERG) K+
channels by cultivation at reduced temperature. BMC. Biotechnol. 7: 93.
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Finlayson, K. et al. (2001). [3H]dofetilide binding to HERG transfected membranes: a potential high throughput
preclinical screen. Eur. J. Pharmacol. 430(1): 147-8.
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