A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel
Section snippets
Materials
BacMam hERG, FluxOR thallium assay kit, and cell culture reagents (Dulbecco’s modified Eagle’s medium [DMEM], Opti-MEM, penicillin/streptomycin, and TrypLE) were purchased from Invitrogen (Carlsbad, CA, USA). U-2 OS, CKO-K1, HEK293, and HeLa cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). The LOPAC 1280 library collection was purchased from Sigma–Aldrich (St. Louis, MO, USA). hERG
Assay principle
Thallium ions are used as surrogate ions in this assay to measure activity of the hERG potassium channel. This is possible because of the selective permeability of all potassium ion channels for thallium and the strong driving force for thallium entry into the cells when the channels are opened. In brief, FluxOR dye is loaded into the cells prior to the experiment (Fig. 1A). FluxOR dye contains aminomethyl (AM) ester groups that render the molecule membrane permeant from the extracellular
Discussion
Unlike other ion channels that interact only with ligands of specific structural classes, the hERG potassium ion channel can be blocked or modulated by a broad spectrum of structurally diverse compounds. Thus, the ligand binding assay has clear limitations on the assessment of compound activity at hERG channels because competition with a labeled ligand is limited to structurally similar compounds. This means that binding/displacement assays that pick up compounds occupying the same binding site
Acknowledgments
This research was supported by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research and the Intramural Research Programs of the National Human Genome Research Institute. The authors thank Paul Shinn for assistance with compound management. The authors also thank Shouming Du of Hamamatsu for technical support with the FDSS 7000 kinetic plate reader; Michael O’Grady, Matt Robers, and George Hanson of Invitrogen/Life Technologies and Blake Anson of Cellular Dynamics for
References (44)
Human ether-a-go-go-related (HERG) gene and ATP-sensitive potassium channels as targets for adverse drug effects
Pharmacol. Therapeut.
(2006)- et al.
A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome
Cell
(1995) - et al.
A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel
Cell
(1995) - et al.
QT interval prolongation by non-cardiovascular drugs: issues and solutions for novel drug development
Pharm. Sci. Technol. Today
(1999) - et al.
Validation of a [3H]astemizole binding assay in HEK293 cells expressing HERG K+ channels
J. Pharmacol. Sci.
(2004) - et al.
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
(2004) - et al.
A comparison of the receptor binding and HERG channel affinities for a series of antipsychotic drugs
Eur. J. Pharmacol.
(2002) - et al.
A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels
J. Biomol. Screen.
(2002) - et al.
Evaluation of a high-throughput fluorescence assay method for HERG potassium channel inhibition
J. Biomol. Screen.
(2005) - et al.
Rb+ flux through hERG channels affects the potency of channel blocking drugs: correlation with data obtained using a high-throughput Rb+ efflux assay
J. Biomol. Screen.
(2004)