High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Summary

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Abstract

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain^1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered³. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels^4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.

The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology⁶. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).

Here, we describe how to develop a fluorescence-based thallium (Tl⁺) flux assay of Kir channel function for high-throughput compound screening^7,8,9,10.The assay is based on the permeability of the K⁺ channel pore to the K⁺ congener Tl⁺. A commercially available fluorescent Tl⁺ reporter dye is used to detect transmembrane flux of Tl⁺ through the pore. There are at least three commercially available dyes that are suitable for Tl⁺ flux assays: BTC, FluoZin-2, and FluxOR^7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl⁺ binding. We began working with FluoZin-2 before FluxOR was available^7,8 and have continued to do so^9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl⁺ readily permeates most K⁺ channels, the assay should be adaptable to most K⁺ channel targets.

Protocol

1. Generation of Stable Polyclonal Cell Lines

1. The establishment of a high quality stable cell line expressing the Kir channel of interest is an important first step toward developing a robust high-throughput screening assay. Constitutive K⁺ channel overexpression can lead to activation of cell death pathways, stable cell line degeneration and loss of assay performance. To avoid these potential problems and provide a convenient internal control for assay development (see below), a tetracycline-inducible expression system is recommended⁸.
2. Culture the parental T-REx-HEK293 cells using standard techniques in B-medium (DMEM gr....

Results

The use of a tetracycline-inducible expression system provides a convenient internal control for distinguishing Tl⁺ flux through endogenous pathways and the Kir channel of interest. Figure 1 shows some examples of cell plating maps used in different types of experiments. The positions of wells containing uninduced or tetracycline-induced cells are indicated with different colors. Figure 2A shows the source plate map used to determine the optimal Tl⁺ concentration fo.......

Discussion

Data treatment: Once the data are collected, a common step in the analysis involves normalizing each well's fluorescence response, F, to its initial value at the beginning of the experiment, F₀. This is commonly referred to as the "static ratio" and symbolized "F/F₀^"_. In cases where F₀ is dominated by the indicator dye the static ratio operation will substantially correct for many factors such as disuniformities in illumination, signal collection, an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pcDNA5/TO	Invitrogen	V1033-20	Tetracycline-inducible expression vector
T-REx-HEK293 cells	Invitrogen	R71007	Tetracycline-inducible cell line
Lipofectamine LTX/Plus Reagent	Invitrogen	15338100	Transfection reagent
FBS	ATLANTA Biologicals	S11550	Cell culture media
DMEM	Invitrogen	11965	Cell culture media
Hygromycin B	Invitrogen	10687-010	Cell culture media
Blasticidin S	Invitrogen	R210-01	Cell culture media
Penicillin/Streptomycin	Invitrogen	15140	Cell culture media
HBSS-divalent free	Mediatech	21022CV	Cell washing
Trypsin-0.25%	Mediatech	25053CI	Cell dissociation
Tetracycline-HCl	Sigma	T9823	Induction reagent
Dialyzed FBS	ATLANTA Biologicals	S12650	Plating media
FluoZin-2	Invitrogen	F24189	Fluorescent dye
Pluronic F-127	Invitrogen	P-3000MP	Dye loading
HBSS	Invitrogen	14175	Assay buffer
HEPES	Invitrogen	15630	Assay buffer
NaHCO3	Sigma	S6297	Tl+ stimulus buffer
MgSO4	Sigma	M2643	Tl+ stimulus buffer
CaSO4∙2H2O	Sigma	C3771	Tl+ stimulus buffer
D-Glucose	Sigma	G7528	Tl+ stimulus buffer
Thallium sulfate	Aldrich	204625	Tl+ stimulus buffer
HEPES	Sigma	H4034	Tl+ stimulus buffer
DMSO	Sigma	D4540	Solvent
Eight-channel electronic pipettor	Biohit	E300	Cell plating in 384-well plates
BD PureCoat amine-coated 384-well plates	BD Biosciences	356719	Assay microplates
Echo qualified 384-Well polypropylene microplate (384PP)	Labcyte	P-05525	Compound source microplates
384-well polypropylene microplates	Greiner Bio-One	781280
Multidrop Combi reagent dispenser	Thermo Scientific	5840300
ELx405 microplate washer	BioTek	ELx405HT	Automated cell washing
Echo liquid handler	Labcyte	Labcyte Echo 550
Bravo automated liquid handling platform	Agilent Technologies	Standard model
Hamamatsu FDSS 6000	Hamamatsu		Kinetic imaging plate reader
Table 1. List of Materials and Reagents.