A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

Summary

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K⁺ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Abstract

G protein-gated inward rectifier K⁺ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue^1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (G_i and G_o) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K⁺ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.

GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders³. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations³.

Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K⁺ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K⁺ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry⁴ or radiotracer analysis⁵, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

Protocol

1. Preparation of Cells

1. Grow pituitary AtT20 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% horse serum and maintain cultures at 37 °C in a humidified atmosphere of 5% CO₂.
2. Maintain the culture by subculturing the cells every 5 to 7 days using a standard trypsinization procedure.
3. Coat the wells of black, clear bottom, 96-well plates with 50 μL of poly-l-lysine. Allow the wells to dry for 30 min in the incubator.
4. Plate the cells in the 96-well plates containing 200 μL media at density of 30,000 cells per well and store the cells in the incubator for 3-4 days.
5. Use an aspi....

Discussion

While membrane potential-sensitive fluorescent dyes have been used to identify drugs that modulate ion channels^9,10, this is the first report of their application for neuronal GIRK channel drug discovery. The GIRK channel fluorescent assay presented here provides a fast, reliable and real-time method for the screening of ligand-gated K⁺ channels. The assay can be modified for use with a wide range of cells including immortalized cells lines (HEK293, CHO, etc.) expressing an exogenous recombinant GI.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Cellgro	10-013
Horse serum	Invitrogen	16050-114
96-well plates	Corning	3603
Poly-l-lysine	Sigma-Aldrich	P4707
Somatostatin	Sigma-Aldrich	S9129
Carbachol	Sigma-Aldrich	C4382
HLB 021-152	AnaSpec	89300
Versette automated liquid handler	Thermo Fisher Scientific, Inc.	650-01
Synergy2 fluorescent plate reader	BioTek
Gen5 analysis software	BioTek
Table 1. Table of specific reagents and equipment.