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1Department of Pharmacology, Physiology & Neuroscience, University of South Carolina, School of Medicine
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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.
Vazquez, M., Dunn, C. A., Walsh, K. B. A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels. J. Vis. Exp. (62), e3850, doi:10.3791/3850 (2012).
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 tissue1,2. 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 (Gi and Go) 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.
1. Preparation of Cells
2. Preparation of Test Compounds and Cell Treatment
3. Activation of GIRK Channels, Fluorescent Measurement and Analysis
4. Representative Results
An example of the fluorescence signal measured using the GIRK channel assay is shown in Figure 2. Addition of the GPCR ligand somatostatin (200 nM) to the AtT20 cells caused a rapid, time-dependent decrease in the HLB 021-152 fluorescent signal (Figure 2). In contrast, addition of control solution produced a small instantaneous rise in the fluorescence that with time returned to the baseline (Figure 2). Comparison of peak fluorescent values in 96-well plates injected with control and somatostatin solutions gave a Z'-factors in the range of 0.5 to 0.7. For further quantification, the control record was subtracted from the somatostatin record and the resulting somatostatin-sensitive signal analyzed (Figure 2).
The assay provides a method for quickly identifying drugs that modulate GIRK channels. For example, drugs that inhibit the GIRK channel should reduce GPCR ligand-mediated decreases in fluorescence by preventing K+ efflux from the cells. As shown in Figure 3, treatment of the cells with tertiapin-Q, a toxin that blocks GIRK channels7, produced an inhibition of the somatostatin-mediated fluorescent change. Propafenone, an anti-arrhythmic drug that blocks GIRK channels in the heart8, also inhibited the fluorescent change (Figure 4). The assay might also be useful for identifying activators of the GIRK channel. However, application of ethanol (100 & 200 mM), an activator of the GIRK channel2,3, caused no significant change in the fluorescent signal when compared to control solution (p > 0.5).
Figure 1. Experimental design of the GIRK channel fluorescent assay. AtT20 cells are incubated in buffer containing a membrane potential-sensitive fluorescent dye (D). The dye molecules enter into the cells and become fluorescent upon binding to intracellular proteins (top panel). Binding of somatostatin (Som) to its GPCR stimulates the G inhibitory protein (Gi) causing the activation of the GIRK channel (bottom panel). The subsequent efflux of K+ out of the cells causes the membrane potential to become more negative and the dye molecules to exit the cells. As a result the fluorescent signal decreases (bottom panel).
Figure 2. Representative HLB 021-152 fluorescent signal measured over time in the AtT20 cells during addition of somatostatin or control solution. The ratio of the fluorescent intensity (F/FO) was calculated by dividing the signal in the presence (F) of somatostatin (or control solution) by the baseline signal measured before (FO) addition of somatostatin (or control solution). Each point represents the mean ± SE obtained in 5-6 wells. Somatostatin or control solution was added at time zero (↓). Addition of the control solution caused a small transient increase in the fluorescent signal. This resulted from a temperature change caused by injection of the solution.
Figure 3. Treatment of the AtT20 cells with tertiapin-Q (500 nM) inhibits the somatostatin-mediated decrease in the fluorescent signal by binding to and blocking the GIRK channels. Each point represents the mean ± SE obtained in 4-6 wells. Somatostatin was added at time zero (↓).
Figure 4. Treatment of the AtT20 cells with propafenone (20 μM) also inhibits the somatostatin-mediated decrease in the fluorescent signal. Each point represents the mean ± SE obtained in 5-6 wells. Somatostatin was added at time zero (↓).
While membrane potential-sensitive fluorescent dyes have been used to identify drugs that modulate ion channels9,10, 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 GIRK channel11 or clonal cell lines (AtT20, PC-12) expressing an endogenous channel. In addition, the assay may be adapted for screening drugs in embryonic stem cells and primary cell cultures. As used with the AtT20 cells, the assay also provides the extra benefit of allowing the study of multiple GPCR ligands (somatostatin and carbachol) on the GIRK channel. As with any fluorescent assay, control experiments must be carried out to eliminate artifacts due to compound auto-fluorescence and to identify errors resulting from direct interactions of the compounds with the dye molecules. Alternate approaches such as the thallium influx assay11 and automated patch clamp procedure12 should also be considered.
No conflicts of interest declared.
This work was supported by US Public Health Service award NS-071530.
|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.|
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