Name: a fluorescent screening assay for identifying modulators of girk channels
Uploaded: 2012-04-24T13:00:00
Description: Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

This work was supported by US Public Health Service award NS-071530.

1. Preparation of Cells
<ol>
 <li> Grow pituitary AtT20 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% horse serum and maintain cultures at 37 &deg;C in a humidified atmosphere of 5% CO2.</li>
 <li>Maintain the culture by subculturing the cells every 5 to 7 days using a standard trypsinization procedure.</li>
 <li> Coat the wells of black, clear bottom, 96-well plates with 50 &mu;L of poly-l-lysine. Allow the wells to dry for 30 min in the incubator.</li>
 <li>Plate the cells ...

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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 GI...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>CellGro</td>
 <td>10-013</td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>Invitrogen</td>
 <td>16050-114 </td>
 </tr>
 <tr>
 <td>96-well plates</td>
 <td>Corning</td>
 <td>3603</td>
 </tr>
 <tr>
 <td>Poly-l-lysine</td>
 <td>Sigma-Aldrich</td>
 <td>P4707</td>
 </tr>
 <tr>
 <td>Somatostatin</td>
 <td>Sigma-Aldrich</td>
 <td>S9129</td>
 </tr>
 <tr>
 <td>Carbachol</td>
 <td>Sigma-Aldrich</td>
 <td>C4382</td>
 </tr>
 <tr>
 <td>HLB 021-152</td>
 <td>AnaSpec</td>
 <td>89300</td>
 </tr>
 <tr>
 <td>Versette automated liquid handler</td>
 <td>ThermoFisher</td>
 <td>650-01</td>
 </tr>
 <tr>
 <td>Synergy2 fluorescent plate reader</td>
 <td>Biotek</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gen5 analysis software</td>
 <td>Biotek</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table 1. Table of specific reagents and equipment.

a fluorescent screening assay for identifying modulators of girk channels

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.

Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

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.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

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