GIRK चैनल की Modulators की पहचान के लिए एक फ्लोरोसेंट जांच परख
Summary
दवाओं के कि प्रोटीन gated जी आवक सही करनेवाला कश्मीर साथ बातचीत की पहचान के लिए एक वास्तविक समय स्क्रीनिंग प्रक्रिया<sup> +</sup> चैनल (GIRK) में वर्णित है. परख झिल्ली क्षमता संवेदनशील फ्लोरोसेंट GIRK चैनल गतिविधि को मापने के रंगों का इस्तेमाल करता है. इस तकनीक को सेल लाइनों की एक संख्या पर इस्तेमाल के लिए अनुकूलनीय है.
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 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.
Protocol
Discussion
जबकि संभावित संवेदनशील झिल्ली फ्लोरोसेंट रंजक के लिए दवाओं है कि आयन चैनल मिलाना 9,10 की पहचान के लिए इस्तेमाल किया गया है, इस neuronal GIRK चैनल दवाओं की खोज के लिए अपने आवेदन की पहली रिपोर्ट है. GIRK चैनल फ्लोर?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
यह काम अमेरिका के सार्वजनिक स्वास्थ्य सेवा पुरस्कार एन एस 071,530 द्वारा समर्थित किया गया था.
Materials
| Name of the reagent | Company | Catalogue number |
| 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 | ThermoFisher | 650-01 |
| Synergy2 fluorescent plate reader | Biotek | |
| Gen5 analysis software | Biotek |
Table 1. Table of specific reagents and equipment.
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