Name: high-throughput screening for small-molecule modulators of inward rectifier potassium channels
Uploaded: 2013-01-27T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels at JoVE.com

यह काम स्वास्थ्य 1R21NS073097-01 और 1R01DK082884 (JSD) और राष्ट्रीय संस्थानों अनुदान PIER11VCTR के लिए फाउंडेशन अनुदान के राष्ट्रीय संस्थानों से धन के द्वारा समर्थित किया गया.

1. स्थिर पॉलीक्लोनल सेल लाइन्स का सृजन <ol><li> एक उच्च गुणवत्ता स्थिर सेल लाइन ब्याज की कीर चैनल व्यक्त की स्थापना एक मजबूत उच्च throughput स्क्रीनिंग परख के विकास की ओर एक महत्वपूर्ण पहला कदम है. विधान कश्मीर +</...

<ol>
	<li>Ehrlich, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inward+rectifier+potassium+currents+as+a+target+for+atrial+fibrillation+therapy.">Inward rectifier potassium currents as a target for atrial fibrillation therapy.</a> J. Cardiovasc. Pharmacol. 52 (2), 129 (2008).</li><li>Bhave, G., Lonergan, D., Chauder, B. A., Denton, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+modulators+of+inward+rectifier+K++channels:+recent+advances+and+future+possibilities.">Small-molecule modulators of inward rectifier K+ channels: recent advances and future possibilities.</a> Future Med. Chem. 2 (5), 757 (2010).</li><li>Swartz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tarantula+toxins+interacting+with+voltage+sensors+in+potassium+channels.">Tarantula toxins interacting with voltage sensors in potassium channels.</a> Toxicon. 49 (2), 213 (2007).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-affinity+inhibitor+for+inward-rectifier+K++channels.">A novel high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 37 (38), 13291 (1998).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+stable+form+of+tertiapin:+a+high-affinity+inhibitor+for+inward-rectifier+K++channels.">Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 38 (43), 14286 (1999).</li><li>Roy, A., McDonald, P. R., Sittampalam, S., Chaguturu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+access+high+throughput+drug+discovery+in+the+public+domain:+a+Mount+Everest+in+the+making.">Open access high throughput drug discovery in the public domain: a Mount Everest in the making.</a> Curr. Pharm. Biotechnol. 11 (7), 764 (2010).</li><li>Weaver, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thallium-sensitive,+fluorescence-based+assay+for+detecting+and+characterizing+potassium+channel+modulators+in+mammalian+cells.">A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.</a> J. Biomol. Screen. 9 (8), 671 (2004).</li><li>Lewis, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+reveals+a+small-molecule+inhibitor+of+the+renal+outer+medullary+potassium+channel+and.">High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and.</a> 76 (5), 1094 (2009).</li><li>Bhave, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+selective+small-molecule+inhibitor+of+Kir1.1,+the+renal+outer+medullary+potassium+channel.">Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.</a> Mol. Pharmacol. 79 (1), 42 (2011).</li><li>Raphemot, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterization,+and+structure-activity+relationships+of+an+inhibitor+of+inward+rectifier+potassium+(Kir)+channels+with+preference+for+Kir2.3,+Kir3.x,+and+Kir7.1.">Discovery, characterization, and structure-activity relationships of an inhibitor of inward rectifier potassium (Kir) channels with preference for Kir2.3, Kir3.x, and Kir7.1.</a> Front Pharmacol. 2, 75 (2012).</li><li>Niswender, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+of+Gi/o-linked+G+protein-coupled+receptor+coupling+to+potassium+channels+provides+new+insights+into+the+pharmacology+of+the+group+III+metabotropic+glutamate+receptors.">A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors.</a> Mol. Pharmacol. 73 (4), 1213 (2008).</li></ol>

डेटा उपचार: एक बार डेटा एकत्र कर रहे हैं, इस विश्लेषण में एक आम कदम शामिल प्रत्येक अच्छी तरह से प्रतिदीप्ति प्रतिक्रिया, एफ, प्रयोग, एफ 0 की शुरुआत में अपने प्रारंभिक मूल्य सामान्य. यह आमतौर पर &...

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.
The formula in step 7.1 of the Protocol has been updated from:
Z prime = 1- (3SDp + 3SDn)/|meanp + meann |
to:
Z prime = 1- (3SDp + 3SDn)/|meanp - meann |

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.

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

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> कैटलॉग संख्या </td><td> टिप्पणियां </td></tr><tr><td> pcDNA5/TO </td><td> Invitrogen </td><td> V1033-20 </td><td> टेट्रासाइक्लिन-inducible अभिव्यक्ति वेक्टर </td></tr><tr><td> T-rex HEK293 कोशिकाओं </td><td> Invitrogen </td><td> R71007 </td><td> टेट्रासाइक्लिन-inducible सेल लाइन </td></tr><tr><td> Lipofectamine अभिकर्मक / LTX प्लस </td><td> Invitrogen </td><td> 15338100 </td><td> अभिकर्मक अभिकर्मक </td></tr><tr><td> FBS </td><td> अटलांटा बायोलॉजिकल </td><td> S11550 </td><td> सेल संस्कृति मीडिया </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td><td> सेल संस्कृति मीडिया </td></tr><tr><td> Hygromycin बी </td><td> Invitrogen </td><td> 10687-010 </td><td> सेल संस्कृति मीडिया </td></tr><tr><td> एस Blasticidin </td><td> Invitrogen </td&gt; <td> R210-01 </td><td> सेल संस्कृति मीडिया </td></tr><tr><td> पेनिसिलिन स्ट्रेप्टोमाइसिन / </td><td> Invitrogen </td><td> 15140 </td><td> सेल संस्कृति मीडिया </td></tr><tr><td> मुफ्त HbSS द्विसंयोजक </td><td> Mediatech </td><td> 21022CV </td><td> सेल धोने </td></tr><tr><td> ट्रिप्सिन 0.25% </td><td> Mediatech </td><td> 25053CI </td><td> सेल हदबंदी </td></tr><tr><td> टेट्रासाइक्लिन - एचसीएल </td><td> सिग्मा </td><td> T9823 </td><td> प्रेरण अभिकर्मक </td></tr><tr><td> Dialyzed FBS </td><td> अटलांटा बायोलॉजिकल </td><td> S12650 </td><td> चढ़ाना मीडिया </td></tr><tr><td> FluoZin-2 </td><td> Invitrogen </td><td> F24189 </td><td> फ्लोरोसेंट रंजक </td></tr><tr><td> F-127 Pluronic </td><td> Invitrogen </td><td> पी 3000MP </td><td> डाई loading </td></tr><tr><td> HbSS </td><td> Invitrogen </td><td> 14175 </td><td> परख बफर </td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630 </td><td> परख बफर </td></tr><tr><td> 3 NaHCO </td><td> सिग्मा </td><td> S6297 </td><td> Tl + प्रोत्साहन बफर </td></tr><tr><td> 4 MgSO </td><td> सिग्मा </td><td> M2643 </td><td> Tl + प्रोत्साहन बफर </td></tr><tr><td> 4 CASO • 2H 2 हे </td><td> सिग्मा </td><td> C3771 </td><td> Tl + प्रोत्साहन बफर </td></tr><tr><td> D-ग्लूकोज </td><td> सिग्मा </td><td> G7528 </td><td> Tl + प्रोत्साहन बफर </td></tr><tr><td> थालियम सल्फेट </td><td> Aldrich </td><td> 204625 </td><td> Tl + प्रोत्साहन बफर </td></tr><tr><td> HEPES </td><td> सिग्मा </td><td> H4034 </td><td> Tl + प्रोत्साहन बफर </td></tr><tr><td> DMSO </td><td> सिग्मा </td><td> D4540 </td><td> विलायक </td></tr><tr><td> आठ चैनल इलेक्ट्रॉनिक pipettor </td><td> Biohit </td><td> E300 </td><td> 384 में सेल चढ़ाना -अच्छी तरह प्लेटें </td></tr><tr><td> BD amine लेपित प्लेटें 384 अच्छी तरह PureCoat </td><td> बी.डी. बायोसाइंसेज </td><td> 356719 </td><td> परख microplates </td></tr><tr><td> इको 384 खैर polypropylene microplate (384PP) अर्हता प्राप्त </td><td> Labcyte </td><td> पी 05,525 </td><td> यौगिक स्रोत microplates </td></tr><tr><td> 384 अच्छी तरह polypropylene microplates </td><td> जैव एक Greiner </td><td> 781280 </td><td></td></tr><tr><td> Multidrop कोम्बी अभिकर्मक मशीन </td><td> थर्मो वैज्ञानिक </td><td> 5840300 </td><td></td></tr><tr><td> ELx405 microplate वॉशर </td><td> Biotek </td><td> ELx405HT </td><td> स्वचालित सेल धोने </td></tr><tr><td> तरल हैंडलर इको </td><td> Labcyte </td><td> Labcyte इको 550 </td><td></td></tr><tr><td> ब्रावो स्वचालित तरल संभालने मंच </td><td> टेक्नोलॉजीज </td><td> मानक मॉडल </td><td></td></tr><tr><td> Hamamaत्सू FDSS 6000 </td><td> हामामत्सु </td><td></td><td> काइनेटिक इमेजिंग प्लेट पाठक </td></tr></tbody></table> तालिका 1. सामग्री और अभिकर्मकों की सूची.

high-throughput screening for small-molecule modulators of inward rectifier potassium channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. 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 channels4,5, 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 screening7,8,9,10.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 FluxOR7,8. 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 available7,8 and have continued to do so9,10. 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.

एक टेट्रासाइक्लिन-inducible अभिव्यक्ति प्रणाली का उपयोग अंतर्जात रास्ते और ब्याज की कीर चैनल के माध्यम से Tl प्रवाह + भेद के लिए एक सुविधाजनक आंतरिक नियंत्रण प्रदान करता है. चित्रा 1 से पता चलता है स...

Watch this Scientific Journal Video about High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels at JoVE.com

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

आवक सही करनेवाला (कीर) पोटेशियम उच्च throughput यौगिक स्क्रीनिंग के लिए चैनलों की गतिविधि को मापने के लिए एक मात्रात्मक प्रतिदीप्ति परख के लिए विकासशील और मान्य तरीके प्रस्तुत किया है.

आवक करनेवाला पोटेशियम चैनल के छोटे अणु Modulators के लिए उच्च throughput स्क्रीनिंग

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

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