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

Эта работа была поддержана финансирование от Национального института здоровья гранты 1R21NS073097-01 и 1R01DK082884 (JSD) и Фонд грантов PIER11VCTR Национальные институты.

1. Генерация Стабильный Поликлональные клеточных линий <ol><li> Создание высокого качества стабильной клеточной линии, выражающие Кир канала интересов является важным первым шагом на пути создания надежной высокопроизводительного скрининга анализа. Учредительный K + канала гипер...

<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>

Обработка данных: После того как данные собраны, общий шаг анализа заключается в нормализации флуоресценции ответ каждого колодца, F, к своему исходному значению в начале эксперимента, F 0. Это обычно упоминается как &quot;статическое отношение&quot; и символизирует &quot;F / F <s...

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> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии </td></tr><tr><td> pcDNA5/TO </td><td> Invitrogen </td><td> V1033-20 </td><td> Тетрациклин-индуцируемой экспрессии вектора </td></tr><tr><td> T-REX-НЕК293 </td><td> Invitrogen </td><td> R71007 </td><td> Тетрациклин-индуцируемый клеточной линии </td></tr><tr><td> Lipofectamine LTX / Plus реагентов </td><td> Invitrogen </td><td> 15338100 </td><td> Трансфекции реагента </td></tr><tr><td> FBS </td><td> АТЛАНТА биологические </td><td> S11550 </td><td> СМИ Клеточные культуры </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td><td> СМИ Клеточные культуры </td></tr><tr><td> Гигромицина </td><td> Invitrogen </td><td> 10687-010 </td><td> СМИ Клеточные культуры </td></tr><tr><td> Бластицидин S </td><td> Invitrogen </td&gt; <td> R210-01 </td><td> СМИ Клеточные культуры </td></tr><tr><td> Пенициллина / стрептомицина </td><td> Invitrogen </td><td> 15140 </td><td> СМИ Клеточные культуры </td></tr><tr><td> HBSS-двухвалентных бесплатно </td><td> Mediatech </td><td> 21022CV </td><td> Сотовые стиральные </td></tr><tr><td> Трипсин-0,25% </td><td> Mediatech </td><td> 25053CI </td><td> Клеточная диссоциация </td></tr><tr><td> Тетрациклин-HCl </td><td> Сигма </td><td> T9823 </td><td> Индукционная реагентов </td></tr><tr><td> Диализу FBS </td><td> АТЛАНТА биологические </td><td> S12650 </td><td> Покрытие СМИ </td></tr><tr><td> FluoZin-2 </td><td> Invitrogen </td><td> F24189 </td><td> Флуоресцентный краситель </td></tr><tr><td> Pluronic F-127 </td><td> Invitrogen </td><td> P-3000MP </td><td> Краска загрузки </td></tr><tr><td> HBSS </td><td> Invitrogen </td><td> 14175 </td><td> Буфер для анализа </td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630 </td><td> Буфер для анализа </td></tr><tr><td> NaHCO 3 </td><td> Сигма </td><td> S6297 </td><td> Tl + стимул буфер </td></tr><tr><td> MgSO 4 </td><td> Сигма </td><td> M2643 </td><td> Tl + стимул буфер </td></tr><tr><td> CaSO 4 • 2H 2 O </td><td> Сигма </td><td> C3771 </td><td> Tl + стимул буфер </td></tr><tr><td> D-глюкозы </td><td> Сигма </td><td> G7528 </td><td> Tl + стимул буфер </td></tr><tr><td> Таллия сульфат </td><td> Aldrich </td><td> 204625 </td><td> Tl + стимул буфер </td></tr><tr><td> HEPES </td><td> Сигма </td><td> H4034 </td><td> Tl + стимул буфер </td></tr><tr><td> ДМСО </td><td> Сигма </td><td> D4540 </td><td> Растворитель </td></tr><tr><td> Восьмиканальный электронных дозаторов </td><td> Biohit </td><td> E300 </td><td> Сотовые покрытия в 384 -луночных </td></tr><tr><td> BD PureCoat амина покрытием 384-луночных планшетов </td><td> BD Biosciences </td><td> 356719 </td><td> Анализ микропланшетов </td></tr><tr><td> Эхо квалифицированы 384-Ну полипропилена планшет (384PP) </td><td> Labcyte </td><td> P-05525 </td><td> Микропланшеты соединение источника </td></tr><tr><td> 384-луночных микропланшетов полипропилена </td><td> Greiner Bio-One </td><td> 781280 </td><td></td></tr><tr><td> Multidrop реагента Combi дозатором </td><td> Thermo Scientific </td><td> 5840300 </td><td></td></tr><tr><td> ELx405 микропланшетов шайбы </td><td> BioTek </td><td> ELx405HT </td><td> Автоматизированная стиральных клетки </td></tr><tr><td> Эхо жидкость обработчик </td><td> Labcyte </td><td> Labcyte Echo 550 </td><td></td></tr><tr><td> Браво автоматизированной обработки жидких платформы </td><td> Agilent Technologies </td><td> Стандартное исполнение </td><td></td></tr><tr><td> HamamaТГУ FDSS 6000 </td><td> Hamamatsu </td><td></td><td> Кинетическая изображений ридер </td></tr></tbody></table> Таблица 1. Перечень материалов и реагентов.

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.

Применение тетрациклина-индуцируемой экспрессии система обеспечивает удобный внутреннего контроля для различения Tl + поток через эндогенный пути и Кира канал интерес. Рисунок 1 показывает некоторые примеры ячейки покрытия карт, используемых в различных типах эксперим...

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

Методы разработку и утверждение количественного анализа флуоресценции для измерения активности калия внутрь выпрямителя (Кир) каналы высокой пропускной скрининга соединений представлены.

Высокопроизводительного скрининга для малых молекул модуляторы калиевых каналов входящего Rectifier

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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