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

这项工作是从美国国立卫生补助（1R21NS073097-01）和1R01DK082884（JSD）为国家机构授予PIER11VCTR的和基金会的资金支持。

1。生成稳定的多克隆细胞系<ol><li>高品质的稳定表达细胞系感兴趣的KIR通道的建立，是一个发展一个强大的高通量筛选分析的重要的第一步。构K +通道的过度表达可导致细胞死亡的途径，稳定的细胞系的检测性能的变性和损失的激活。为了避免这些潜在的问题，并提供了一种方便的内部控制，研发分析（见下文），四环素诱导表达系统建议8。 </li><li>文化的父母T-REX-HEK293细胞用?...

<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>

数据处理：一旦数据被收集，在分析中的一个共同的步骤涉及到的各孔的荧光响应到其初始值，在开始的实验中，F 0，F，正常化。这通常被称为“静态比”和符号“F / F 0”。静态比操作将在F 0的情况下，主要是由指示剂染料基本上纠正许多因素，如照明，信号采集disuniformities，和细胞数。染料信号弱的情况下，或在系统中的的背景荧光或反射高，静...

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> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> pcDNA5/TO </td><td> Invitrogen公司</td><td> V1033-20 </td><td>四环素诱导表达载体</td></tr><tr><td> T-REX-HEK293细胞</td><td> Invitrogen公司</td><td> R71007 </td><td>四环素诱导的细胞系</td></tr><tr><td>脂质体LTX /加试剂</td><td> Invitrogen公司</td><td> 15338100 </td><td>转染试剂</td></tr><tr><td> FBS </td><td>亚特兰大生物</td><td> S11550 </td><td>细胞培养基</td></tr><tr><td> DMEM </td><td> Invitrogen公司</td><td> 11965 </td><td>细胞培养基</td></tr><tr><td>潮霉素B </td><td> Invitrogen公司</td><td> 10687-010 </td><td>细胞培养基</td></tr><tr><td>杀稻瘟小号</td><td> Invitrogen公司</td&gt; <td> R210-01 </td><td>细胞培养基</td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15140 </td><td>细胞培养基</td></tr><tr><td> HBSS价免费</td><td>敏达</td><td> 21022CV </td><td>细胞洗涤</td></tr><tr><td>胰蛋白酶-0.25％ </td><td>敏达</td><td> 25053CI </td><td>细胞分离</td></tr><tr><td>四环素盐酸</td><td>西格玛</td><td> T9823 </td><td>感应试剂</td></tr><tr><td>透析FBS </td><td>亚特兰大生物</td><td> S12650 </td><td>电镀媒体</td></tr><tr><td> FluoZin-2 </td><td> Invitrogen公司</td><td> F24189 </td><td>荧光染料</td></tr><tr><td>聚醚F-127 </td><td> Invitrogen公司</td><td> P-3000MP </td><td>染料吸附量</td></tr><tr><td> HBSS </td><td> Invitrogen公司</td><td> 14175 </td><td>分析缓冲液</td></tr><tr><td> HEPES </td><td> Invitrogen公司</td><td> 15630 </td><td>分析缓冲液</td></tr><tr><td> 碳酸氢钠 </td><td>西格玛</td><td> S6297 </td><td> TL +刺激缓冲</td></tr><tr><td> 硫酸镁 </td><td>西格玛</td><td> M2643 </td><td> TL +刺激缓冲</td></tr><tr><td>硫酸钙4·2H 2 O </td><td>西格玛</td><td> C3771 </td><td> TL +刺激缓冲</td></tr><tr><td> D-葡萄糖</td><td>西格玛</td><td> G7528 </td><td> TL +刺激缓冲</td></tr><tr><td>硫酸铊</td><td> Aldrich公司</td><td> 204625 </td><td> TL +刺激缓冲</td></tr><tr><td> HEPES </td><td>西格玛</td><td> H4034 </td><td> TL +刺激缓冲</td></tr><tr><td> DMSO </td><td>西格玛</td><td> D4540 </td><td>溶剂</td></tr><tr><td>八通道电子移液器</td><td>百得</td><td> E300 </td><td>在384细胞电镀 -孔板</td></tr><tr><td> BD PureCoat胺涂层的384孔板</td><td> BD Biosciences公司</td><td> 356719 </td><td>检测微孔板</td></tr><tr><td>回声合格384孔微孔板聚丙烯（384PP） </td><td> Labcyte公司</td><td> P-05525 </td><td>复合源微孔板</td></tr><tr><td> 384孔聚丙烯微孔板</td><td>格雷纳生物之一</td><td> 781280 </td><td></td></tr><tr><td>多点COMBI试剂分配器</td><td> Thermo Scientific的</td><td> 5840300 </td><td></td></tr><tr><td> ELx405洗板机</td><td>百特</td><td> ELx405HT </td><td>自动细胞清洗</td></tr><tr><td>回声液体处理</td><td> Labcyte公司</td><td> Labcyte公司回声550 </td><td></td></tr><tr><td> Bravo自动化液体处理平台</td><td>安捷伦科技公司</td><td>标准模型</td><td></td></tr><tr><td> HamamaTSU FDSS 6000 </td><td>滨松</td><td></td><td>动力学成像板读卡器</td></tr></tbody></table> 表1中 。材料与试剂名单。

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

具体内向整流钾通道（基里巴斯）家庭成员的各种疾病，包括高血压，房颤，疼痛1,2推测的药物靶标。然而，在大多数情况下，对理解他们的治疗潜力甚至连最基本的生理功能方面取得的进展已经放缓缺乏良好的药理工具。事实上，分 ​​子药理学的内向整流家庭的已经远远落后的S4家族的电压门控钾通道（KV），其中一些已发现的的纳摩尔亲和力和高度选择性的肽毒素调节3。蜂毒的毒素tertiapin和其衍生物是有效的抑制剂的Kir1.1的Kir3通道4,5，但肽是使用有限的治疗以及实验由于它们的抗原特性和生物利用度差，代谢稳定性和组织外显率。强效的发展改进的药理特性和选择性的小分子探针将是一个关键Kir通道的生理和治疗潜力的充分认识。 分子库探针生产中心网（MLPCN）的支持由美国国立卫生研究院（NIH）共同基金创造了机会学术界的科学家开始探测发现运动的分子靶点和信号转导通路需要更好的药理学6。 MLPCN提供研究获得行业规模筛选中心，药物化学和信息支持开发小分子探针，以阐明基因和基因网络的功能。关键的一步在进入到MLPCN是一个强大的目标或途径的具体分析，是适合高通量筛选（HTS）的发展。 在这里，我们介绍了如何开发基于荧光的铊（TL +）通量阿萨y的高通量化合物筛选7,8,9,10珥通道功能。该测定是基于的通透性上的K +通道孔的K +同源物铊+。市售的荧光铊+报告染料是用来检测通过孔隙的Tl +跨膜通量。有至少三个市售染料适合铊+磁通检测：BTC，FluoZin-2，7,8 FluxOR。该协议描述试验发展，使用FluoZin-2。虽然最初开发和销售锌指标，FluoZin-2是一个强大的和剂量依赖性增加，荧光发射后，TL +结合。我们开始工作前与FluoZin-2 FluxOR是7,8，并继续这样做9,10。然而，分析发展中的步骤基本上是相同的所有三种染料，用户应确定染料是最适合其特定的N电火工品。我们还讨论了检测的性能基准测试，必须达到被认为是进入到MLPCN的。由于铊+容易渗透K +通道，该法应适用于K +通道的目标。

四环素诱导表达系统的使用提供了一个方便的内部控制，用于区分铊+通量通过内源性途径和珥感兴趣通道的图1示出了一些实施例的不同类型的实验中使用的细胞接种地图。含有未诱导或四环素诱导的细胞的孔的位置用不同的颜色表示图2A显示了使用，以确定最佳的铊+浓度的检测开发和筛选化合物的源板图。渐变的颜色表示的3倍稀释系列，范围从100％到0....

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

开发和验证测量活动的高通量化合物筛选的内向整流钾通道（基里巴斯）荧光定量检测方法。

内向整流钾通道的小分子调节剂的高通量筛选

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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