内向整流钾通道的小分子调节剂的高通量筛选
Summary
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。
Abstract
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。
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the reagent | Company | Catalog number | Comments |
| pcDNA5/TO | Invitrogen | V1033-20 | Tetracycline-inducible expression vector |
| T-REx-HEK293 cells | Invitrogen | R71007 | Tetracycline-inducible cell line |
| Lipofectamine LTX/Plus Reagent | Invitrogen | 15338100 | Transfection reagent |
| FBS | ATLANTA Biologicals | S11550 | Cell culture media |
| DMEM | Invitrogen | 11965 | Cell culture media |
| Hygromycin B | Invitrogen | 10687-010 | Cell culture media |
| Blasticidin S | Invitrogen | R210-01 | Cell culture media |
| Penicillin/Streptomycin | Invitrogen | 15140 | Cell culture media |
| HBSS-divalent free | Mediatech | 21022CV | Cell washing |
| Trypsin-0.25% | Mediatech | 25053CI | Cell dissociation |
| Tetracycline-HCl | Sigma | T9823 | Induction reagent |
| Dialyzed FBS | ATLANTA Biologicals | S12650 | Plating media |
| FluoZin-2 | Invitrogen | F24189 | Fluorescent dye |
| Pluronic F-127 | Invitrogen | P-3000MP | Dye loading |
| HBSS | Invitrogen | 14175 | Assay buffer |
| HEPES | Invitrogen | 15630 | Assay buffer |
| NaHCO3 | Sigma | S6297 | Tl+ stimulus buffer |
| MgSO4 | Sigma | M2643 | Tl+ stimulus buffer |
| CaSO4•2H2O | Sigma | C3771 | Tl+ stimulus buffer |
| D-Glucose | Sigma | G7528 | Tl+ stimulus buffer |
| Thallium sulfate | Aldrich | 204625 | Tl+ stimulus buffer |
| HEPES | Sigma | H4034 | Tl+ stimulus buffer |
| DMSO | Sigma | D4540 | Solvent |
| Eight-channel electronic pipettor | Biohit | E300 | Cell plating in 384-well plates |
| BD PureCoat amine-coated 384-well plates | BD Biosciences | 356719 | Assay microplates |
| Echo qualified 384-Well polypropylene microplate (384PP) | Labcyte | P-05525 | Compound source microplates |
| 384-well polypropylene microplates | Greiner Bio-One | 781280 | |
| Multidrop Combi reagent dispenser | Thermo Scientific | 5840300 | |
| ELx405 microplate washer | BioTek | ELx405HT | Automated cell washing |
| Echo liquid handler | Labcyte | Labcyte Echo 550 | |
| Bravo automated liquid handling platform | Agilent Technologies | Standard model | |
| Hamamatsu FDSS 6000 | Hamamatsu | Kinetic imaging plate reader |
Table 1. List of Materials and Reagents.
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