Name: a fluorescent screening assay for identifying modulators of girk channels
Uploaded: 2012-04-24T13:00:00
Description: Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

这项工作得到了美国公共卫生服务奖NS-071530。

1。单元的制备<ol><li>垂体AtT20细胞生长在杜尔贝科的改良Eagle培养基（DMEM培养基）10％马血清和保持在5％ 二氧化碳培养箱的气氛中，在37°C培养为辅。 </li><li>保持文化的传代细胞，每5至7天，使用标准的胰蛋白酶消化过程。 </li><li>被毛黑色，清澈见底的井，50μL聚-L-赖氨酸的96孔板。允许井干在孵化器30分钟。 </li><li>板30000每孔细胞密度和细胞储存在3-4天的孵化器含有200μL的媒体在96板细...

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虽然膜电位敏感荧光染料已被用来确定调节离子通道的药物，9,10，这是他们的神经GIRK通道药物发现中的应用的第一次报告。这里GIRK通道荧光检测提供了快速，可靠，实时的方法，筛选配体门控K +通道。与细胞的永生细胞株（HEK293细胞，CHO等）表达一个外生重组GIRK通道11或克隆细胞株（AtT20，PC-12）表达的内源性通道的广泛使用，可以修改该法。此外，该法可适应在胚胎干...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM培养液</td><td> CellGro </td><td> 10-013 </td></tr><tr><td>马血清</td><td> Invitrogen公司</td><td> 16050-114 </td></tr><tr><td> 96孔板</td><td>康宁</td><td> 3603 </td></tr><tr><td>聚-L-赖氨酸</td><td> Sigma-Aldrich公司</td><td> P4707 </td></tr><tr><td>生长抑素</td><td> Sigma-Aldrich公司</td><td> S9129 </td></tr><tr><td>卡巴</td><td> Sigma-Aldrich公司</td><td> C4382 </td></tr><tr><td> HLB值021-152 </td><td> AnaSpec </td><td> 89300 </td></tr><tr><td> versette自动化液体处理程序</td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> synergy2荧光酶标仪</td><td>百特</td><td></td></tr><tr><td> GEN5分析oftware </td><td>百特</td><td></td></tr></tbody></table> 表1。表特定的试剂和设备。

a fluorescent screening assay for identifying modulators of girk channels

G蛋白门控内向整流钾 （GIRK）通道功能广泛的激素和神经递质的细胞介质，并在大脑，心脏，骨骼肌和内分泌组织1,2表示。 GIRK通道成为激活后的配体结合（神经递质，激素，药物等），其细胞膜结合，G蛋白偶联受体（GPCRs）。这种结合使G蛋白（G I GØ），随后结合并激活GIRK通道的刺激。一旦打开了GIRK通道允许的K运动导致细胞静息膜电位变得更负+。因此，GIRK通道激活的神经元减少自发性动作电位的形成和抑制兴奋性神经递质的释放。在心脏，激活GIRK通道的抑制起搏器的活性，从而减缓心率。 <p为开发新的治疗药物，治疗神经痛，吸毒，心律失常和其他疾病3类=“jove_content”的GIRK通道代表的新的目标。然而，这些通道的药理学仍然人迹罕至。虽然一些药物，包括抗心律失常药物，抗精神病药和抗抑郁药阻止GIRK通道，这种抑制是没有选择性的，发生在相对 ​​较高的药物浓度3。 在这里，我们描述了一个实时的筛选试验，确定新的调制GIRK通道。在这个实验中，神经AtT20细胞，表达GIRK通道，装有膜电位敏感荧光染料，如双（1,3 - dibutylbarbituric酸）的三甲oxonol [DiBAC 4（3）]或021-152 HLB值（ 图1 ）。染料分子成为强烈荧光以下的摄取进入细胞内（ 图1）。治疗GPCR的配体细胞刺激GIRK通道打开。由此产生的K +外流的细胞，导致膜电位变得更负，荧光信号降低（ 图1）。因此，GIRK通道通过调节钾离子流出的药物可以用荧光酶标仪测定。不同于其他离子通道筛选试验，如原子吸收光谱法4或放射性分析5，GIRK通道荧光检测提供了一种快速，实时和廉价的甄别程序。

Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

一个确定药物相互作用与G蛋白门控内向整流钾实时甄别<sup&gt; -</sup&gt;（GIRK）通道描述。该法采用膜电位敏感荧光染料来衡量GIRK通道活性。这项技术是适应的细胞株上使用。

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Research

JoVE Journal

Biology

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

科研

生物学

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

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

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, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...