MOLECULAR CLONING OF EPITHELIAL K CHANNELS

上皮 K 通道的分子克隆

• 批准号: 6176258
• 负责人: HENRY SACKIN
• 金额: $ 26.03万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6176258
• 关键词: Xenopus; Xenopus oocyte; acidity /alkalinity; chimeric proteins; electrochemistry; membrane permeability; membrane potentials; membrane structure; molecular cloning; molecular site; potassium channel; protein sequence; protein structure function; renal tubular transport; site directed mutagenesis; tissue /cell culture; voltage /patch clamp; voltage gated channel

The inward rectifier family (Kir) of channels play an important role in K transport in a variety of tissues including the heart, macrophage and kidney. The IRK (Kir2) subfamily of K channels are characterized by strong inward rectification, with little current in the outward direction. In contrast, the ROMK (Kir1) subfamily of K channels exhibit a much weaker inward rectification, with significant outward current. ROMK is predominantly expressed in the kidney where it mediates K secretion into the lumen of the cortical collecting tubule (CCT) and recycles K for the triple cotransporter at the luminal membrane of the thick ascending limb of Henle (TALH). Members of both the Kir1 and Kir2 subfamilies have been cloned and sequenced. Evidence suggests that both these channels consist of 4 subunits, each with 2 transmembrane spanning segments. Recent crystallographic information (Doyle et. al. Science 280: 69-77, 1998) about a bacterial K channel (KcsA) has revealed it to be a tetrameric channel with each of the 4 subunits possessing 2 membrane spanning segments, similar to the topology of inward rectifiers. This discovery provides a unique opportunity to employ electrophysiological methods to understand how specific structural elements of inward rectifiers control K permeation and gating through IRK and ROMK, using the KcsA channel as a model. In the proposed experiments, I will utilize the techniques of patch-clamp recording, 2- electrode voltage clamp, site directed mutagenesis, and chimeric constructs to focus on 4 aspects of permeation through inward rectifier channels: (1) ion binding within the channel, (2) control by pore-lining residues, (3) regulation by external K, and (4) control of permeation by the C-terminus. Results of these experiments, together with the known crystal structure of KcsA, should provide basic information about: (1) how K traverses inward rectifier channels, (2) which regions of the channel control permeation, and (3) how these regions interact with each other. This would be relevant not only to K handling by the kidney, but also to K transport by the entire (Kir) family of ion channels.

通道的内向整流剂家族（KIR）在包括心脏，巨噬细胞和肾脏在内的各种组织中的K运输中起着重要作用。 K通道的IRK（KIR2）亚家族的特征是强烈的内向整流，向外方向几乎没有电流。 相比之下，k通道的romk（KIR1）亚家族表现出弱的内部整流，并且外在电流明显。罗克主要在肾脏中表达，在肾脏中，它将K分泌到皮质收集小管（CCT）的腔内，并回收K为亨尔（TALH）厚的升高肢体的升上升肢的腔膜上的三重共转运蛋白。 KIR1和KIR2亚家族的成员都被克隆并测序。 证据表明，这两个通道由4个亚基组成，每个亚基都有2个跨膜跨越段。 关于细菌K通道（KCSA）的最新晶体学信息（Doyle等人科学280：69-77，1998）已揭示了它是一个四聚体通道，其中四个具有跨越2个膜的亚基，类似于内向整流器的拓扑。 这一发现提供了一个独特的机会，可以使用电生理方法来了解内向整流器的特定结构元素如何使用KCSA通道作为模型来控制K渗透和通过IRK和ROMK进行门控。在拟议的实验中，我将利用斑块钳记录的技术，2-电极电压夹，位点，定向诱变和嵌合构建体，集中于通过内在整流器通道的渗透到4个方面：（1）通过孔中的孔子控制（2）通过（2）通过（2）通过（2）通过（3）控制（3）k-enter（3）k的（3）k- k的k和4。 这些实验的结果以及KCSA的已知晶体结构应提供有关：（1）K如何遍历内向整流器通道，（2）哪些区域的频道控制渗透的区域以及（3）这些区域如何相互作用。 这不仅与肾脏的处理有关，而且与整个（KIR）离子频道家族的K运输有关。