MOLECULAR CLONING OF EPITHELIAL K CHANNELS

上皮 K 通道的分子克隆

• 批准号: 6380822
• 负责人: HENRY SACKIN
• 金额: $ 26.81万
• 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/6380822
• 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.

内向整流通道家族（Kir）在包括心脏、巨噬细胞和肾脏在内的多种组织中的钾转运中起重要作用。 K通道的IRK（Kir 2）亚家族的特征是强烈的内向整流，向外方向的电流很小。 相比之下，K通道的ROMK（Kir 1）亚家族表现出弱得多的内向整流，具有显著的外向电流。ROMK主要在肾脏中表达，在肾脏中其介导K分泌到皮质集合小管（CCT）的管腔中，并在Henle氏粗升支（TALH）的管腔膜处将K转化为三重共转运蛋白。 Kir 1和Kir 2亚家族的成员已被克隆和测序。 有证据表明，这两个通道由4个亚基组成，每个亚基有2个跨膜段。 最近的晶体学信息（Doyle et.第280章：你是我的女人69-77，1998）揭示了它是四聚体通道，4个亚基中的每一个具有2个跨膜区段，类似于内向整流器的拓扑结构。 这一发现提供了一个独特的机会，采用电生理学的方法来了解特定的结构元素的内向整流器控制K渗透和门控通过IRK和ROMK，使用KcsA通道作为模型。在拟进行的实验中，我将利用膜片钳记录技术、双电极电压钳技术、定点突变技术和嵌合构建体技术，重点研究内向整流通道渗透的4个方面：（1）通道内的离子结合，（2）孔衬残基的控制，（3）外部K的调节，（4）C末端的渗透控制。 这些实验的结果，连同已知的KcsA晶体结构，应该提供基本信息：（1）K如何穿越内向整流通道，（2）通道的哪些区域控制渗透，以及（3）这些区域如何相互作用。 这不仅与肾脏的钾处理有关，而且与整个（Kir）离子通道家族的钾转运有关。