MOLECULAR CLONING OF EPITHELIAL K CHANNELS

内向整流家族（Kir）通道在包括心脏、巨噬细胞和肾脏在内的多种组织中的K转运中发挥重要作用。K通道的IRK （Kir2）亚族具有向内整流强，向外电流小的特点。相比之下，K通道的ROMK （Kir1）亚族表现出更弱的内向整流，具有显著的外向电流。ROMK主要在肾脏中表达，它介导钾分泌到皮质集合小管（CCT）的管腔，并在Henle厚升肢管腔膜（TALH）为三重共转运体循环K。Kir1和Kir2亚家族的成员已被克隆并测序。有证据表明，这两个通道都由4个亚基组成，每个亚基有2个跨膜跨越段。最近关于细菌K通道（KcsA）的晶体学信息（Doyle et al. Science 280: 69-77, 1998）显示它是一个四聚体通道，4个亚基中的每一个都有2个膜跨越段，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，可以使用电生理学方法来了解内向整流器的特定结构元件如何通过IRK和ROMK控制K渗透和门控，并使用KcsA通道作为模型。在拟议的实验中，我将利用膜片钳记录、2电极电压钳、位点定向诱变和嵌合结构等技术，重点研究通过内向整流通道渗透的四个方面：(1)通道内的离子结合，(2)孔衬里残基的控制，(3)外部K的调节，以及(4)c端对渗透的控制。这些实验的结果，加上已知的KcsA晶体结构，应该提供以下基本信息：(1)K如何穿过向内整流通道，(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。这不仅与肾脏处理钾有关，而且与整个（Kir）离子通道家族的钾运输有关。

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.