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Studies of inwardly rectifying K^+ channels and an approach to develop new methods for assaying K^+ channel activity.

内向整流 K^ 通道的研究以及开发用于测定 K^ 通道活性的新方法的方法。

基本信息

批准号：
08557008
负责人：
HORIO Yoshiyuki
金额：
$ 8.19万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

Inwardly rectifying K^+ channels (Kir channels) consist of more than 15 members and they are highy selective for K^+. They have, been estimated to possess two transmembrane domains and one pore-forming (H5) region. Amino acid sequence of H5 region of Kir channels has high similarity to each other and contains K^+ channel signature sequence of Gly-Tyr(Phe)-Gly. Each Kir channel consists of four subunits. Functions of Kir channels have been considered stabilizing and decreasing membrane potential, inhibiting excitability of cell, transport of K^+, and secretion of insulin. To evaluate Kir channel activity, patch-clamp method and measurement of influx of radioactive Rb have been used. But these methods have some defects. We planed to develop a new method to assay Kir channels, and at the same time, we planed to further study expression, distribution and function of Kir channels. Furthermore, we started to determine the chromosomal localization of some Kir channels to know the interaction of Kir channels and diseases. Our achievements are as follows. (1) Kir4.l/K_<AB>-2is expressed in glial cells and gastric parietal cells, the function of which is transport of K4. (2) new sulfonylurea receptor subunit (SUR2B) was cloned and identified as smooth vascular K_<ATP> channel subunit. (3) Kir3.2b, Kir1.lf, and cTBAK-1 were cloned. The chromosomal localization of Kir4.1/K_<Ab>-2, Kir5.1, Kir6.1, SUR2 and cTBAK-1 were identified. (4) We tried to develop measuring K^+ channel activity using PBFI.

内纠偏K^+通道（Kir通道）由超过15个成员组成，它们对K^+具有高选择性。据估计，它们具有两个跨膜结构域和一个成孔区（H5）。Kir通道的H5区氨基酸序列具有较高的相似性，包含Gly-Tyr(Phe)-Gly的K^+通道特征序列。每个Kir通道由四个子单元组成。Kir通道的功能被认为是稳定和降低膜电位，抑制细胞的兴奋性，K^+的运输和胰岛素的分泌。为了评价Kir通道的活性，使用了膜片钳法和放射性Rb内流的测量。但这些方法都存在一定的缺陷。我们计划开发一种新的方法来检测Kir通道，同时我们计划进一步研究Kir通道的表达、分布和功能。此外，我们开始确定一些Kir通道的染色体定位，以了解Kir通道与疾病的相互作用。我们的成就如下。(1) Kir4。l/K_<AB>-2在胶质细胞和胃壁细胞中表达，其功能是转运K4。(2)克隆了新的磺酰脲受体亚基（SUR2B），并鉴定为光滑血管K_<ATP>通道亚基。(3) Kir3.2b, Kir1。克隆了cTBAK-1和cTBAK-1。鉴定出Kir4.1/K_<Ab>-2、Kir5.1、Kir6.1、SUR2和cTBAK-1的染色体定位。(4)我们尝试开发使用PBFI测量K^+通道活性的方法。