Molecular basis of the substate behavior in the inwardly rectifying K channels

内向整流 K 通道亚状态行为的分子基础

基本信息

批准号：
09470013
负责人：
MATSUDA Hiroko
金额：
$ 2.75万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470013/
关键词：
K channel Inward rectification IRK1 Mg ion

项目摘要

To investigate tlie molecular basis of the sublevels induced in the outward current during block by internal Mg^<2+>, single-channel currents through inwardly rectifying K^+ (IRK1) channels were studied. cDNA encoding a functional murine IRK1 channel was transfected into COS-1 cells using the liposome method, and voltage clamp experiments were done after 48-72 h. Intracellular Mg^<2+>2 at micromolar concentrations induced sublevels in the outward current at one-third and two-thirds of the unitary amplitude in wild-type channels. Replacing Asp 172 with Asn (D172N) and Gln (0172Q) abolished these sublevels, i.e. the channel showed only the fully open and blocked states. Both mutations reduced the Mg^<2+> sensitivity of the channel at 2 muM Mg^<2+>. However, the Mg^<2+> sensitivity did not differsignificantly at higher concentrations (10 muM) and voltages (+70 mV), suggesting that a binding site other than D172 is critical in Mg^<2+> blockade. Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behavior. Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two-thirdsamplitude, while those from tetramers with three D172N mutant subunits showed no sublevels. These findings suggest that differences in Mg^<2+> binding patterns lead to different conductive states in a single-barrelled channel. A set of four aspartate carboxyates can interact simultaneously with two Mg^<2+> ions (this is possible only in wild-type channels), inducing the one-third level in the case where another binding site is free of Mg^<2+>2. If only one Mg^<2+> ion binds to the D172 site (this is possible in channels with D172 more than two) and another binding site is free, the two-thirds level appears.

为了研究内部Mg^2+阻断过程中外向电流感应的亚能级的分子基础，研究了通过内向整流K^+(IRK1)通道的单通道电流。采用脂质体法将编码功能性小鼠IRK1通道的cDNA转染至COS-1细胞中，48-72小时后进行电压钳实验。微摩尔浓度的细胞内Mg 2+ 2 诱导外向电流的亚水平为野生型通道中单一振幅的三分之一和三分之二。用 Asn (D172N) 和 Gln (0172Q) 替换 Asp 172 废除了这些子级别，即通道仅显示完全打开和阻塞状态。两种突变均降低了通道在2μM Mg 2+ 下的Mg 2+ 敏感性。然而，Mg 2+ 敏感性在较高浓度(10μM)和电压(+70mV)下没有显着差异，表明D172以外的结合位点在Mg 2+ 阻断中是关键的。 D172E 表达的通道显示了亚能级，表明负电荷对于亚态行为是不可或缺的。具有1个和2个D172N突变亚基的IRK1串联四聚体的通道主要显示具有三分之二振幅的亚水平，而具有3个D172N突变亚基的四聚体的通道则不显示亚水平。这些发现表明Mg^2+结合模式的差异导致单管通道中不同的导电状态。一组四个天冬氨酸羧酸盐可以同时与两个 Mg^<2+> 离子相互作用（这仅在野生型通道中可能），在另一个结合位点不含 Mg^<2 的情况下诱导三分之一水平+>2。如果只有一个Mg 2+ 离子与D172位点结合(这在具有多于两个D172的通道中是可能的)并且另一个结合位点是自由的，则出现三分之二水平。