Voltage- and K ion-dependent gating of inwardly rectifying K channels

内向整流 K 通道的电压和 K 离子依赖性门控

基本信息

批准号：
15390067
负责人：
MATSUDA Hiroko
金额：
$ 7.49万
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：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15390067/
关键词：
Inwardly rectifying K^+ channel K ion

项目摘要

To know the significance of the negative charges in the extracellular loop of the inwardly rectifying K^+ (Kir2.1) channels, single point mutants (D112N, D114N, D152N and E153Q) and double points mutants (D112N/D114N and D152N/E153Q) were constructed and transfected into COS-1 and HEK-293 cells. Single-channel currents similar to those through wild-type Kir2.1 channels were recorded from cells transfected with each single point mutant and D112N/D114N. Cells transfected with D152N/E153Q did not show inwardly rectifying K^+ currents, although fluorescence images confirmed that channel proteins produced by D152N/E153Q were transported to the cell surface. Tandem tetramers with one E153Q subunit and three double mutant subunits, E153Q-(D152N/E153Q)3, expressed Kir2.1 channels. Tandem tetramers with one D152N subunit and three double mutant subunits, D152N-(D152N/E153Q)3, did not express Kir2.1 channels but those with two D152N subunits and two double mutant subunits, (D152N)2-(D152N/E153Q)2, expressed Kir2.1 channels. In addition, outward currents through wild-type and D172N/E224S channels were recorded from outside-out macropatches in the absence of external K^+. These results suggest that one negative charge of D152 or two negative charges of E153 and K^+ either in the external solution or coming from the inside of cells are required for Kir2.1 channels to function. These negative charges may be involved in stabilizing a half hydrated K^+ ion near the external entrance to the selectivity filter revealed by the structural study of the KcsA K^+ channel.

To know the significance of the negative charges in the extracellular loop of the inwardly rectifying K^+ (Kir2.1) channels, single point mutants (D112N, D114N, D152N and E153Q) and double points mutants (D112N/D114N and D152N/E153Q ）构造并转染到COS-1和HEK-293细胞中。单通道电流与通过野生型KIR2.1通道相似，从每个点突变体和D112N/D114N转染的细胞中记录。用D152N/E153Q转染的细胞未显示内部整流的K^+电流，尽管荧光图像证实了由D152N/E153Q产生的通道蛋白转运到细胞表面。带有一个E153Q亚基和三个双突变亚基的串联四聚体E153Q-（D152N/E153Q）3表示KIR2.1通道。带有一个D152N亚基和三个双突变亚基D152N-（D152N/E153Q）3的串联四聚体，没有表达KIR2.1通道，但具有两个D152N亚基和两个双突变体亚基，（D152N）2-（D152N/E153Q） 2，表示Kir2.1通道。此外，在没有外部K^+的情况下，通过野生型和D172N/E224S通道进行外向电流记录。这些结果表明，在外部溶液中或来自细胞内部的d152或两个负电荷的一个负电荷是Kir2.1的功能。这些负电荷可能参与稳定一半水合的K^+离子在选择性滤波器外部入口附近，这是通过KCSA K^+通道的结构研究所揭示的。