Voltage-dependent gating and block by internal spermine in the inwardly rectifying K channels

内向整流 K 通道中的电压依赖性门控和内部精胺阻断

基本信息

批准号：
11470013
负责人：
MATSUDA Hiroko
金额：
$ 7.23万
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：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470013/
关键词：
K channel Inward rectification spermine

项目摘要

To investigate the mechanism of inward rectification, single-channel currents through inwardly rectifying K^+ (IRK1 ; Kir 2.1) channels were studied. cDNA encoding a wild-type (WT) IRK1 channel, D172N and tandem tetramer WT-(D172N)2-WT was transfected into COS-1 cells using the liposome method, and voltage clamp experiments were done after 48-72 h. Steady-state outward currents were recorded and open probability was calculated. The activation curve was fitted with single Boltzmann equation. The voltage of half-activation was 33.4 mV (WT), 51.2 mV (WT-(D172N)2-WT) and 76.6 mV (D172N). Open-and zero-current tirne histograms were constructed on current records at +42 mV The open-time histogram was fitted with a single exponential function. The mean open time was 53.1 ms (WT), 46.2 ms (WT-(D172N)2-WT) and 39.4 ms (D172N) in the control. Internal spermine reduced the open time in a concentration dependent manner: 37.8 ms (1 nM) and 11.7 ms (10 nM) in WT channels ; 34.7 ms (1 nM), 10.9 ms (10 nM) and 1.6 ms (100 nM) in WT-(D172N)2-WT channels ; 31.6 ms (1 nM), 11.7 ms (10 nM) and 1.7 ms (100 nM) in D172N channels. More than two exponential functions were necessary to fit the zero-cunent time histogram except in D172N channels with 10 and 100 nM spermine. These results suggest that intrinsic gating does exist separately from the spermine block. On the assumption of the state model (blocked state --- Open state --- closed state 1 --- closed state 2), blocking rates were estimatcd in each channel. They were 6.2-7.7 s^<-1> (1 nM), 64.7-70.1 s^<-1> (10 nM) and 563-603 s^<-1> (100 nM) and did not depend on the number of D172N mutant.

为了研究内向整流的机理，研究了通过内向整流K^+（IRK1; KIR 2.1）通道的单通道电流。使用脂质体方法将编码野生型（WT）IRK1通道，D172N和串联四聚体WT-（D172N）2-WT转染到COS-1细胞中的cDNA，在48-72 h后进行电压夹实验。记录了稳态外电流并计算开放概率。激活曲线拟合了单个玻尔兹曼方程。半激活的电压为33.4 mV（WT），51.2 mV（WT-（D172N）2-WT）和76.6 mV（D172N）。在+42 MV的当前记录上构建了开放和零电流的Tirne直方图，开放时间直方图拟合了单个指数函数。在控件中，平均开放时间为53.1 ms（WT），46.2 ms（WT-（D172N）2-WT）和39.4 ms（D172N）。内部精子以浓度依赖性方式减少开放时间：WT通道中的37.8 ms（1 nm）和11.7 ms（10 nm）； WT-（D172N）2-WT通道中的34.7 ms（1 nm），10.9 ms（10 nm）和1.6 ms（100 nm）； D172N通道中的31.6 ms（1 nm），11.7 ms（10 nm）和1.7 ms（100 nm）。除了10和100 nm精子的D172N通道中，需要两个以上的指数函数以适合零单位直方图。这些结果表明，固有的门控确实与精子块分开存在。根据状态模型的假设（阻止状态 - 开放状态---封闭状态1 ---封闭状态2），在每个通道中估计了阻塞率。它们为6.2-7.7 s^<-1>（1 nm），64.7-70.1 s^<-1>（10 nm）和563-603 s^<-1>（100 nm），不取决于D172N突变体的数量。