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制，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的cDNA转染至COS-1细胞中，并在48-72小时后进行电压钳实验。记录稳态外向电流并计算开路概率。激活曲线用单一玻尔兹曼方程拟合。半激活电压为33.4mV(WT)、51.2mV(WT-(D172N)2-WT)和76.6mV(D172N)。根据 +42 mV 的电流记录构建开路和零电流时间直方图。开路时间直方图采用单指数函数拟合。对照中的平均开放时间为 53.1 ms (WT)、46.2 ms (WT-(D172N)2-WT) 和 39.4 ms (D172N)。内部精胺以浓度依赖性方式缩短开放时间：WT 通道中为 37.8 ms (1 nM) 和 11.7 ms (10 nM)； WT-(D172N)2-WT 通道中为 34.7 ms (1 nM)、10.9 ms (10 nM) 和 1.6 ms (100 nM)； D172N 通道中为 31.6 ms (1 nM)、11.7 ms (10 nM) 和 1.7 ms (100 nM)。除了具有 10 和 100 nM 精胺的 D172N 通道外，需要两个以上的指数函数来拟合零电流时间直方图。这些结果表明，内在门控确实与精胺块分开存在。假设状态模型（阻塞状态---打开状态---关闭状态1---关闭状态2），估计每个通道中的阻塞率。它们是 6.2-7.7 s^<-1> (1 nM)、64.7-70.1 s^<-1> (10 nM) 和 563-603 s^<-1> (100 nM)，并且不依赖于数量D172N 突变体。