Voltage-dependent gating and block by internal spermine of the murine inwardly rectifying K+ channel, Kir2.1
Voltage-dependent gating and block by internal spermine of the murine inwardly rectifying K+ channel, Kir2.1
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DOI：
10.1113/jphysiol.2003.038844
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发表时间：
2003-04-15
影响因子：
5.5
通讯作者：
Omori, K
中科院分区：
文献类型：
--
作者：
Matsuda, H;Oishi, K;Omori, K
通过记录通过内向整流K+通道（Kir2.1）的单通道电流来研究内向整流的机制。用脂质体法将编码野生型（WT）通道、用Asn（D172 N）替换Asp 172的突变体和串联四聚体WT-（D172 N）2-WT的cDNA转染到COS-1细胞中，48-72 h后，在150 mm的内部和外部K+下以由内而外的构型记录单通道电流。外向电流的稳态开放概率随去极化增大而减小。活化曲线用单一Boltzmann方程拟合。在不存在精胺的情况下，半激活电压为+35.9mV（WT）、+55.0mV（WT-（D172 N）2-WT）和+76.7mV（D172 N）。构建了开路时间和零电流时间直方图。开放时间直方图用单指数函数拟合。需要两个指数函数来拟合关闭时间直方图。在每个通道中，1-100 nm的精胺以浓度依赖的方式减少外向电流的开放时间，并产生一个阻断状态，而不影响内向电流，表明精胺作为开放通道阻断剂。标准化稳态开放概率-精胺浓度曲线由饱和动力学拟合，希尔系数为1。在线性序贯状态模型的假设下，估计了各信道的解阻塞率和阻塞率。解阻断率依赖于D172 N突变亚基的数量，但阻断率没有。结果表明，剂量门独立于精胺阻滞工作，D172参与内在门控和精胺阻滞。
The mechanism of inward rectification was investigated by recording single-channel currents through an inwardly rectifying K+ channel (Kir2.1). cDNA encoding a wild-type (WT) channel, a mutant replacing Asp 172 with Asn (D172N), and a tandem tetramer WT-(D172N)2-WT, was transfected into COS-1 cells using the liposome method, and after 48-72 h single-channel currents were recorded in the inside-out configuration at 150 mm internal and external K+. Steady-state open probability of outward currents decreased with larger depolarizations. The activation curve was fitted with a single Boltzmann equation. The voltages of half-activation in the absence of spermine were +35.9 mV (WT), +55.0 mV (WT-(D172N)2-WT) and +76.7 mV (D172N). Open-time and zero-current-time histograms were constructed. The open-time histogram was fitted with a single exponential function. Two exponential functions were necessary to fit the closed-time histogram. In each channel, internal spermine at a concentration of 1-100 nm reduced the open time of the outward currents in a concentration-dependent manner and produced one blocked state without affecting the inward currents, suggesting that spermine acts as an open channel blocker. The normalized steady-state open probability-spermine concentration curve was fitted by saturation kinetics with a Hill coefficient of 1. On the assumption of the linear sequential state model, the unblock and blocking rates were estimated in each channel. Unblock rates depended on the number of D172N mutant subunits, but blocking rates did not. The results suggest that dosing gates work independently of the spermine block and D 172 is involved in both intrinsic gating and the spermine block.