Molecular properties of Kcv, a virus encoded K+ channel
Molecular properties of Kcv, a virus encoded K+ channel
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DOI：
10.1021/bi061530w
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发表时间：
2007-01-30
期刊：
影响因子：
2.9
通讯作者：
Parcej, David
中科院分区：
文献类型：
--
作者：
Pagliuca, Cinzia;Goetze, Tom Alexander;Parcej, David
微型病毒K+通道Kcv代表了所有K+通道的孔模块。与野生型基因相比，GC含量较高的Kcv合成基因在毕赤酵母中异源表达，纯化后的蛋白被功能重构为脂质体。通过SDS-PAGE进行的生化分析显示该通道四聚体具有显著的阳离子选择性稳定性。只有渗透到Kcv中的阳离子能够保护低聚物在高温下不被分解成单体。单个Kcv通道的电生理特性表明，饱和电导（Lambda(max)）为360 pS；在极端电压下，单通道电流-电压关系强整流，电导斜率为负。该通道对K+具有高度选择性，可被Ba2+阻断，也可被Na+和Cs+以侧特异性方式阻断。该通道传导Rb+，但结果，该通道被转移到一个过度活跃的状态。我们得出结论，导电途径中阳离子的特异性结合相互作用是通道稳定性和功能的重要决定因素。
The miniature viral K+ channel Kcv represents the pore module of all K+ channels. A synthetic gene of Kcv with an elevated GC content compared to that of the wild-type gene was expressed heterologously in Pichia pastoris, and the purified protein was functionally reconstituted into liposomes. Biochemical assays reveal a remarkable cation selctive stability of the channel tetramer via SDS-PAGE. Only cations, which permeate Kcv, were able to protect the oligomer against disassembly into monomers at high temperatures. Electrophysiological characterization of the single Kcv channel reveals a saturating conductance (Lambda(max)) of 360 pS; the single-channel current-voltage relation was strongly rectifying with a negative slope conductance at extreme voltages. The channel was highly selective for K+ and was blocked by Ba2+ and in a side specific manner by Na+ and Cs+ also. The channel conducted Rb+, but as a consequence, the channel was shifted into a hyperactive state. We conclude that specific binding interactions of cations in the conductive pathway are an important determinant of channel stability and function.