Transfer of Kv3.1 Voltage Sensor Features to the Isolated Ci-VSP Voltage-Sensing Domain
Transfer of Kv3.1 Voltage Sensor Features to the Isolated Ci-VSP Voltage-Sensing Domain
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DOI：
10.1016/j.bpj.2012.07.031
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发表时间：
2012-08-22
影响因子：
3.4
通讯作者：
Knoepfel, Thomas
中科院分区：
文献类型：
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作者：
Mishina, Yukiko;Mutoh, Hiroki;Knoepfel, Thomas
对跨膜电压变化作出反应的膜蛋白在调节活细胞的功能方面是至关重要的。电压门控离子通道的电压敏感结构域(VSD)被广泛研究以阐明其电压敏感机制，但其结构-功能关系的许多方面仍不清楚。在这里，我们将来自四聚体电压激活钾通道KV3.1的同源氨基酸基序移植到肠黄瓜电压敏感磷酸酶(Ci-VSP)的单体VSD中，以探索KV3.1亚基的哪些部分依赖于Kv通道的四聚体结构，以及KV3.1的哪些特性可以转移到单体Ci-VSP支架上。通过将荧光蛋白附着到这些嵌合的VSD上，我们获得了一个光学读数来建立膜的运输和电压依赖的结构重排的动力学。我们发现，从10个氨基酸到大约100个氨基酸的基序可以很容易地从KV3.1移植到Ci-VSP中，形成有效整合到质膜和感应电压中的工程化VSD。这些工程设计的VSD的一些功能特征让人想起KV3.1通道，表明这些特性不需要Kv亚基之间或Kv通道的电压传感和孔域之间的相互作用。
Membrane proteins that respond to changes in transmembrane voltage are critical in regulating the function of living cells. The voltage-sensing domains (VSDs) of voltage-gated ion channels are extensively studied to elucidate voltage-sensing mechanisms, and yet many aspects of their structure-function relationship remain elusive. Here, we transplanted homologous amino acid motifs from the tetrameric voltage-activated potassium channel Kv3.1 to the monomeric VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) to explore which portions of Kv3.1 subunits depend on the tetrameric structure of Kv channels and which properties of Kv3.1 can be transferred to the monomeric Ci-VSP scaffold. By attaching fluorescent proteins to these chimeric VSDs, we obtained an optical readout to establish membrane trafficking and kinetics of voltage-dependent structural rearrangements. We found that motifs extending from 10 to roughly 100 amino acids can be readily transplanted from Kv3.1 into Ci-VSP to form engineered VSDs that efficiently incorporate into the plasma membrane and sense voltage. Some of the functional features of these engineered VSDs are reminiscent of Kv3.1 channels, indicating that these properties do not require interactions between Kv subunits or between the voltage sensing and the pore domains of Kv channels.