Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape.
Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape.
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DOI：
10.1085/jgp.201812075
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发表时间：
2018-07-02
期刊：
影响因子：
--
通讯作者：
Ahern CA
中科院分区：
文献类型：
--
作者：
Infield DT;Lee EEL;Galpin JD;Galles GD;Bezanilla F;Ahern CA
激活电压门控通道是由于精氨酸残基在S4片段上向外移动。Infield等人使用体内无义抑制用瓜氨酸取代Shaker的S4精氨酸残基，并发现R4上需要带正电荷才能实现电压依赖性失活。电压门控阳离子通道的电压依赖性激活是由蛋白质电压传感器内含精氨酸螺旋向外运动引起的。钾通道中的电压感应残基已被广泛表征，但目前的功能方法不允许区分精氨酸侧链的静电和空间贡献。在这里，我们使用化学误酰化和体内无义抑制来编码瓜氨酸，一种中性的和精氨酸的近等构类似物，到Shaker钾通道的电压传感器中。我们对工程通道进行了功能表征，并将它们与在相同位置上携带常规突变的通道进行了比较。我们观察到对电压敏感性和门控动力学的影响，从而能够解剖残基结构与正电荷在通道功能中的作用。在某些位置，与自然突变相比，瓜氨酸替代对通道激活的影响较轻。相反，用瓜氨酸取代第四个S4精氨酸会导致电导-电压关系和通道动力学发生实质性变化，这表明该位置需要正电荷才能有效地使电压传感器失活和通道关闭。瓜氨酸的编码有望提高其他膜蛋白中位于拥挤跨膜环境中的精氨酸残基的研究精度。此外，该方法还可促进瓜氨酸化的体内研究。
Activation of voltage-gated channels results from the outward movement of arginine residues on the S4 segments. Infield et al. use in vivo nonsense suppression to replace Shaker's S4 arginine residues with citrulline and reveal that a positive charge is required on R4 for voltage-dependent deactivation. Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance–voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.
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影响因子：
10.8
作者：
Fert-Bober, Justyna;Giles, John T.;Van Eyk, Jennifer E.
通讯作者：
Van Eyk, Jennifer E.
影响因子：
16.2
作者：
Aggarwal, SK;MacKinnon, R
通讯作者：
MacKinnon, R
DOI：
10.1073/pnas.1103397108
发表时间：
2011-04-19
影响因子：
11.1
作者：
Lacroix, Jerome J.;Bezanilla, Francisco
通讯作者：
Bezanilla, Francisco
影响因子：
2.9
作者：
Choudhury, Ambar K.;Golovine, Serguei Y.;Hecht, Sidney M.
通讯作者：
Hecht, Sidney M.
离子通道温度依赖性门控的分子框架。
影响因子：
64.5
作者：
Chowdhury S;Jarecki BW;Chanda B
通讯作者：
Chanda B