Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents
Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents
复制标题
使用甲硫代磺酸盐试剂通过取代半胱氨酸可达性方法探测 ClC-0 的孔
DOI：
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Tsung
中科院分区：
文献类型：
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作者：
Chia;Tsung
ClC通道是一类含有两个离子渗透孔的蛋白质分子。尽管这些跨膜蛋白在多种生理功能中发挥着重要作用，但人们对它们的分子运作机制却知之甚少。高分辨率x射线晶体学技术最近揭示了两种细菌ClC通道的结构，但脊椎动物ClC通道孔隙是否与细菌同源物相似尚不清楚。为了研究鱼雷ClC-0通道的孔隙结构，我们采用了取代半胱氨酸接近法（SCAM），并使用带电荷的甲烷硫代磺酸（MTS）化合物对引入的半胱氨酸进行修饰。从这种方法中得出了几个结论。首先，ClC-0中由Y512C到E526C的MTS修饰模式，与细菌ClC通道中形成螺旋R的残基相对应，确实与提出的螺旋结构一致。其次，带负电荷的MTS化合物比带正电荷的MTS化合物更容易进入ClC-0孔，这是由孔内固有静电势调节的孔性质。最后，试图将引入的半胱氨酸修饰到选择性滤波器的细胞内位置，并没有导致开放状态通道的MTS修饰率增加，这表明ClC-0的快速门不能位于细胞内的Cl -选择性滤波器位置。因此，谷氨酸侧链是ClC-0通道的快速通道的说法适用于ClC-0，揭示了细菌和脊椎动物之间ClC通道在结构和功能上的保守性。
ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged MTS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl− selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species.
DOI：
10.1073/pnas.81.9.2772
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
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作者：
MILLER, C;WHITE, MM
通讯作者：
WHITE, MM