Extracellular blockade of K(+) channels by TEA: results from molecular dynamics simulations of the KcsA channel.
Extracellular blockade of K(+) channels by TEA: results from molecular dynamics simulations of the KcsA channel.
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DOI：
10.1085/jgp.118.2.207
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发表时间：
2001-08
期刊：
影响因子：
--
通讯作者：
Roux B
中科院分区：
文献类型：
--
作者：
Crouzy S;Bernèche S;Roux B
TEA是一种经典的K⁺通道阻断剂。诱变研究表明，TEA的外部阻断作用强烈依赖于位于Shaker蛋白449位的芳香族残基的存在，该位置靠近孔道的细胞外入口（Heginbotham, L., 和R. MacKinnon. 1992.《神经元》8:483 - 491）。数据表明TEA同时与四个单体的芳香族残基相互作用。利用X射线晶体学测定KcsA通道的三维结构（Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, 和R. MacKinnon. 1998.《科学》280:69 - 77）引发了一些目前关于这些观察结果的解释尚未解决的问题。特别是，KcsA中Tyr82侧链的中心（对应于Shaker中的449位）形成一个边长为11.8 Å的正方形，这个距离太大，不允许一个TEA分子同时与四个芳香族侧链相互作用。在本文中，通过在含盐水溶液的明确磷脂双层中对KcsA的原子模型进行分子动力学模拟，研究了TEA的外部阻断作用。观察到，与实验结果定性相符的是，TEA与野生型KcsA通道（含Tyr82）的外侧结合时是稳定的，但与酪氨酸残基被苏氨酸取代的突变通道结合时是不稳定的。利用伞形采样模拟计算TEA相对于孔道的自由能分布，以定量表征细胞外阻断作用。发现与实验非常吻合的是，TEA与含四个酪氨酸残基的通道结合更稳定，稳定程度为2.3 kcal/mol。对于野生型KcsA通道，TEA（呈扁平扁球体形状）作为理想的塞子堵塞孔道。相反，在突变通道的情况下，它明显更偏离中心且倾斜。酪氨酸残基赋予的稳定性增强并非源于π - 阳离子相互作用，而似乎是由于TEA的水合结构差异。最后，表明实验观察到的TEA阻断的电压依赖性（传统上根据TEA沿孔道轴的物理位置来解释）必须通过与孔道中的离子耦合间接产生。
TEA is a classical blocker of K+ channels. From mutagenesis studies, it has been shown that external blockade by TEA is strongly dependent upon the presence of aromatic residue at Shaker position 449 which is located near the extracellular entrance to the pore (Heginbotham, L., and R. MacKinnon. 1992. Neuron. 8:483–491). The data suggest that TEA interacts simultaneously with the aromatic residues of the four monomers. The determination of the 3-D structure of the KcsA channel using X-ray crystallography (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69–77) has raised some issues that remain currently unresolved concerning the interpretation of these observations. In particular, the center of the Tyr82 side chains in KcsA (corresponding to position 449 in Shaker) forms a square of 11.8-Å side, a distance which is too large to allow simultaneous interactions of a TEA molecule with the four aromatic side chains. In this paper, the external blockade by TEA is explored by molecular dynamics simulations of an atomic model of KcsA in an explicit phospholipid bilayer with aqueous salt solution. It is observed, in qualitative accord with the experimental results, that TEA is stable when bound to the external side of the wild-type KcsA channel (with Tyr82), but is unstable when bound to a mutant channel in which the tyrosine residue has been substituted by a threonine. The free energy profile of TEA relative to the pore is calculated using umbrella sampling simulations to characterize quantitatively the extracellular blockade. It is found, in remarkable agreement with the experiment, that the TEA is more stably bound by 2.3 kcal/mol to the channel with four tyrosine residues. In the case of the wild-type KcsA channel, TEA (which has the shape of a flattened oblate spheroid) acts as an ideal plug blocking the pore. In contrast, it is considerably more off-centered and tilted in the case of the mutant channel. The enhanced stability conferred by the tyrosine residues does not arise from Π–cation interactions, but appears to be due to differences in the hydration structure of the TEA. Finally, it is shown that the experimentally observed voltage dependence of TEA block, which is traditionally interpreted in terms of the physical position of the TEA along the axis of the pore, must arise indirectly via coupling with the ions in the pore.