Probing ion-channel pores one proton at a time
Probing ion-channel pores one proton at a time
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DOI：
10.1038/nature04293
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发表时间：
2005-12-15
期刊：
影响因子：
64.8
通讯作者：
Grosman, C
中科院分区：
文献类型：
--
作者：
Cymes, GD;Ni, Y;Grosman, C
尽管膜蛋白的结构和功能通常依赖于可电离残基，但它们在生理条件下的电离状态在很大程度上无法得到实验估计。为了深入了解局部微环境对可电离残基质子亲和力的影响，我们沿着烟碱型乙酰胆碱受体跨膜孔的α螺旋衬里设计了单个赖氨酸、组氨酸和精氨酸。我们可以在单个侧链上的单个质子的电生理水平上检测到单个质子结合-解锁反应，作为经过的阳离子电流的短暂的阻断-解锁事件。对这些涨落的动力学分析得到了与位置有关的质子转移速率，由此可以计算出相应的pK(A)值和pK(A)的位移。在这里，我们提出了一个自洽的，逐个残基的描述，根据保持工程基础侧链相对于主体水质子化所需的过剩自由能，描述了明渠构象中孔衬跨膜α-螺旋(M2)周围的微环境。与闭合通道数据的比较导致我们认为M2的旋转是最小的，如果有的话。M2的旋转经常被引用作为Cys-loop受体门控机制的标志。
Although membrane proteins often rely on ionizable residues for structure and function, their ionization states under physiological conditions largely elude experimental estimation. To gain insight into the effect of the local microenvironment on the proton affinity of ionizable residues, we have engineered individual lysines, histidines and arginines along the alpha-helical lining of the transmembrane pore of the nicotinic acetylcholine receptor. We can detect individual proton binding - unbinding reactions electrophysiologically at the level of a single proton on a single side chain as brief blocking - unblocking events of the passing cation current. Kinetic analysis of these fluctuations yields the position-dependent rates of proton transfer, from which the corresponding pK(a) values and shifts in pK(a) can be calculated. Here we present a self-consistent, residue-by-residue description of the microenvironment around the pore-lining transmembrane alpha-helices (M2) in the open-channel conformation, in terms of the excess free energy that is required to keep the engineered basic side chains protonated relative to bulk water. A comparison with closed-channel data leads us to propose that the rotation of M2, which is frequently invoked as a hallmark of the gating mechanism of Cys-loop receptors, is minimal, if any.