Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy
Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy
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DOI：
10.1016/s0006-3495(03)74611-6
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发表时间：
2003-09-01
影响因子：
3.4
通讯作者：
Lu, HP
中科院分区：
文献类型：
--
作者：
Harms, GS;Orr, G;Lu, HP
复杂的构象变化影响和调节离子通道的动力学。这种构象变化是随机的，而且往往是不均匀的，这使得通过集合平均实验或单通道电流记录来表征它们非常困难，如果不是不可能的话，这些电流记录报告了开合事件，但没有具体探测相关的构象变化。在这里，我们报道了我们使用一种新的方法-膜片钳荧光显微镜来研究离子通道的构象变化，该方法同时结合单分子荧光光谱和单通道电流记录来探测单个离子通道的开闭跃迁和构象动力学。我们展示了膜片钳荧光显微镜通过测量在缓冲溶液下在膜片钳微吸管顶端形成的脂质双层中的革兰西丁离子通道的构象变化。通过测量单对荧光共振能量转移和荧光自猝灭，我们观察到单对荧光共振能量转移和自猝灭的效率广泛分布，这反映了构象的广泛分布。我们的结果强烈地表明，在脂质双层中，甘利胶通道的多种构象状态与其闭合和开放状态之间存在着迄今无法检测到的相关性。
Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.