Visualizing ion channel dynamics at the plasma membrane.
Visualizing ion channel dynamics at the plasma membrane.
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DOI：
10.1016/j.hrthm.2008.02.015
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发表时间：
2008-06
期刊：
影响因子：
5.5
通讯作者：
James W. Smyth;R. Shaw
中科院分区：
文献类型：
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作者：
James W. Smyth;R. Shaw
心脏离子通道在本质上具有令人惊讶的动态性，并且不断形成，运输到质膜的特定亚区，插入质膜，然后去除以降解或再循环。由于这些运动会影响通道的可用性，离子通道的功能不仅取决于通道的生物物理特性，还取决于通道的运输。用荧光和其他基因编码蛋白标记感兴趣蛋白的分子技术的发展，以及全内反射显微镜（TIRF）等先进成像方式的发展，为了解质膜附近蛋白质的细胞内运动及其动力学创造了新的机会。在这篇文章中，我们介绍了离子通道生物学家使用荧光和非荧光融合蛋白的方法，在哺乳动物细胞中克隆和表达融合蛋白的技术，以及活细胞高分辨率显微镜成像技术。为了说明这一点，本文提供了一种稳定的细胞系的原始数据，该细胞系能够诱导表达连接蛋白43，该连接蛋白43被标记为绿色荧光蛋白，并通过宽视场荧光和TIRF显微镜观察其分布。随着荧光显微镜技术的革命性进步，离子通道生物学家正在进入一个研究通道功能的新领域，即了解通道运输的机制。
Cardiac ion channels are surprisingly dynamic in nature, and are continuously formed, trafficked to specific subregions of plasma membrane, inserted in the plasma membrane, and removed to be degraded or recycled. Because of these movements, which affect channel availability, ion channel function is dependent on not just channel biophysical properties but channel trafficking as well. The development of molecular techniques to tag proteins of interest with fluorescent and other genetically encoded proteins, and of advanced imaging modalities such as total internal reflection microscopy (TIRF), have created new opportunities to understand the intracellular movement of proteins near the plasma membrane and their dynamics therein. In this article we present approaches for ion channel biologists to the use of fluorescent and nonfluorescent fusion proteins, techniques for cloning and expression of fusion proteins in mammalian cells, and imaging techniques for live-cell high-resolution microscopy. For illustration, original data are presented on creation of a stable cell line capable of inducible expression of connexin 43 tagged to green fluorescent protein and its distribution viewed with both wide-field epifluorescence and TIRF microscopy. With revolutionary advances in fluorescence microscopy, ion channel biologists are now entering a new realm of studying channel function, which is to understand the mechanisms of channel trafficking.