Gandhi Chris S, Olcese Riccardo
Division of Chemistry and Chemical Engineering, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.
Methods Mol Biol. 2008;491:213-31. doi: 10.1007/978-1-59745-526-8_17.
Ion channels are the cell's gatekeepers. These proteins selectively allow ionic current to flow down its electrochemical gradient. In some cases, specialized chemical or voltage sensing domains respond to environmental changes and signal the cell to adjust its internal chemistry in response to its surroundings. Because of their importance in cell function, channels have been the focus of intense study at the functional and structural level. Here we describe the optical technique voltage-clamp fluorometry (VCF) which is used to monitor the functional state and probe the structural rearrangements that take place as ion channels are activated by voltage. VCF combines electrophysiology, molecular biology, chemistry, and fluorescence into a single technique. Our focus is on voltage-gated ion channels, but the technique described can be applied to other proteins. We describe the cut open vaseline gap configuration (COVG) for VCF recording.
离子通道是细胞的守门人。这些蛋白质选择性地允许离子电流沿其电化学梯度流动。在某些情况下，特殊的化学或电压感应结构域会对环境变化做出反应，并向细胞发出信号，使其根据周围环境调整内部化学组成。由于它们在细胞功能中具有重要性，通道一直是功能和结构层面深入研究的焦点。在这里，我们描述了光学技术电压钳荧光测定法（VCF），该技术用于监测功能状态，并探测离子通道被电压激活时发生的结构重排。VCF将电生理学、分子生物学、化学和荧光结合成一种单一技术。我们关注的是电压门控离子通道，但所描述的技术也可应用于其他蛋白质。我们描述了用于VCF记录的切开凡士林间隙配置（COVG）。
