Seward Elizabeth P, Wykes Robert C
School of Biosciences, University of Sheffield, Sheffield, UK.
Department of Clinical and Experimental Epilepsy, Queen Square Institute of Neurology, University College London, London, UK.
Methods Mol Biol. 2023;2565:187-202. doi: 10.1007/978-1-0716-2671-9_13.
Research using membrane capacitance (C) measurements in adrenal chromaffin cells has transformed our understanding of the molecular mechanisms controlling regulated exocytosis. This is in part due to the exquisite temporal resolution of the technique, and the possibility of combining quantification of exo-/endocytosis at the whole-cell level, with the ability to simultaneously monitor and control the calcium signals triggering vesicle fusion. In this regard, experiments performed with C measurements complement amperometry experiments that give a measure of secreted transmitter and the behavior of the fusion pore, and fluorescent microscopy studies used to monitor vesicle and protein dynamics in imaged regions of the cell. In this chapter, we provide a detailed account of the methodology used to perform whole-cell patch clamp measurements of C in combination with voltage-clamp recordings of voltage-gated calcium channels to quantify stimulus-secretion coupling in chromaffin cells. Stimulus protocols developed for investigation of functionally distinct releasable vesicle pools are also described.
利用肾上腺嗜铬细胞的膜电容（C）测量进行的研究，改变了我们对控制调节性胞吐作用分子机制的理解。部分原因在于该技术具有极高的时间分辨率，以及在全细胞水平上能够将胞吐/内吞作用的量化与同时监测和控制触发囊泡融合的钙信号的能力相结合。在这方面，用C测量进行的实验补充了安培法实验，后者可测量分泌的递质和融合孔的行为，以及用于监测细胞成像区域中囊泡和蛋白质动态的荧光显微镜研究。在本章中，我们详细介绍了结合电压门控钙通道的电压钳记录来进行C的全细胞膜片钳测量，以量化嗜铬细胞中刺激-分泌偶联的方法。还描述了为研究功能上不同的可释放囊泡池而开发的刺激方案。
