Grimm Christiane, Vierock Johannes, Hegemann Peter, Wietek Jonas
Experimental Biophysics, Institute of Biology, Humboldt-Universität zu Berlin.
Experimental Biophysics, Institute of Biology, Humboldt-Universität zu Berlin;
J Vis Exp. 2017 May 22(123):55497. doi: 10.3791/55497.
Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable - ideally monochromatic - light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.
在过去十年中，视紫红质通道蛋白在神经科学研究中变得不可或缺，在该研究领域，它们被用作非侵入性操纵靶细胞电活动过程的工具。在此背景下，视紫红质通道蛋白的离子选择性尤为重要。本文描述了通过在HEK293细胞上进行的电生理膜片钳记录，对最近鉴定出的苏氏原滴虫阴离子传导视紫红质通道蛋白的氯离子选择性进行的研究。测量光门控光电流的实验程序需要一个快速可切换的——理想情况下是单色的——光源，该光源耦合到常规膜片钳装置的显微镜中。概述了实验前的准备程序，包括缓冲溶液的制备、液接电位的考虑、细胞的接种和转染以及膜片吸管的拉制。在转染后24小时至48小时进行电流-电压关系的实际记录，以确定不同氯离子浓度下的反转电位。最后，根据氯离子传导的理论考虑对电生理数据进行分析。
