Bazzone Andre, Costa Wagner Steuer, Braner Markus, Călinescu Octavian, Hatahet Lina, Fendler Klaus
Department of Biophysical Chemistry, Max Planck Institute of Biophysics.
J Vis Exp. 2013 May 11(75):e50230. doi: 10.3791/50230.
The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire. After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage. The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis. This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.
我们所展示的电生理方法基于一种固体支撑膜（SSM），它由化学吸附在镀金传感器芯片上的十八烷硫醇层和顶部的磷脂酰胆碱单层组成。该组件安装在一个含有参比电极（氯化银线）的比色皿系统中。在吸附含有感兴趣膜蛋白的膜片段或蛋白脂质体后，采用快速溶液交换来诱导膜蛋白的转运活性。在单溶液交换方案中，需要两种溶液，一种是非激活溶液，一种是激活溶液。流动由法拉第笼内的压缩空气以及阀门和管道系统控制。通过SSM与蛋白脂质体或膜片段之间的电容耦合获得电转运活性的动力学。因此，该方法仅产生瞬态电流。峰值电流代表稳定的转运活性。随时间变化的转运体电流可通过电路分析重建。该方法特别适用于原核转运体或细胞内膜的真核转运体，这些转运体无法通过膜片钳或电压钳方法进行研究。
