Young Victoria C, Rayaprolu Vamseedhar, Kohout Susy C
Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ, USA.
Pacific Northwest Cryo-EM Center, Oregon Health and Sciences University, Portland, OR, USA.
Bio Protoc. 2025 Feb 20;15(4):e5212. doi: 10.21769/BioProtoc.5212.
Voltage clamp fluorometry (VCF) is a powerful technique in which the voltage of a cell's membrane is clamped to control voltage-sensitive membrane proteins while simultaneously measuring fluorescent signals from a protein of interest. By combining fluorescence measurements with electrophysiology, VCF provides real-time measurement of a protein's motions, which gives insight into its function. This protocol describes the use of VCF to study a membrane protein, the voltage-sensing phosphatase (VSP). VSP is a 3 and 5 phosphatidylinositol phosphate (PIP) phosphatase coupled to a voltage sensing domain (VSD). The VSD of VSP is homologous to the VSD of ion channels, with four transmembrane helices (S1-S4). The S4 contains the gating charge arginine residues that sense the membrane's electric field. Membrane depolarization moves the S4 into a state that activates the cytosolic phosphatase domain. To monitor the movement of S4, the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) is attached extracellularly to the S3-S4 loop. Using VCF, the resulting fluorescence signals from the S4 movement measure the kinetics of activation and repolarization, as well as the voltage dependence of the VSD. This protocol details the steps to express VSP in oocytes and then acquire and analyze the resulting VCF data. VCF is advantageous as it provides voltage control of VSP in a native membrane while quantitatively assessing the functional properties of the VSD. Key features • Voltage clamp fluorometry using oocytes expressing the voltage-sensing phosphatase of • This protocol uses the fluorophore tetramethylrhodamine-6-maleimide (TMRM). • This protocol details the procedure for a two-electrode voltage clamp using the Dagan CA-1B amplifier.
电压钳荧光测定法（VCF）是一项强大的技术，通过将细胞膜电压钳制来控制电压敏感的膜蛋白，同时测量来自目标蛋白的荧光信号。通过将荧光测量与电生理学相结合，VCF可实时测量蛋白质的运动，从而深入了解其功能。本方案描述了使用VCF研究膜蛋白——电压感应磷酸酶（VSP）的方法。VSP是一种与电压感应结构域（VSD）偶联的3,5-磷脂酰肌醇磷酸（PIP）磷酸酶。VSP的VSD与离子通道的VSD同源，有四个跨膜螺旋（S1-S4）。S4包含感知膜电场的门控电荷精氨酸残基。膜去极化会使S4进入激活胞质磷酸酶结构域的状态。为监测S4的运动，将对环境敏感的荧光团四甲基罗丹明-6-马来酰亚胺（TMRM）细胞外连接到S3-S4环上。使用VCF，S4运动产生的荧光信号可测量激活和复极化的动力学以及VSD的电压依赖性。本方案详细介绍了在卵母细胞中表达VSP，然后获取并分析所得VCF数据的步骤。VCF的优势在于它能在天然膜中对VSP进行电压控制，同时定量评估VSD的功能特性。关键特性• 使用表达电压感应磷酸酶的卵母细胞进行电压钳荧光测定法• 本方案使用荧光团四甲基罗丹明-6-马来酰亚胺（TMRM）。• 本方案详细介绍了使用Dagan CA-1B放大器进行双电极电压钳的操作步骤。
