Bourdin Benoîte, Segura Emilie, Tétreault Marie-Philippe, Lesage Sylvie, Parent Lucie
Département de Physiologie Moléculaire et Intégrative, Montreal Heart Institute Research Centre.
Département de Microbiologie, Infectiologie, Immunologie, Centre de recherche de l'Hôpital Maisonneuve-Rosemont.
J Vis Exp. 2016 Sep 28(115):54732. doi: 10.3791/54732.
Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 10 to 10 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.
阳离子选择性通道的遗传或新发突变可能导致心源性猝死。这些多次跨膜蛋白的质膜运输改变，无论通道门控有无变化，通常被认为在这一过程中起重要作用。因此，开发一种大规模量化心脏离子通道相对细胞表面表达变化的方法变得至关重要。本文提供了一个详细方案，用于使用双色荧光细胞术测定tsA - 201细胞中心脏L型钙通道CaV1.2和膜相关亚基的相对总表达和细胞表面表达。与其他基于显微镜或免疫印迹的定性方法相比，流式细胞术实验快速、可重复且为大容量测定，能在单个流程中对具有相似细胞特征的大量活细胞样本（范围从10到10个细胞）给出可量化的终点结果。构建体被设计为在细胞内C末端组成性表达mCherry（从而允许快速评估总蛋白表达），并表达面向细胞外的血凝素（HA）表位，以便使用抗HA荧光偶联抗体估计膜蛋白的细胞表面表达。为避免假阴性，还在透化细胞中进行实验以确认HA表位的可及性和正确表达。详细步骤包括：（1）标记DNA构建体的设计，（2）脂质介导的构建体转染到tsA - 201细胞中，（3）非透化和透化细胞的培养、收获和染色，以及（4）荧光信号的采集和分析。此外，还解释了流式细胞术的基本原理，并深入讨论了实验设计，包括荧光团的选择、HA抗体的滴定和对照实验。这种特定方法提供了离子通道总表达和细胞表面表达的客观相对定量，可扩展用于研究离子泵和质膜转运体。
