Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Beno&#238;te Bourdin; Emilie Segura; Marie-Philippe T&#233;treault; Sylvie Lesage; Lucie Parent

doi:10.3791/54732

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using...

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Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

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11:32 min

September 28, 2016

DOI: 10.3791/54732-v

Benoîte Bourdin¹, Emilie Segura¹, Marie-Philippe Tétreault¹, Sylvie Lesage², Lucie Parent¹

¹Département de Physiologie Moléculaire et Intégrative,Montreal Heart Institute Research Centre, ²Département de Microbiologie, Infectiologie, Immunologie,Centre de recherche de l'Hôpital Maisonneuve-Rosemont

Overview

This article presents a method for quantifying the cell surface expression of membrane proteins using flow cytometry assays. The technique is particularly relevant for studying cardiac arrhythmias linked to genetic mutations affecting ion channel trafficking.

Key Study Components

Area of Science

Cardiac arrhythmias
Ion channel biology
Flow cytometry techniques

Background

Inherited cardiac arrhythmias are often due to mutations in ion channels.
These mutations can disrupt the surface delivery of ion channels.
Flow cytometry allows for the analysis of large volumes of live cells.
Identifying suitable insertion sites for epitope tagging is crucial for successful experiments.

Purpose of Study

To develop a method for quantifying membrane protein expression.
To facilitate the diagnosis and therapy of cardiac ventricular arrhythmias.
To improve the understanding of ion channel trafficking defects.

Methods Used

Utilization of flow cytometry assays for quantification.
Double tagging of DNA constructs with mCherry at the intracellular C-terminus.
Assessment of cell surface protein expression in tsA-201 cells.
Demonstration of the procedure by a research assistant.

Main Results

Successful quantification of total and cell surface protein expression.
Identification of effective epitope insertion sites.
Demonstration of the method's applicability to live cell analysis.
Insights into the relationship between genetic mutations and ion channel function.

Conclusions

The method provides a reliable approach for studying membrane proteins.
It has potential implications for understanding cardiac arrhythmias.
Future applications may enhance therapeutic strategies for affected individuals.

Frequently Asked Questions

What is the main advantage of using flow cytometry in this study?

Flow cytometry allows for quantifiable analysis of large volumes of live cells in a single experiment.

How does this method contribute to cardiac arrhythmia research?

It helps in understanding the trafficking defects of cardiac ion channels associated with genetic mutations.

What is the significance of tagging DNA constructs with mCherry?

Tagging with mCherry enables visualization and quantification of protein expression at the cell surface.

Who demonstrated the procedure in this study?

The procedure was demonstrated by Benoite Bourdin, a research assistant from the lab.

What challenges do researchers face when using this method?

Identifying suitable insertion sites for epitope tagging without affecting protein function can be challenging.

What type of cells were used in this study?

The study utilized tsA-201 cells for expressing recombinant ion channels.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

The overall goal of this method is to quantify the cell surface expression of membrane proteins using a fluorescent spaced assay. The main advantage of this technique is that flow cytometry assays deliver quantifiable end points on large volumes of live cells in a single experiment. The implications of this technique extend toward a diagnosis and a therapy of cardiac ventricular arrhythmias because these pathologies are often associated with genetic mutations that cause defects in the trafficking of cardiac ion channels.

Generally individuals new to this method struggle to identify a suitable extrorsal insertion site for the epitope that does not hurt their protein function and generate a strong fluorescent signal in the presence of the conjugated antibody. Demonstrating this procedure will be Benoite Bourdin, a research assistant from my lab. To begin, doubley tag DNA constructs with mCherry at the intracellular C-terminus.

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