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Published on: November 29, 2014
Model system for the analysis of cell surface expression of human ABCA1
Ildikó Kasza1, Zoltán Hegyi, Katalin Szabó
1Membrane Biology Research Group, Hungarian Academy of Sciences, Department of Biophysics and Radiation Biology, Semmelweis University, Diószegi u 64, H-1113, Budapest, Hungary. kasza@kkk.org.hu
Background:
The ABCA1 protein plays a pivotal role in reverse cholesterol transport, by mediating the generation of HDL particles and removing cellular cholesterol. Both the proper expression of ABCA1 in the plasma membrane and the internalization along with apoA-I are required for function. Therefore, we developed a model system to investigate the effect of clinically relevant drugs on the cell surface appearance of ABCA1.
Results:
By retroviral transduction system, we established stable mammalian cell lines expressing functional and non-functional ABCA1 variants, tagged with an extracellular hemagglutinin epitope. After characterization of the expression, proper localization and function of different ABCA1 variants, we followed quantitatively their cell surface expression by immunofluorescent staining, using flow cytometry. As expected, we found increased cell surface expression of ABCA1 after treatment with a calpain inhibitor, and observed a strong decrease in plasma membrane ABCA1 expression upon treatment with a trans-Golgi transport inhibitor, Brefeldin A. We tested cholesterol level lowering drugs and other potential inhibitors of ABCA1. Here we demonstrate that ezetimibe affects ABCA1 cell surface expression only in the case of a functional ABCA1.
Conclusions:
Our model system allows a quantitative detection of cell surface expression of ABCA1, screening of substrates or specific inhibitors, and investigating transport regulation.
