Abstract
BackgroundThe 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.ResultsBy 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.ConclusionsOur model system allows a quantitative detection of cell surface expression of ABCA1, screening of substrates or specific inhibitors, and investigating transport regulation.
Highlights
The ABCA1 protein plays a pivotal role in reverse cholesterol transport, by mediating the generation of high-density lipoprotein (HDL) particles and removing cellular cholesterol
It is commonly accepted that the ATP-binding cassette protein, ABCA1 plays a pivotal role in the initial steps of the reverse cholesterol transport pathway by mediating the interactions of amphiphilic apolipoproteins with cellular lipids to generate nascent HDL particles removing excess cellular cholesterol [3,4]
Treatment with 50 μM ALLN substantially increased, whereas 5 mg/ml Brefeldin A (BFA) reduced the cell surface expression level of both wild type (HA-WT) and mutant (HA-motif mutant ABCA1 (MM)) HA-ABCA1 variants, as compared to cells treated with vehicle only. (C, D) Cell surface expression of wild type and MM mutant HA-ABCA1 variants in HEK293H cells treated with 50 μM ALLN, 5 mg/ml BFA, 10 μg/ml apoA-I, 10 μM cylcosporin A (CsA), or 50 μM EZ for 4 h
Summary
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. It is commonly accepted that the ATP-binding cassette protein, ABCA1 plays a pivotal role in the initial steps of the reverse cholesterol transport pathway by mediating the interactions of amphiphilic apolipoproteins (e.g., apoA-I) with cellular lipids to generate nascent HDL particles removing excess cellular cholesterol [3,4]. Certain types of calcium channel blockers (CCBs), e.g., verapamil, nifedipine, have been found to be anti-atherogenic in clinical trials [17,18] When their effects on ABCA1 expression were investigated, contradictory results were obtained. These agents either increased ABCA1 mRNA levels or elevated the protein expression without affecting the mRNA level, depending on the cellular test system used [19,20]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
