Abstract 18439: Membrane Microdomains Modulate Ligand Binding Activity of Oligomeric ABCA1 and ApoA-I-Mediated Lipid Removal: Impact on Nascent HDL Genesis
The apoA-I/ABCA1 system plays a pivotal role in the removal of excess intracellular cholesterol and HDL biogenesis. However, the nature and specifics of apoA-I/ABCA1 interaction with plasma membrane microdomains and its relationship to nascent HDL formation remains poorly understood. In this study, using chemical cross-linking combined to sucrose gradient fractionation, we obtained evidence that oligomeric ABCA1 was found exclusively associated with non-raft microdomains (NRMD) isolated from fibroblasts and BHK cells stably overexpressing ABCA1. Similarly, following incubation of 125I-apoA-I with cells expressing ABCA1 at 37°C for short periods of time, both the 125I-apoA-I/ABCA1 complex, as well as the 125I-apoA-I associated with cells were localized to NRMD. In addition, a significant portion of 125I-apoA-I without ABCA1 was found associated with the less dense non-raft fractions. Next, we examined the distribution of cholesterol (Chol), phosphatidylcholine (PC) and sphingomyelin (SM) between different membrane microdomains and monitored the removal of these lipids by apoA-I. The majority of Chol and SM were found associated with raft microdomains (RMD), whereas PC was distributed between raft and non-raft domains. Chol and SM were removed by apoA-I from both raft and NRMD. In contrast, PC was selectively removed by apoA-I from NRMD. Finally, we examined the modulatory role of Chol on the ligand binding activity of ABCA1. Depletion of Chol from fibroblasts membrane using cholesterol-methyl-β-cyclodextrin (MCD) resulted in a drastic reduction in apoA-I binding to ABCA1. Conversely, replenishment of membranes with Chol led to recovery of apoA-I binding activity of ABCA1. Importantly, MCD cell treatment did not alter neither the integrity of RMD nor the levels and localization of oligomeric ABCA1 within the NRMD. Taken together, these results provide strong evidence that NRMD structures constitute functional platforms for apoA-I binding to ABCA1, thereby allowing the insertion of apoA-I to adjacent PC-rich membrane microdomains. Our data also suggests that the majority of Chol molecules containing nascent HDL originate from RMD and support the concept that membrane Chol environment regulates ABCA1-mediated nascent HDL genesis pathway.
- © 2010 by American Heart Association, Inc.