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Effects of membrane cholesterol modulation on mu opioid receptor signaling
Author(s) -
Levitt Erica,
Clark Mary J,
Thomson Jennifer A,
Traynor John R
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.727.5
Subject(s) - lipid raft , adenylyl cyclase , chemistry , sphingomyelin , hek 293 cells , cholesterol , damgo , receptor , microbiology and biotechnology , g protein coupled receptor , signal transduction , agonist , biochemistry , opioid receptor , biology
Methyl‐β‐cyclodextrin (MβCD) is used to deplete membrane cholesterol leading to disruption of lipid rafts. Previously, we have shown that MβCD alters mu opioid‐mediated acute and chronic signaling to adenylyl cyclase in HEK293 cells. The effects of MβCD are due to the removal of membrane cholesterol, since treatment of HEK293 cells expressing the FLAG‐tagged mu receptor (HEK FLAG‐mu) with MβCD that is pre‐complexed to cholesterol restored signaling of the mu agonist, DAMGO. Although, we have confirmed that mu receptors are present in cholesterol‐enriched raft membranes using detergent and non‐detergent based methods, they are also present in non‐raft membranes. Here we use cholesterol modulators to test the hypothesis that the effects observed following cholesterol removal by MβCD are indeed due to disruption of lipid rafts rather than alterations in general membrane environment. Treatment of HEK FLAG‐mu cells with other cholesterol modulators, including cholesterol oxidase (0.5U/ml, 1h) and sphingomyelinase (0.1U/ml, 2h), to disrupt lipid rafts without affecting membrane viscosity, did not alter the ability of DAMGO to interact acutely or chronically with adenylyl cyclase. Therefore, modulation of mu opioid signaling by MβCD appears to be due to the general effects of cholesterol depletion on membrane environment rather than disruption of lipid rafts. Supported by DA04087, DA07267, GM07767, DA22377.