Abstract
BackgroundEthanol affects the signaling of several important neurotransmitter and neuromodulator systems in the CNS. It has been recently proposed that ethanol alters the dynamic lateral organization of proteins and lipids in the plasma membrane, thereby affecting surface receptor-mediated cellular signaling. Our aims are to establish whether pharmacologically relevant levels of ethanol can affect the lateral organization of plasma membrane and cytoskeletal proteins at the nanoscopic level, and investigate the relevance of such perturbations for mu-opioid receptor (MOP) function.Methodology/Principal FindingsWe used Photoactivated Localization Microscopy with pair-correlation analysis (pcPALM), a quantitative fluorescence imaging technique with high spatial resolution (15–25 nm) and single-molecule sensitivity, to study ethanol effects on protein organization in the plasma membrane. We observed that short (20 min) exposure to 20 and 40 mM ethanol alters protein organization in the plasma membrane of cells that harbor endogenous MOPs, causing a rearrangement of the lipid raft marker glycosylphosphatidylinositol (GPI). These effects could be largely occluded by pretreating the cells with the MOP antagonist naltrexone (200 nM for 3 hours). In addition, ethanol induced pronounced actin polymerization, leading to its partial co-localization with GPI.Conclusions/SignificancePharmacologically relevant levels of ethanol alter the lateral organization of GPI-linked proteins and induce actin cytoskeleton reorganization. Pretreatment with the MOP antagonist naltrexone is protective against ethanol action and significantly reduces the extent to which ethanol remodels the lateral organization of lipid-rafts-associated proteins in the plasma membrane. Super-resolution pcPALM reveals details of ethanol action at the nanoscale level, giving new mechanistic insight on the cellular and molecular mechanisms of its action.
Highlights
Alcohol is one of the most widely used and abused psychoactive substances, with a large negative effect on health and public safety
It is widely assumed that ethanol increases the activity of the endogenous opioid system through the release of opioid peptides and that anticraving/antihedonic effects of naltrexone are achieved through antagonizing the effect of opioid peptides acting at the mu-opioid receptor (MOP)
The aim of this study is to establish whether pharmacologically relevant concentrations of ethanol (20 and 40 mM) change the lateral organization of plasma membrane and cytoskeletal proteins at the nanoscale level, and whether action of the opioid receptor antagonist naltrexone at endogenous MOPs is protective against such changes
Summary
Alcohol is one of the most widely used and abused psychoactive substances, with a large negative effect on health and public safety. It is widely assumed that ethanol increases the activity of the endogenous opioid system through the release of opioid peptides and that anticraving/antihedonic effects of naltrexone are achieved through antagonizing the effect of opioid peptides acting at the mu-opioid receptor (MOP). This hypothesis is not fully validated experimentally [6]. Our aims are to establish whether pharmacologically relevant levels of ethanol can affect the lateral organization of plasma membrane and cytoskeletal proteins at the nanoscopic level, and investigate the relevance of such perturbations for mu-opioid receptor (MOP) function
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