Effects of Ethanol and Opioid Receptor Antagonists Naltrexone and LY2444296 on the Organization of Cholesterol- and Sphingomyelin-Enriched Plasma Membrane Domains

Excessive consumption of alcohol (ethanol (EtOH)) can lead to alcohol use disorder (AUD). While AUD can be managed through behavioral interventions, there is a great need for pharmacological remedies for this condition. To date, only three pharmacotherapeutic treatments for AUD have been approved by both the U.S. Food and Drug Administration and the European Medicines Agency. This is partly due to an incomplete understanding of the intricate molecular mechanisms through which EtOH affects cellular functions. Here, we focus on EtOH effects on nanoscale organization and the lateral dynamics of molecules in the plasma membrane, the primary cellular component that is affected by this compound. In a cell culture model natively expressing opioid receptors, important targets for medications aimed at preventing relapses in AUD, we used methods with single-molecule sensitivity to characterize the lateral organization and dynamics of cholesterol (Chol)- and sphingomyelin (SM)-enriched domains. Our data reveal that EtOH triggered a reorganization of Chol/SM-enriched domains and increased the plasma membrane fluidity. The general opioid receptor antagonist naltrexone (NTX), approved for relapse prevention in AUD, caused reorganization of Chol/SM-enriched domains, while pretreatment with NTX warded off EtOH-induced plasma membrane reorganization. In contrast, the selective kappa-opioid receptor antagonist LY2444296, at concentrations tested, showed a modest effect on Chol/SM-enriched domain organization and did not protect against EtOH-induced changes in plasma membrane organization. While the significance of these findings for AUD treatment remains uncertain at this stage, our study reveals that the action of NTX is not only limited to blocking opioid receptor activity by preventing agonist binding to the orthosteric binding site, but also is protective against short- and long-range EtOH-induced plasma membrane reorganization. By protecting against EtOH-induced changes in the lateral organization and dynamics of lipids in the plasma membrane, NTX may affect physiological outcomes through previously unrecognized noncanonical mechanisms.