Mechanisms of Alcohol-Altered Membrane Fusion

Abstract

Many studies on the effects of ethanol in humans have focused on its interactions with membrane protein receptors, however we recently showed that short-chain alcohols dramatically alter the rate of liposome fusion to a planar membrane (Paxman et al. 2017. Biophys J. 112:121-132.). To understand the molecular mechanism(s) whereby alcohols alter fusion, we utilized the nystatin/ergosterol fusion assay and recorded fusion rates in response to increasing doses of alcohols on one or both sides of the membrane (BLM). Addition of ethanol typically excited fusion when applied on the cis (liposome) side, and inhibited on the trans side. Other short-chain alcohols followed a similar pattern, with the exception of glycerol. Since symmetric and cis addition allow alcohol access to both membranes, we anticipated similar results. However, a decrease was generally observed with symmetric addition. To isolate the effect of alcohol on just the vesicle membrane, we altered the osmotic gradient to drive alcohol out of the planar membrane which increased fusion rates. Differential scanning calorimetry (DSC) was used to observe how short-chain alcohols shift the melting temperature (Tm) of DPPC liposomes. As expected, at low doses (0.4-4% v/v) alcohols decrease Tm by several degrees Celsius. Surprisingly, at higher doses, Tm increases toward control. However, these effects are not observed with glycerol, which did not significantly change Tm. We believe this is due to glycerol's size and polarity preventing its insertion into the lipid tails. We are developing a kinetic and a molecular model to fit these data. By utilizing this fusion assay, we hope to determine the potential effects of alcohol on neuronal exocytosis.