Brady Hunt and Dixon Woodbury, Physiology and Developmental Biology
Introduction
My research focus has been to investigate how ethanol’s pharmacological effects could be tied to it’s interactions with a key component of all cellular membranes, lipid bilayers.
Methodology
To investigate the effect of ethanol on fusion rate, we used the Nystatin/Ergosterol (NYS/ERG) fusion assay. We measured fusions per minute using planar membranes containing PE/PC/CHL and vesicles containing PE/PC/PS/ERG. For our initial experiments we used high concentrations of ethanol added to either side of the bilayer. We also utilized differential scanning calorimetry to see what effect ethanol may have on the phase behavior or the lipids exposed to ethanol. Solutions of Potassium Chloride buffer and DPPC vesicles were analyzed with and without ethanol.
Results
The fusion assay showed that addition of ethanol to the cis side of the bilayer (same side as vesicles) caused and increase in fusion rate. Compared to a control, 1% ethanol caused a 50% increase in fusion rate. At 4% fusion rates increased as much as 300%. However, no significant increase was observed when added to the trans side. Similar results were observed with methanol. It also appears that the 2-3 fold increase in fusion rate occurs regardless of whether ethanol is added before or after vesicles. The DPPC vesicles with ethanol were shown to have a transition state ~0.5°C lower than without ethanol. This is consistent with our hypothesis that adding ethanol alters lipid fluidity and/or phase behavior.
Discussion
When I received funding for this research, we had observed an effect with ethanol on fusion only once. Since that time we have been able to replicate this effect in over 30 experiments. We have moved past these basic experiments and begun to explore more deeply a mechanism that could explain the result. Unfortunately, our doses of ethanol are 7 times higher than doses that cause human drunkenness, so it seems like a stretch that our effect could be linked to pharmacological effects of ethanol. But because it is such a strong effect, it will be interesting to refine our experimental methods and investigate it further at lower concentrations.
Conclusion
Planar lipid bilayers exposed to 4% ethanol exhibit a 3 fold increase in fusion rates.