Investigating the cut-off effect of n-alcohols on lipid movement: a biophysical study

Cellular membranes are responsible for absorbing the effects of external perturbants for the cell's survival. Such perturbants include small ubiquitous molecules like n-alcohols which were observed to exhibit anesthetic capabilities, with this effect tapering off at a cut-off alcohol chain length. To explain this cut-off effect and complement prior biochemical studies, we investigated a series of n-alcohols (with carbon lengths 2-18) and their impact on several bilayer properties, including lipid flip-flop, intervesicular exchange, diffusion, membrane bending rigidity and more. To this end, we employed an array of biophysical techniques such as time-resolved small angle neutron scattering (TR-SANS), small angle X-ray scattering (SAXS), all atomistic and coarse-grained molecular dynamics (MD) simulations, and calcein leakage assays. At an alcohol concentration of 30 mol% of the overall lipid content, TR-SANS showed 1-hexanol (C6OH) increased transverse lipid diffusion, i.e. flip-flop. As alcohol chain length increased from C6 to C10 and longer, lipid flip-flop slowed by factors of 5.6 to 32.2. Intervesicular lipid exchange contrasted these results with only a slight cut-off at alcohol concentrations of 30 mol% but not 10 mol%. SAXS, MD simulations, and leakage assays revealed changes to key bilayer properties, such as bilayer thickness and fluidity, that correlate well with the effects on lipid flip-flop rates. Finally, we tie our results to a defect-mediated pathway for alcohol-induced lipid flip-flop.