Diffusion Rate of Cholesterol through a Single Lipid Bilayer*

Our lab has measured the rate and energetics of the intramembrane diffusion (flip-flop) of the steroid-lipid cholesterol using small angle neutron scattering (SANS) and found that the diffusion rate was much slower (hundreds of minutes) than the accepted value (under a second). Our work showed that the discrepancy was due to artifacts produced by differences between cholesterol and its analogues or by the use of compounds, like cyclodextrin, that, our group argued, disrupts the membrane. Our results were based on measurements in which cholesterol flip-flop within the membrane occurred while cholesterol was also diffusing between different membranes (exchange). So, our team has been taking steps to eliminate exchange from the measurement, thus measuring the flip-flop diffusion rate directly. We used a new method to craft an asymmetric distribution of cholesterol in the lipid bilayers of unilamellar vesicles to measure the rate at which cholesterol diffuses through the bilayer. We used orthogonal cholesterol sensors, nuclear magnetic resonance spectroscopy (NMR), and SANS in order to follow the change of cholesterol’s distribution within the bilayers as a function of time and temperature.