(749a) Intra- and Inter-Membrane Cholesterol Transport in Model Membrane Systems: A Small Angle Neutron Scattering Study

Cholesterol is essential for a myriad of biological functions, but its excess is toxic. Cholesterol levels are maintained by various cholesterol metabolic pathways which depend critically on its intracellular transport and distribution. Any disorder in intracellular cholesterol distribution can lead to diseases, ranging from neurodegenerative Niemann Pick TYPE-C and Alzheimer, to cholelithiasis and atherosclerosis. Unfortunately, the understanding of intracellular transport of cholesterol has lagged behind the other aspects of cholesterol metabolism due to technical limitations of the methods being used. These limitations have resulted in huge inconsistencies in reported cholesterol transport rates. This is due to the fact that these measurements are not done in-situ, but rather requires the biochemical isolation of cholesterol-donor and cholesterol-acceptor vesicles to estimate the amount of cholesterol transfer at any given time. Another important limitation has been the need for a fluorescent tag on cholesterol which has a significant effect on the resulting rates of transfer. These methods also require extraneous compounds such as cyclodextrine or cholesterol-oxidase to perform these measurements. To circumvent such potential limitations, this work employed Time-Resolved Small Angle Neutron Scattering (in-situ TR-SANS) technique for first time in-situ measurement of Intra- and Inter-membrane cholesterol exchange rates in 100nm lipid vesicles without any requirement of biochemical tag and presence of any extraneous compound. Interestingly our approach finds that trans-membrane flipping rates of cholesterol are much slower (half-life of few hours) without any extraneous particles or fluorescent tags in contrast to high flipping rates reported in literature (half-life of few seconds). We also found that replacing cholesterol with fluorescent cholesterol or adding low concentration of extraneous compounds can significantly accelerate the transport rates. Molecular dynamics simulations have also been performed to investigate the energetic and kinetic behavior of the cholesterol. We found that simulation results are in agreement with our SANS results, providing a more detailed thermodynamic description at the molecular level. Such a synergistic approach combining TR-SANS and MD simulation will provide new insight into the ongoing efforts of understanding cholesterol traffic and related disorders.