The orientation of cholesterol's hydroxy group affects its membrane dynamics and intracellular transport.

The brain, though less than 10% of body mass, contains about 25% of total cholesterol (CHL), emphasizing CHL's key role in neuronal function. Many actions of CHL are stereospecific, as shown by differences from its 3α-hydroxy epimer, epicholesterol (epiCHL). How this minor structural change alters membrane properties and sterol transport remains unclear. Here, we compare fluorescent analogs of CHL (cholestatrienol [CTL]) and epiCHL (epicholestatrienol [epiCTL]), which closely mimic their natural counterparts. Biophysical membrane properties, such as flip-flop, acyl-chain ordering, and interbilayer transfer, depend on the orientation of the 3-hydroxy group. Similarly, transport by sterol transport proteins (STPs) and intracellular trafficking of the sterols in human astrocytes are stereospecific. Treatment with 25-hydroxycholesterol increases the uptake of both epimers, but only CTL shows enhanced esterification and lipid droplet storage. These findings demonstrate that subtle CHL structural changes affect cellular homeostasis and establish epiCTL as a useful probe of sterol stereospecificity and trafficking.