Probing Red Cell Membrane Cholesterol Movement with Cyclodextrin

Abstract

We probed the kinetics with which cholesterol moves across the human red cell bilayer and exits the membrane using methyl-beta-cyclodextrin as an acceptor. The fractional rate of cholesterol transfer (% s(-1)) was unprecedented, the half-time at 37 degrees C being ~1 s. The kinetics observed under typical conditions were independent of donor concentration and directly proportional to acceptor concentration. The rate of exit of membrane cholesterol fell hyperbolically to zero with increasing dilution. The energy of activation for cholesterol transfer was the same at high and low dilution; namely, 27-28 Kcal/mol. This behavior is not consistent with an exit pathway involving desorption followed by aqueous diffusion to acceptors nor with a simple one-step collision mechanism. Rather, it is that predicted for an activation-collision mechanism in which the reversible partial projection of cholesterol molecules out of the bilayer precedes their collisional capture by cyclodextrin. Because the entire membrane pool was transferred in a single first-order process under all conditions, we infer that the transbilayer diffusion (flip-flop) of cholesterol must have proceeded faster than its exit, i.e., with a half-time of <1 s at 37 degrees C.

The sterol transporting heterodimer ABCG5/ABCG8 requires bile salts to mediate cholesterol efflux

“…4 shows that apoA1 and HDL failed to induce Abcg5/g8 specific efflux. A similar result was observed for methyl-b-cyclodextrin, which in contrast to apoA1 or HDL does not interact with proteins but takes up cholesterol directly from the membrane [22]. Since this cholesterol acceptor is very potent and may overrule the importance of Abcg5/g8 we have used limiting amounts of the acceptor and carried out kinetic studies.…”

Effect of Cholesterol Reduction on Receptor Signaling in Neurons

“…MBCD depletes cholesterol from plasma membranes by sequestering cholesterol in its hydrophobic pocket (27). This effect is rapid.…”

Scrambling of Phospholipids Activates Red Cell Membrane Cholesterol

“…While the lateral and transverse distributions of cholesterol are apparently each in rapid diffusional equilibrium, the tendencies of the sterol population at each leaflet to project into the neighboring aqueous space do not come to equilibrium but rather remain different from one another. The transfer of membrane cholesterol to aqueous acceptors such as cyclodextrin appears to depend on this bobbing motion (6). In particular, it is during these transient projections that sterol molecules are captured by collision with the acceptors.…”

“…Cholesterol equilibrates rapidly between the two leaflets of the PM bilayer; however, its transbilayer distribution is debated (6). The transverse distribution of the PM PLs is asymmetrical, with a preponderance of PC and SM in the exofacial leaflet and anionic PL, mostly PE and PS, in the cytoplasmic leaflet (7).…”