Stability and Stoichiometry of Bilayer Phospholipid–Cholesterol Complexes: Relationship to Cellular Sterol Distribution and Homeostasis

Abstract

Does cholesterol distribute among intracellular compartments by passive equilibration down its chemical gradient? If so, its distribution should reflect the relative cholesterol affinity of the constituent membrane phospholipids as well as their ability to form stoichiometric cholesterol complexes. We tested this hypothesis by analyzing the reactivity to cholesterol oxidase of large unilamellar vesicles (LUVs) containing biological phospholipids plus varied cholesterol. The rates of cholesterol oxidation differed among the various phospholipid environments by roughly four orders of magnitude. Furthermore, accessibility to the enzyme increased by orders of magnitude at cholesterol thresholds that suggested stoichiometries of association of 1:1, 2:3 or 1:2 cholesterol:phospholipid (mol:mol). Cholesterol accessibility above the threshold was still constrained by its particular phospholipid environment. One phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine, exhibited no threshold. The analysis suggested values for the relative stabilities of the cholesterol-phospholipid complexes and for the fractions of bilayer cholesterol not in complexes at the threshold equivalence points; predictably, the saturated phosphorylcholine species had the lowest stoichiometries and the strongest affinities for cholesterol. These results were in general agreement with the equilibrium distribution of cholesterol between the various LUVs and methyl-β-cyclodextrin. In addition, the properties of the cholesterol in intact human red blood cells matched predictions made from LUVs of the corresponding composition. These results support a passive mechanism for the intracellular distribution of cholesterol that can provide a signal for its homeostatic regulation.

Switch-like Responses of Two Cholesterol Sensors Do Not Require Protein Oligomerization in Membranes

“…Theoretical studies have shown that the cooperative formation of oligomers of such complexes can further sharpen the sigmoidal change in chemical activity, making it more threshold-like (2,30). This simple, Biophysical Journal 108(6) 1459-1469 intuitive model of complex formation has been extremely useful in accounting for many physical chemical properties of membranes (2,(29)(30)(31)(32)(33); however, such complexes have not been isolated. This may not be surprising, because molecular complexes in liquids have been described with relatively well-defined structures but very short lifetimes (<10 ps) (34).…”

“…This may not be surprising, because molecular complexes in liquids have been described with relatively well-defined structures but very short lifetimes (<10 ps) (34). A well-defined specific structure for phospholipidcholesterol complexes may be unlikely, since sharp changes in chemical activities are observed for a wide variety of phospholipid and sterol structures (2,31,35). Other models that consider nonrandom arrangements of cholesterol in the bilayer could also result in sharp changes in its chemical activity (36,37).…”

“…The chemical activity of membrane cholesterol likely controls its surface accessibility to other soluble molecules such as cholesterol oxidase and cyclodextrin (30,31,42). Of particular interest is a recent report that the cholesterol-binding site in Scap, the mammalian cholesterol sensor, is located not in its transmembrane region but in a membrane-associated loop that projects into the lumen of the ER (43).…”

Box C/D Small Nucleolar RNA (snoRNA) U60 Regulates Intracellular Cholesterol Trafficking

“…The cholesterol activation hypothesis (6) posits that the tendency of PM cholesterol to become more exposed at the aqueous-membrane interface and thereby increase accessibility to extramembrane acceptors is dependent on the lipid composition of the membrane. Increased saturation of PM phospholipid acyl chains could account for increased PM cholesterol retention: the saturated acyl chains interacting more strongly with cholesterol and positioning cholesterol closer to the bilayer center, thereby shielding cholesterol's hydroxyl group from interaction with extracellular membrane acceptors (6,23,29,32). How reduced U60 expression might specifically alter the PM lipid environment will require further understanding of the noncanonical functions of the U60 snoRNA.…”

Artificial biomembrane based on DPPC — Investigation into phase transition and thermal behavior through ellipsometric techniques

“…This effect occurred during first, second or third heating cycle, proving Y-Type to be a much more stable structure than X-type bilayer. This is why this deposition type is the most widely used for phospholipid membrane formation [37][38][39][40][41].…”