Abstract
Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7β-hydroxycholesterol (7β-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane—bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7β-OH-chol which represents ring-oxidized sterols.
Highlights
Permeation of small molecules across cell membranes is of crucial importance to life
Fluorescence quenching experiments were performed on oxysterol-containing POPC Large Unilamellar Vesicles (LUVs) at 37 °C
An NBD fluorescent lipid analogue incorporated in the lipid bilayer of the LUVs was irreversibly quenched by dithionite added to the liposomal dispersion
Summary
Permeation of small molecules across cell membranes is of crucial importance to life. One of the underlying molecular mechanisms used by cells to control permeation through membranes is the level of cholesterol. Unbiased molecular dynamics (MD) simulations showed that the rate of spontaneous water translocation through a membrane decreases by a factor of 7 when the content of cholesterol is increased from 0 to 33 mol %.7. This is in agreement with experimental studies that reported a 4-fold reduction of water permeability through a bilayer containing Subsequent investigations described this effect in detail,[2−5] showing that the effect of cholesterol in lipid membrane structures is exceptionally prominent at the level of the cholesterol ring.[3,6]
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