PubMed · 10839711
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J C Wootton. 1999. Cholesterol.. https://doi.org/10.1089/jwh.1.1999.8.555
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Temperature-dependent internal content release from liposomes was examined using di-oleoylphosphatidylcholine (DOPC)/cholesterol liposomes with encapsulated Pluronic F127 molecules. The interaction of Pluronic F127 with the lipid bilayer at elevated temperature causes the release of encapsulated contents. Content release was measured using fluorescent markers of two different sizes: small, carboxyfluorescein (CF), and large, bovine serum albumin-conjugated fluorescein iso-thiocyanate (BSA-FITC). Release of CF was studied using fluorescence de-quenching, while that of BSA-FITC was studied using fluorescence emission quenching due to fluorescence resonance energy transfer (FRET). Temperature-controlled complete internal content release was achieved at a precise temperature by controlling the concentration of the encapsulated Pluronic. Increasing cholesterol % in the liposome composition resulted in a sharper transition with temperature in content release. The onset temperature of content release increased with decrease in Pluronic concentration. For the same Pluronic concentration, the onset temperature also depended on the size of the encapsulated marker and was higher for larger markers. We have established that onset of content release is determined by the critical micellar temperature (CMT) of the Pluronic. Temperature-sensitive liposomes, made stealth using di-stearoyl(polyethylene glycol 5000) phosphatidylethanolamine (DSPEG5000PE) in conjunction with Pluronic F127, had similar temperature sensitivity and efficiency in content release compared to the non-stealth liposomes.
The role of membrane composition in modulating the rate of G protein-receptor complex formation was examined using rhodopsin and transducin (G(t)) as a model system. Metarhodopsin II (MII) and MII-G(t) complex formation rates were measured, in the absence of GTP, via flash photolysis for rhodopsin reconstituted in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC) bilayers, with and without 30 mol% cholesterol. Variation in bilayer lipid composition altered the lifetime of MII-G(t) formation to a greater extent than the lifetime of MII. MII-G(t) formation was fastest in 18:0,22:6PC and slowest in 18:0,18:1PC/30 mol% cholesterol. At 37 degrees C and a G(t) to photolyzed rhodopsin ratio of 1:1 in 18:0,22:6PC bilayers, MII-G(t) formed with a lifetime of 0.6 +/- 0.06 ms, which was not significantly different from the lifetime for MII formation. Incorporation of 30 mol% cholesterol slowed the rate of MII-G(t) complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-G(t) formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-G(t) formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system.
The visual transduction system was used as a model to investigate the effects of membrane lipid composition on receptor-G protein coupling. Rhodopsin was reconstituted into large, unilamellar phospholipid vesicles with varying acyl chain unsaturation, with and without cholesterol. The association constant (K(a)) for metarhodopsin II (MII) and transducin (G(t)) binding was determined by monitoring MII-G(t) complex formation spectrophotometrically. At 20 degrees C, in pH 7.5 isotonic buffer, the strongest MII-G(t) binding was observed in 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC), whereas the weakest binding was in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) with 30 mol% cholesterol. Increasing acyl chain unsaturation from 18:0,18:1PC to 18:0,22:6PC resulted in a 3-fold increase in K(a). The inclusion of 30 mol% cholesterol in the membrane reduced K(a) in both 18:0,22:6PC and 18:0,18:1PC. These findings demonstrate that membrane compositions can alter the signaling cascade by changing protein-protein interactions occurring predominantly in the hydrophilic region of the proteins, external to the lipid bilayer. These findings, if extended to other members of the superfamily of G protein-coupled receptors, suggest that a loss in efficiency of receptor-G protein binding is a contributing factor to the loss of cognitive skills, odor and spatial discrimination, and visual function associated with n-3 fatty acid deficiency.