Manipulation of Cholesterol Levels in Rod Disk Membranes by Methyl- -cyclodextrin

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter fv. The level of MII formed showed a positive linear correlation with fv over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonistor antagonistbound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.