Abstract
The combined effects of ethanol and osmolytes on both the extent of formation of metarhodopsin II (MII), which binds and activates transducin, and on acyl chain packing were examined in rod outer segment disc membranes. The ethanol-induced increase in MII formation was amplified by the addition of neutral osmolytes. This enhancement was linear with osmolality. At 360 milliosmolal, the osmolality of human plasma, 50 mM ethanol was 2.7 times more potent than at 0 osmolality, demonstrating the importance of water activity in in vitro experiments dealing with ethanol potency. Ethanol disordered acyl chain packing, and increasing osmolality enhanced this acyl chain disordering. Prior osmotic stress data showed a release of 35 +/- 2 water molecules upon MII formation. Ethanol increases this number to 49 water molecules, suggesting that ethanol replaces 15 additional water molecules upon MII formation. Amplification of ethanol effects on MII formation and acyl chain packing by osmolytes suggests that ethanol increases the equilibrium concentration of MII both by disordering acyl chain packing and by disrupting rhodopsin-water hydrogen bonds, demonstrating a direct effect of ethanol on rhodopsin. At physiologically relevant levels of osmolality and ethanol, about 90% of ethanol's effect is due to disordered acyl chain packing.
Highlights
From the Section of Fluorescence Studies, Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland 20853
This experimental strategy is based on the fact that the effect of a neutral osmolyte on the water activity of aqueous compartments in equilibrium with a protein depends on the degree to which it is excluded from the protein-associated water
We examined the combined effects of ethanol and osmotic stress on both the activating conformational change of the G proteincoupled receptor rhodopsin and on acyl chain packing of the ROS disc membrane
Summary
REDUCED WATER ACTIVITY AMPLIFIES THE EFFECT OF ETHANOL ON METARHODOPSIN II FORMATION*. The combined effects of ethanol and osmolytes on both the extent of formation of metarhodopsin II (MII), which binds and activates transducin, and on acyl chain packing were examined in rod outer segment disc membranes. The osmotic stress protocol [10] is an established method for determining changes in the number of bound water molecules associated with a specific enzymatic process This experimental strategy is based on the fact that the effect of a neutral osmolyte on the water activity of aqueous compartments in equilibrium with a protein depends on the degree to which it is excluded from the protein-associated water. The available structural information for rhodopsin coupled with its central role in visual transduction and its relationship to neurotrans-
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