Cholesterol stabilizes the structure of the nicotinic acetylcholine receptor reconstituted in lipid vesicles

Abstract

A technique of heat inactivation of α-bungarotoxin binding sites, has been used to probe structural alteration of the nicotinic acetylcholine receptor when reconstituted into soybean lipid vesicles containing different amounts of added cholesterol. The profiles of heat inactivation of α-bungarotoxin binding sites are gradually shifted to higher temperatures, as the cholesterol/phospholipid molar ratio in the reconstituted vesicles is increased from 0 to 0.4, thus, indicating that presence of cholesterol within the lipid matrix produces a structural stabilization of the reconstituted acetylcholine receptor protein. The observed stabilization of receptor structure induced by cholesterol is such that, depending upon the different conditions used to form the reconstituted vesicles by detergent dialysis procedures, the profiles of heat inactivation for the reconsutituted receptor vesicles at a cholesterol/phospholipid molar ratio of 0.4 become undistinguishable from that exhibited by native acetylcholine receptor membranes isolated from the electric organ of Torpedo. Increasing the cholesterol concentration in the reconstituted vesicles also induces a decrease in the apparent ‘fluidity’ of the membrane, which correlates very closely with the observed stabilization of the receptor protein. Such a correlation, however, does not necessarily imply that changes in receptor structure are caused by pertubations of the membrane ‘fluidity’. This conclusion is based on experiments using local anesthetics, well known to cause alteration of membrane lipid dynamics, but unable to modify the characteristic heat-inactivation profiles from native acetylcholine receptor membranes. As a possible alternative to the above observations, it is suggested that the effects of cholesterol on receptor structure could be exerted through direct interaction with the receptor protein. Also, since similarly high concentrations of cholesterol have been reported to be required for optimal cation-gating activity of reconstituted acetylcholine receptor, we interpret our data as indicative of a correlation between structural and functional alterations of the acetylcholine receptor induced by the presence of cholesterol within the membrane bilayer.