Phospholipase A 2 hydrolysis of membrane phospholipids causes structural alteration of the nicotinic acetylcholine receptor

Abstract

Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the α-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A 2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of α-bungarotoxin-binding sites are shifted to lower temperaturex by approx. 7 and 5 °C, respectively, upon exposure to phospholipase A 2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A 2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3( n −3 ) causes detectable destabilization of the α-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A 2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A 2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A 2 hydrolysis products.