THERMAL-STABILITY OF TORPEDO-CALIFORNICA ACETYLCHOLINE-RECEPTOR IN A CHOLESTEROL LIPID ENVIRONMENT

Controlled heating of acetylcholine receptor (AChR) vesicles inactivat es the alpha-bungarotoxin (alpha-Bgtx) binding sites with a T-50 (temp erature at which 50% of the initial capacity to bind alpha-Bgtx remain s) of 60+/-0.2 degrees C. The same value was obtained for receptor rec onstituted in lipid vesicles from Torpedo electroplax where the % mold composition of cholesterol to phospholipid was 30. However, when the reconstitution was carried out in dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidic acid (DOPA) vesicles (3:1 molar ratio), T-50 of t he curves decreased to 56+/-0.2 degrees C and no carbamylcholine stimu lated Na-22(+) flux was detected. Inclusion of cholesterol in the DOPC -DOPA vesicles increased the toxin binding site stability. The maximal T-50 of the toxin binding curves was 63+/-0.1 degrees C when the % mo l cholesterol/mol DOPC:DOPA in the vesicles was 33. Under these condit ions AChR was able to translocate ions, a property that was lost upon heating at 46 degrees C. Preincubation of AChR in the presence of d-tu bocurarine, tetracaine or procaine did not affect T-50 values of toxin binding. However, a slight increment in thermal stability was found w hen the receptor was preincubated in the presence of carbamylcholine. The results show that cholesterol requirements for protecting against thermal inactivation of toxin binding and ion gating properties are di fferent and the carbamylcholine-bound receptor may have a different co nformation.