DOES GENERAL ANESTHETIC-INDUCED DESENSITIZATION OF THE TORPEDO ACETYLCHOLINE-RECEPTOR CORRELATE WITH LIPID DISORDERING

We have tested the hypothesis that general anesthetics stabilize the d esensitized state of the nicotinic acetylcholine receptor by disorderi ng its surrounding lipids. Acetylcholine receptor-rich postsynaptic me mbranes from the electroplaques of Torpedo were used in this study to obtain the highest possible receptor specific activity in native membr anes. We examined 18 general anesthetics, including six inhalation age nts, eight 1-alcohols, the enantiomers of 2-octanol, and two intraveno us general anesthetics (pentobarbital and ethylcarbamate). The degree of desensitization after preincubation with the general anesthetics wa s determined by brief exposure to [H-3]acetylcholine, making use of th e facts that desensitized receptors have much higher affinity than do those in the resting state and that interconversion between the states is slow. All of the general anesthetics desensitized the receptor wit hin minutes, exhibiting steep concentration-response curves with Hill coefficients generally within the range of 2-4. At the highest general anesthetic concentrations, almost all receptors were desensitized. Th e concentrations that desensitized half of the resting state receptors varied by >3000-fold. The 2-octanol enantiomers were without stereose lectivity. Membrane order was examined in parallel by using spin-label ed fatty acids doped into the native membranes. The spin label 5-doxyl palmitate reported from the most ordered part of the bilayer near the aqueous interface, whereas 12-doxylstearate reported from the less ord ered region nearer the center of the bilayer. The spin label deeper in the membranes was 3 times more sensitive to a given anesthetic than w as the other probe. At both depths in the membrane general anesthetics decreased lipid order linearly with increasing concentration. The ran ge of disordering potencies (change in order parameter induced by a un it concentration of general anesthetic in the aqueous phase) was 5333 for 5-doxylpalmitate and 7143 for 12-doxylstearate, but the range of d isordering compared at equally desensitizing concentrations was reduce d by 875- and 1430-fold, respectively. The average degrees of disorder ing at concentrations that desensitized half of the resting state rece ptors were 1.5% and 4.4%, respectively. It is unlikely that changes in membrane order parameter per se cause desensitization, because the as sociated changes in order parameter can be reproduced by changes in ch olesterol content or temperature that do not cause desensitization. We conclude that, although there is a strong association between anesthe tic-induced membrane disordering and desensitization, more detailed te sts of a mechanistic nature will be necessary to elucidate the mechani sms underlying the Meyer-Overton-type behavior we have observed.