title: Does general anesthetic-induced desensitization of the Torpedo acetylcholine receptor correlate with lipid disordering?
abstract: We have tested the hypothesis that general anesthetics stabilize the desensitized state of the nicotinic acetylcholine receptor by disordering its surrounding lipids. Acetylcholine receptor-rich postsynaptic membranes from the electroplaques of Torpedo were used in this study to obtain the highest possible receptor specific activity in native membranes. We examined 18 general anesthetics, including six inhalation agents, eight 1-alcohols, the enantiomers of 2-octanol, and two intravenous general anesthetics (pentobarbital and ethylcarbamate). The degree of desensitization after preincubation with the general anesthetics was determined by brief exposure to [3H]acetylcholine, making use of the 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 within 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. The concentrations that desensitized half of the resting state receptors varied by > 3000-fold. The 2-octanol enantiomers were without stereoselectivity. Membrane order was examined in parallel by using spin-labeled fatty acids doped into the native membranes. The spin label 5-doxylpalmitate reported from the most ordered part of the bilayer near the aqueous interface, whereas 12-doxylstearate reported from the less ordered region nearer the center of the bilayer. The spin label deeper in the membranes was 3 times more sensitive to a given anesthetic than was the other probe. At both depths in the membrane general anesthetics decreased lipid order linearly with increasing concentration. The range of disordering potencies (change in order parameter induced by a unit concentration of general anesthetic in the aqueous phase) was 5333 for 5-doxylpalmitate and 7143 for 12-doxylstearate, but the range of disordering compared at equally desensitizing concentrations was reduced by 875- and 1430-fold, respectively. The average degrees of disordering at concentrations that desensitized half of the resting state receptors were 1.5% and 4.4%, respectively. It is unlikely that changes in membrane order parameter per se cause desensitization, because the associated changes in order parameter can be reproduced by changes in cholesterol content or temperature that do not cause desensitization. We conclude that, although there is a strong association between anesthetic-induced membrane disordering and desensitization, more detailed tests of a mechanistic nature will be necessary to elucidate the mechanisms underlying the Meyer-Overton-type behavior we have observed.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
authors.forename: L L
authors.initials: LL
authors.lastname: Firestone
authors.forename: J K
authors.initials: JK
authors.lastname: Alifimoff
authors.forename: K W
authors.initials: KW
authors.lastname: Miller
mesh_terms: D000768:Anesthesia, General; D000777:Anesthetics / Q000633:toxicity*; D000818:Animals; D002499:Centrifugation, Density Gradient; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000378:metabolism; D014101:Torpedo
chemical_list: D000777:Anesthetics; D008051:Lipid Bilayers; D011978:Receptors, Nicotinic