Abstract
Enhancement of gamma-aminobutyric acid type A receptor (GABA(A)R)-mediated inhibition is a property of most general anesthetics and a candidate for a molecular mechanism of anesthesia. Intravenous anesthetics, including etomidate, propofol, barbiturates, and neuroactive steroids, as well as volatile anesthetics and long-chain alcohols, all enhance GABA(A)R function at anesthetic concentrations. The implied existence of a receptor site for anesthetics on the GABA(A)R protein was supported by identification, using photoaffinity labeling, of a binding site for etomidate within the GABA(A)R transmembrane domain at the beta-alpha subunit interface; the etomidate analog [(3)H]azietomidate photolabeled in a pharmacologically specific manner two amino acids, alpha1Met-236 in the M1 helix and betaMet-286 in the M3 helix (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599-11605). Here, we use [(3)H]azietomidate photolabeling of bovine brain GABA(A)Rs to determine whether other structural classes of anesthetics interact with the etomidate binding site. Photolabeling was inhibited by anesthetic concentrations of propofol, barbiturates, and the volatile agent isoflurane, at low millimolar concentrations, but not by octanol or ethanol. Inhibition by barbiturates, which was pharmacologically specific and stereospecific, and by propofol was only partial, consistent with allosteric interactions, whereas isoflurane inhibition was nearly complete, apparently competitive. Protein sequencing showed that propofol inhibited to the same extent the photolabeling of alpha1Met-236 and betaMet-286. These results indicate that several classes of general anesthetics modulate etomidate binding to the GABA(A)R: isoflurane binds directly to the site with millimolar affinity, whereas propofol and barbiturates inhibit binding but do not bind in a mutually exclusive manner with etomidate.
Highlights
Our results provide evidence that isoflurane at millimolar concentrations may inhibit binding competitively, propofol and barbiturates act as allosteric inhibitors, and ethanol and octanol at anesthetic concentrations have no effect on [3H]azietomidate binding
Some General Anesthetics Inhibit GABAAR Photolabeling by [3H]Azietomidate—When purified bovine brain GABAAR photolabeled with [3H]azietomidate in the absence or presence of 200 M nonradioactive etomidate was fractionated by SDSPAGE, the 3H incorporated into GABAAR subunit polypeptides of ϳ50 –55 kDa was inhibitable by Ͼ90% in the presence of etomidate and was shown to result from labeling of ␣1Met2364 in ␣M1 and Met-2864 in M3 [14]
We photolabeled bovine brain GABAARs with the photoreactive etomidate analog [3H]azietomidate to determine whether other structural classes of general anesthetics bind to the same site as etomidate
Summary
General Anesthetics Inhibit Etomidate Binding to GABAAR affinity determinants for etomidate and propofol [15] These photolabeling results suggested a GABAAR structural model based on homology with the nicotinic acetylcholine receptor [14, 16] that positioned both photolabeled amino acids within an intersubunit binding pocket at the -␣ interface, two copies of a single class of sites per pentamer. This structural model is supported by cysteine replacement cross-linking data defining the orientation of various GABAAR transmembrane helices within and between subunits [17, 18]. Several structural classes of general anesthetics, including propofol, barbiturates, and volatile agents, but not alcohols, interact, directly or indirectly, with the intersubunit etomidate-binding site in the transmembrane domain of GABAAR proteins
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