title: Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.
abstract: Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl-etomidate).
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Ayman K
authors.initials: AK
authors.lastname: Hamouda
authors.forename: Deirdre S
authors.initials: DS
authors.lastname: Stewart
authors.forename: S Shaukat
authors.initials: SS
authors.lastname: Husain
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D018686:Anesthetics, Intravenous / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D005045:Etomidate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D029941:Fish Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D018722:Nicotinic Agonists / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D011978:Receptors, Nicotinic; D014101:Torpedo / Q000235:genetics / Q000378:metabolism*
chemical_list: D018686:Anesthetics, Intravenous; D029941:Fish Proteins; D018722:Nicotinic Agonists; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D005045:Etomidate