title: Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.
abstract: At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [(3)H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [(3)H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [(3)H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ-α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ-α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts (A.K.H., D.C.C., J.B.C.); Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts (D.S.S.); and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois (P.Y.S., K.S.B.).
authors.forename: Ayman K
authors.initials: AK
authors.lastname: Hamouda
authors.forename: Deirdre S
authors.initials: DS
authors.lastname: Stewart
authors.forename: David C
authors.initials: DC
authors.lastname: Chiara
authors.forename: Pavel Y
authors.initials: PY
authors.lastname: Savechenkov
authors.forename: Karol S
authors.initials: KS
authors.lastname: Bruzik
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001463:Barbiturates / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005260:Female; D008618:Mephobarbital / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium / Q000737:chemistry / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D001463:Barbiturates; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; D014316:Tritium; D008618:Mephobarbital; C032232:barbituric acid