%0 Journal Article %A Ayman K Hamouda %A Deirdre S Stewart %A David C Chiara %A Pavel Y Savechenkov %A Karol S Bruzik %A Jonathan B. Cohen %T Identifying Barbiturate Binding Sites in a Nicotinic Acetylcholine Receptor with [3H]Allyl m-Trifluoromethyldiazirine Mephobarbital, a Photoreactive Barbiturate %D 2014 %R 10.1124/mol.113.090985 %J Molecular Pharmacology %P mol.113.090985 %X At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory γ-aminobutyric acid type-A receptors (GABAAR) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChR). Recently, [3H]R-mTFD-MPAB ([3H]R-5-allyl-1-methyl-5-(m- trifluoromethyl-diazirinylphenyl)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 IC50s of 5 and 10 μM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [3H]ACh to nAChR-rich membranes (EC50, 9 µM) and inhibited binding of the ion channel blocker [3H]tenocyclidine in the nAChR desensitized and resting states with IC50s of 2 and 170 μM, respectively. Photoaffinity labeling identified two binding sites for [3H]R-mTFD-MPAB in the nAChR transmembrane domain: (i) 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; (ii) a site at the γ-α subunit interface, identified by photolabeling of γMet-299 within 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. %U https://molpharm.aspetjournals.org/content/molpharm/early/2014/02/21/mol.113.090985.full.pdf