PT  - JOURNAL ARTICLE
AU  - Ayman K. Hamouda
AU  - Deirdre S. Stewart
AU  - David C. Chiara
AU  - Pavel Y. Savechenkov
AU  - Karol S. Bruzik
AU  - Jonathan B. Cohen
TI  - Identifying Barbiturate Binding Sites in a Nicotinic Acetylcholine Receptor with [<sup>3</sup>H]Allyl <em>m</em>-Trifluoromethyldiazirine Mephobarbital, a Photoreactive Barbiturate
AID  - 10.1124/mol.113.090985
DP  - 2014 May 01
TA  - Molecular Pharmacology
PG  - 735--746
VI  - 85
IP  - 5
4099  - http://molpharm.aspetjournals.org/content/85/5/735.short
4100  - http://molpharm.aspetjournals.org/content/85/5/735.full
SO  - Mol Pharmacol2014 May 01; 85
AB  - 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 [3H]R-mTFD-MPAB ([3H]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 [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 IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [3H]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.