Mol090985 735..746

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 [H]R-mTFD-MPAB ([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 a1b3g2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, Sand R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 mM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [H]ACh to nAChR-rich membranes (EC50 5 9 mM) and inhibited binding of the ion channel blocker [H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 mM, respectively. Photoaffinity labeling identified two binding sites for [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 g–a subunit interface, identified by photolabeling of gMet299 within the gM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFDMPAB 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 g–a subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.