Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR).

Nicotinic acetylcholine receptors (nAChRs) and γ-aminobutyric acid type A receptors (GABAA Rs) are members of the pentameric ligand-gated ion channel superfamily. Drugs acting as positive allosteric modulators of muscle-type α2 βγδ nAChRs, of use in treatment of neuromuscular disorders, have been hard to identify. However, identification of nAChR allosteric modulator binding sites has been facilitated by using drugs developed as photoreactive GABAA R modulators. Recently, R- 1-methyl-5-allyl-5-( m -trifluoromethyl-diazirinylphenyl) barbituric acid ( R-m TFD-MPAB), an anesthetic and GABAA R potentiator, has been shown to inhibit Torpedo α2 βγδ nAChRs, binding in the ion channel and to a γ+ -α- subunit interface site similar to its GABAA R intersubunit binding site. In contrast, S- 1-methyl-5-propyl-5-( m -trifluoromethyl-diazirinylphenyl) barbituric acid ( S-m TFD-MPPB) acts as a convulsant and GABAA R inhibitor. Photolabeling studies established that S-m TFD-MPPB binds to the same GABAA R intersubunit binding site as R-m TFD-MPAB, but with negative rather than positive energetic coupling to GABA binding. We now show that S-m TFD-MPPB binds with the same state (agonist) dependence as R-m TFD-MPAB within the nAChR ion channel, but it does not bind to the intersubunit binding site. Rather, S-m TFD-MPPB binds to intrasubunit sites within the α and δ subunits, photolabeling αVal-218 (αM1), δPhe-232 (δM1), δThr-274 (δM2), and δIle-288 (δM3). Propofol, a general anesthetic that binds to GABAA R intersubunit sites, inhibited [3 H] S-m TFD-MPPB photolabeling of these nAChR intrasubunit binding sites. These results demonstrate that in an nAChR, the subtle difference in structure between S-m TFD-MPPB and R-m TFD-MPAB (chirality; 5-propyl versus 5-allyl) determines selectivity for intra- versus intersubunit sites, in contrast to GABAA Rs, where this difference affects state dependence of binding to a common site.