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

Nicotinic acetylcholine receptors (nAChRs) and γ-aminobutyric acid type A receptors (GABA_ARs) are members of the pentameric ligand-gated ion channel superfamily. Drugs acting as positive allosteric modulators of muscle-type α₂βγδ 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 GABA_AR modulators. Recently, R-1-methyl-5-allyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (R-mTFD-MPAB), an anesthetic and GABA_AR potentiator, has been shown to inhibit Torpedo α₂βγδ nAChRs, binding in the ion channel and to a γ⁺-α^- subunit interface site similar to its GABA_AR intersubunit binding site. In contrast, S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (S-mTFD-MPPB) acts as a convulsant and GABA_AR inhibitor. Photolabeling studies established that S-mTFD-MPPB binds to the same GABA_AR intersubunit binding site as R-mTFD-MPAB, but with negative rather than positive energetic coupling to GABA binding. We now show that S-mTFD-MPPB binds with the same state (agonist) dependence as R-mTFD-MPAB within the nAChR ion channel, but it does not bind to the intersubunit binding site. Rather, S-mTFD-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 GABA_AR intersubunit sites, inhibited [³H]S-mTFD-MPPB photolabeling of these nAChR intrasubunit binding sites. These results demonstrate that in an nAChR, the subtle difference in structure between S-mTFD-MPPB and R-mTFD-MPAB (chirality; 5-propyl versus 5-allyl) determines selectivity for intra- versus intersubunit sites, in contrast to GABA_ARs, where this difference affects state dependence of binding to a common site.