Allosteric site on muscarinic acetylcholine receptors: A single amino acid in transmembrane region 7 is critical to the subtype selectivities of caracurine V derivatives and alkane-bisammonium ligands

Diverse muscarinic allosteric ligands exhibit greatest affinity toward the M-2 receptor subtype and lowest affinity toward M-5. In this study, we evaluated the potencies with which two groups of highly M-2/M-5 selective allosteric agents modulate the dissociation of [H-3]N-methylscopolamine from M-2/M-5 chimeric and point-mutated receptors. These allosteric ligands included two alkane-bisammonium compounds and a series of caracurine V derivatives, which are structurally closely related to (but stereochemically different from) the prototype allosteric ligand alcuronium. Like alcuronium, the caracurine V and alkane-bisammonium compounds displayed significantly increased affinities compared with M-5 toward the chimera that included the M-2 second outer loop (o2) plus surrounding regions. Unlike alcuronium, the compounds had enhanced affinities for a chimera with M-2 sequence in transmembrane region (TM) 7; site-directed mutagenesis in wild-type and chimeric receptors indicated that the threonine residue at M-2(423) was entirely responsible for the sensitivity toward TM7. Subsequent studies demonstrated that this TM7 epitope is likewise present in the M, receptor, as M(2)(436)serine. The M(2)(423)threonine residue is near the M(2)(419)asparagine identified previously to influence gallamine binding. These studies demonstrate that a stereochemical difference can be sufficient to translate into divergent epitope sensitivities. Nonetheless, these allosteric ligands seem to derive affinity from two main regions of the receptor: o2 plus flanking regions and o3/TM7. These two epitopes are sufficient to explain the M-2/M-5 selectivity of the presently investigated compounds; this is the first time that the subtype selectivity of muscarinic allosteric agents has been completely accounted for by distinct receptor epitopes.