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 M2 receptor subtype and lowest affinity toward M5. In this study, we evaluated the potencies with which two groups of highly M2/M5 selective allosteric agents modulate the dissociation of [3H]N-methylscopolamine from M2/M5 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 M5 toward the chimera that included the M2 second outer loop (o2) plus surrounding regions. Unlike alcuronium, the compounds had enhanced affinities for a chimera with M2 sequence in transmembrane region (TM) 7; site-directed mutagenesis in wild-type and chimeric receptors indicated that the threonine residue at M2(423) was entirely responsible for the sensitivity toward TM7. Subsequent studies demonstrated that this TM7 epitope is likewise present in the M4 receptor, as M4(436)serine. The M2(423)threonine residue is near the M2(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 M2/M5 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.