Abstract

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 ⁴³⁶serine. The M₂ ⁴²³threonine residue is near the M₂ ⁴¹⁹asparagine 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.