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

• Published in: Molecular pharmacology Vol. 61; no. 1; pp. 160 - 168
• Main Authors: Buller, Stefan, Zlotos, Darius Paul, Mohr, Klaus, Ellis, John
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.01.2002
• Subjects: Alkaloids - pharmacology; Allosteric Regulation; Animals; COS Cells; Humans; Membrane Proteins - drug effects; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutagenesis; Point Mutation; Receptors, Muscarinic - drug effects; Receptors, Muscarinic - genetics; Receptors, Muscarinic - metabolism; Recombinant Fusion Proteins - metabolism; Transfection
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.61.1.160

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 [ 3 H] 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 4 receptor, as M 4 436 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.