Abstract

The purpose of our study was to investigate the interactions of allosteric antagonists at the individual m1, m2 and m3 muscarinic receptor subtypes. This was achieved through the use of transformed Chinese hamster ovary cells stably expressing the rat m1 or m3 receptor genes. A homogeneous population of the m2 subtype was obtained from rat heart tissue. Our data indicate that the cardioselective antagonists (gallamine, methoctramine, AF-DX 116 and himbacine) display the following rank order of potency for both displacing ligand binding to the primary site on the receptor and allosterically decelerating ligand dissociation: m2 greater than m1 greater than m3. Schild analysis showed the following rank order of the magnitude of gallamine's cooperative interactions with the three receptor subtypes: m3 greater than m1 greater than m2. By comparison, the ion-channel blockers (verapamil, phencyclidine and quinidine) exhibited a rank order of potency for cooperative effects similar to that of cardioselective antagonists; however, these blockers did not show appreciable specificity in their interaction with the receptor primary binding site. There was a lack of correlation between the displacement of ligand binding and the allosteric potencies of the allosteric antagonists at each of the three muscarinic receptor subtypes, thus revealing the complex nature of interaction (both competitive and allosteric) between many of these compounds with the muscarinic receptor. Despite the fact that the majority of allosteric muscarinic antagonists are also K+ channel blockers, the use of pertussis toxin did not support the notion that this channel represents the allosteric site coupled to the receptor.