Characterization of the subtype selectivity of the allosteric modulator heptane-1,7-bis-(dimethyl-3′-phthalimidopropyl) ammonium bromide (C7/3-phth) at cloned muscarinic acetylcholine receptors

The present study investigated the interaction between the muscarinic acetylcholine receptor (mAChR) allosteric modulator heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl) ammonium bromide (C-7/3phth) and the orthosteric antagonist [H-3]N-methylscopolamine ([H-3]NMS) at the five cloned human mAChRs expressed in Chinese hamster ovary cells. Equilibrium binding studies, using two different concentrations of radioligand, showed the interaction between C-7/3phth and [3H]NMS to be characterized by different degrees of negative cooperativity, depending on the receptor subtype. The modulator exhibited the highest affinity (85 nM) for the unoccupied M-2 receptor and the lowest affinity for the unoccupied M-5 receptor, the latter being approximately 100-fold lower. In contrast, the highest degree of negative cooperativity was observed at the M-5 receptor, whereas lowest negative cooperativity was found at the M-1 and M-4 receptors. Non equilibrium dissociation kinetic studies also confirmed the allosteric properties of C-7/3-phth at all five mAChRs and yielded independent estimates of the modulator affinity for the occupied receptor. The latter estimates showed good agreement with those calculated using parameter Values determined from the equilibrium experiments. The present results extend previous findings that C-7/3-phth is a potent allosteric modulator at mAChRs, particularly the M-2 subtype, and also highlight the effects of cooperativity on apparent drug-receptor subtype selectivity. BIOCHEM PHARMACOL 51;2:171-179, 1999. (C) 1998 Elsevier Science Inc.