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 (C₇/3-phth) and the orthosteric antagonist [³H]N-methylscopolamine ([³H]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 C₇/3-phth 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 M₂ receptor and the lowest affinity for the unoccupied M₅ receptor, the latter being approximately 100-fold lower. In contrast, the highest degree of negative cooperativity was observed at the M₅ receptor, whereas lowest negative cooperativity was found at the M₁ and M₄ receptors. Non-equilibrium dissociation kinetic studies also confirmed the allosteric properties of C₇/3-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 C₇/3-phth is a potent allosteric modulator at mAChRs, particularly the M₂ subtype, and also highlight the effects of cooperativity on apparent drug-receptor subtype selectivity. Copyright (C) 1999 Elsevier Science Inc.