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

The present study investigated the interaction between the muscarinic acety lcholine receptor (mAChR) allosteric modulator heptane-1,7-bis-(dimethyl-3' -phthalimidopropyl) ammonium bromide (C-7/3phth) and the orthosteric antago nist [H-3]N-methylscopolamine ([H-3]NMS) at the five cloned human mAChRs ex pressed 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 1 00-fold lower. In contrast, the highest degree of negative cooperativity wa s observed at the M-5 receptor, whereas lowest negative cooperativity was f ound at the M-1 and M-4 receptors. Non equilibrium dissociation kinetic stu dies also confirmed the allosteric properties of C-7/3-phth at all five mAC hRs and yielded independent estimates of the modulator affinity for the occ upied receptor. The latter estimates showed good agreement with those calcu lated using parameter Values determined from the equilibrium experiments. T he present results extend previous findings that C-7/3-phth is a potent all osteric modulator at mAChRs, particularly the M-2 subtype, and also highlig ht the effects of cooperativity on apparent drug-receptor subtype selectivi ty. BIOCHEM PHARMACOL 51;2:171-179, 1999. (C) 1998 Elsevier Science Inc.