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)/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 C(7)/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 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 M2 subtype, and also highlight the effects of cooperativity on apparent drug-receptor subtype selectivity.
Christopoulos
A
A
Division of Neuroscience Research in Psychiatry, University of Minnesota Medical School, Minneapolis 55455, USA.
Sorman
J L
JL
Mitchelson
F
F
El-Fakahany
E E
EE
D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D016015:Logistic Models; D015394:Molecular Structure; D010797:Phthalimides / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000378:metabolism*
D010797:Phthalimides; D000644:Quaternary Ammonium Compounds; D011976:Receptors, Muscarinic; C116689:heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl)ammonium bromide