Abstract
This study investigated the reciprocal cross-interactions between two distinct allosteric sites on the M4 muscarinic acetylcholine receptor (mAChR) in the absence or presence of different orthosteric ligands. Initial studies revealed that two novel benzimidazole allosteric modulators, 17-β-hydroxy-17-α-ethy nyl-delta(4)-androstano[3,2-b]pyrimido[1,2-a]benzimidazole (WIN 62,577) and 17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-b]pyrimido[1,2-a]benzimidazole (WIN 51,708), exhibited different degrees of positive, negative, or close-to-neutral cooperativity with the orthosteric site on M1 or M4 mAChRs, depending on the chemical nature of the orthosteric radioligand that was used [[3H]N-methylscopolamine ([3H]NMS) versus [3H]quinuclidinylbenzilate ([3H]QNB)]. The largest window for observing an effect (negative cooperativity) was noted for the combination of WIN 62,577 and [3H]QNB at the M4 mAChR. Experiments involving the combination of these two ligands with unlabeled agonists [acetylcholine, 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), or xanomeline] revealed low degrees of negative cooperativity between WIN 62,577 and each agonist, whereas stronger negative cooperativity was observed against atropine. It is interesting that when these experiments were repeated using the prototypical modulators heptane-1,7-bis-(dimethyl-3′-phthalimidopropyl)-ammonium bromide (C7/3-phth), alcuronium, or brucine (which act at a separate allosteric site), WIN 62,577 exhibited negative cooperativity with each modulator when the orthosteric site was unoccupied, but this switched to neutral cooperativity when the receptor was occupied by [3H]QNB. Dissociation kinetic experiments using [3H]NMS and combination of C7/3-phth with WIN 62,577 also provided evidence for neutral cooperativity between the two allosteric sites when the orthosteric site is occupied. Together, these results provide insight into the nature of the interaction between two distinct allosteric sites on the M4 mAChR and how this interaction is perturbed upon occupancy of the orthosteric site.
- Received March 20, 2006.
- Accepted May 18, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|