Muscarinic acetylcholine receptors belong to the very numerous class of heptahelical-receptors, but are unusual for this class in that they possess multiple extracellular binding sites. The present studies are designed to better understand muscarinic allosteric interactions from pharmacological and structural viewpoints. Dysfunction of muscarinic cholinergic systems in the brain contributes to the deficits associated with many illnesses, including Alzheimer's Disease. However, treatment with directly-acting muscarinic cholinergic drugs has not proven to be very beneficial to date. This may relate in part to the adverse effects and lack of selectivity of currently available muscarinic drugs. There are theoretical reasons why allosteric drugs can have inherent advantages of selectivity, efficacy, and safety over directly-acting (competitive) agents. For example, the allosterically-acting benzodiazopines enhance the action of the neurotransmitter GABA and are very effective and safe, even though directly-acting GABA agonists and reuptake inhibitors have no clinical use whatsoever. Although it has been known for some time that muscarinic receptors are susceptible to allosteric regulation only very recently have allosteric ligands been discovered that are positively cooperative with the endogenous agonist, acetylcholine. These first-generation ligands have relatively low affinities, but they demonstrate a potential for the development of drugs that are subtype-selective in terms of cooperativity, as well as affinity.
The aims of this application are to apply approaches that we have developed in past studies to better understand the interactions between the receptors and allosteric ligands, especially these new ligands. These tools include: pharmacological model testing, to determine whether ligands interact solely with an allosteric site and whether different allosteric ligands act at a common site; and, molecular genetic techniques, to determine which structural features of the receptors are responsible for the subtype-selective binding and cooperativity of allosteric ligands. The hypothesis behind all of these studies is that subtype-selective ligands interact with specific residues that can be located through the use of mutagenic and chimeric studies. Once candidate residues have been identified by this process, the hypothesis that they are in fact involved in intimate contact with the ligand will be tested by making reciprocal mutations on different subtypes, by investigating the interaction of modified ligands with wild-type and modified receptors, and by inserting specific epitopes into non-muscarinic receptors. We expect that the results of these studies will benefit cholinergic pharmacology and also contribute to an understanding of the potential for allosteric modulation of other heptahelical receptors.
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