The aims of this study are to define the structural features of allosteric sites on muscarinic acetylcholine receptors. These receptors have become a key model system for the molecular study of allosteric drug action at G protein-coupled receptors. Therefore, the knowledge to be gained from the proposed experiments is expected to have benefits ranging far beyond the advancement of muscarinic pharmacology. Allosteric drugs have inherent advantages of selectivity, efficacy, and safety. These advantages are especially important when the desired effect is to enhance the action of a neurotransmitter at a particular receptor in the brain. Allosteric enhancers are able to preserve the pattern of activity that is dictated by the very complex spatio-temporal motif of synaptic activity; by contrast, directly-acting agonists tend to disrupt these complex motifs that are the hallmark of synaptic activity in the brain. Previous studies in our lab have employed chimeric receptors, point-mutated receptor constructs, and molecular modeling to define a working model of the allosteric binding site. This site is predicted to lie within the extracellular loops and closely adjacent regions of the transmembrane domains of the receptor. An accurate model of the binding site is expected to be a key component of future attempts at rational design of therapeutic allosteric drugs. Therefore, in the requested funding period, we will investigate four different types of allosteric agonists, to refine the existing model so that it more accurately defines the specific sites and modes of binding of allosteric ligands that enhance the actions of acetylcholine. Additionally, we will independently test two major hypotheses of the current model: (1) the location of the binding site; and (2) that a single monomeric receptor represents the appropriate model. For (1), we will use new irreversible allosteric ligands that covalently label the site to unequivocally identify amino acid(s) involved in the binding site. For (2), receptors that have been purified in the monomeric or oligomeric state will allow us to evaluate """"""""atypical"""""""" ligands that are known to bind cooperatively to multiple sites and to determine whether the multiple sites lie within one receptor or across an oligomeric complex. We expect these studies to lead to improvements in the cholinergic pharmacology of disorders of the central nervous system, including Alzheimer's Disease, and to contribute to an understanding of the potential for allosteric modulation of other G protein-coupled receptors. ? ?
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