Previous work has led to cloning and sequencing five different muscarinic receptor genes(m1-m5). Expression studies have shown that the encoded receptors display differential ligand binding and functional properties. Our primary research goal was to study the precise anatomical distribution of all five muscarinic receptors and to elucidate the molecular mechanisms responsible for their pharmacological heterogeneity. We demonstrated in mRNA mapping and immunocytochemical studies involving the use of newly developed subtype-specific antibodies that each muscarinic receptor shows a distinct pattern of expression throughout the central and peripheral nervous system. Studies with chimeric muscarinic receptors revealed that a short stretch of 17 amino acids is largely responsible for determining the functional specificity of the individual receptor subtypes. On the other hand, multiple receptor domains appear to be involved in conferring subtype-selectivity on a variety of muscarinic antagonists. By studying the pharmacological properties of a series of m3 receptor point mutants, we have identified six conserved threonine and tyrosine residues which are critical for agonist but not for antagonist binding These residues are positioned at a similar level within different transmembrane domains of the muscarinic receptors and may thus define the plane in which muscarinic agonists bind to their target receptors. These studies should lead to a more rational approach toward the development of novel subtype-selective muscarinic drugs which may have a therapeutic potential in the treatment of several CNS disorders associated with dysfunction of cholinergic systems such as Alzheimer's and Parkinson's disease.
