The cDNAs encoding five subtypes of muscarinic cholinergic receptors have been isolated. Four (m1-m4) are widely, but differentially, expressed in the brain and in peripheral neurons and end organs. Four subtypes of muscarinic receptors have been described pharmacologically and are termed M1-M4. The pharmacologically defined receptors correspond only loosely to the molecularly defined receptors and, especially in the brain where concentrations of drugs are difficult to determine, it is not always clear which subtype of muscarinic receptor mediates a given physiological or behavioral response. Thus, experiments are designed, using a gene knockout strategy, to more clearly define the role of specific subtypes of muscarinic receptor in several in vivo responses to muscarinic receptor stimulation. These experiments should help determine the involvement of the m1 and m4 receptors in such diverse responses as temperature regulation, salivation, tremor, seizure activity, striatal function, circadian rhythm, and memory. In addition, different subtypes appear to couple to distinct G proteins and even subtypes such as m2 and m4 which both inhibit adenylyl cyclase appear to have differential coupling to subtypes of Gi. Chimeric m2/m4 receptors will be generated to determine which portions of each receptor are involved in the selective coupling to the Gi subtypes. Experiments by many investigators have demonstrated that m2 and m3 receptors are often presynaptically or postsynaptically localized, respectively. The applicant has explored a model system of protein trafficking, the MDCK cells, in which it appears that m2 receptors are sent to the apical surface while the m3 receptor is sent to the basolateral surface. It is proposed that this differential trafficking in the MDCK cells is representative of what happens in neurons when one receptor subtype (e.g. m2) is sent down the axon while another (e.g. m3) is sent to the dendrites. Experiments are proposed to test this hypothesis and to determine the structural requirements in each receptor that determine its targeting destination.
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