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Partial Deletion of the Nicotinic Cholinergic Receptor 4 or β2 Subunit Genes Changes the Acetylcholine Sensitivity of Receptor-Mediated ⁸⁶Rb⁺ Efflux in Cortex and Thalamus and Alters Relative Expression of 4 and β2 Subunits

Department of Medical Pharmacology and Center of Excellence on Neurodegenerative Diseases, Institute of Neuroscience, Cellular and Molecular Pharmacology, Consiglio Nazionale delle Ricerche, University of Milan, Milan, Italy (C.G., M.M., F.C., A.G.); and Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado (N.M.M., A.C.C., M.J.M.)

4 and β2 nicotinic cholinergic receptor (nAChR) subunits can assemble in heterologous expression systems as pentameric receptors with different subunit stoichiometries that exhibit differential sensitivity to activation by acetylcholine, yielding biphasic concentration-effect curves. nAChR-mediated ⁸⁶Rb⁺ efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by 4β2^*-nAChR, because deletion of either the 4 or β2 subunit reduces response at least 90%. A relatively larger decrease in the component of ⁸⁶Rb⁺ efflux with lower ACh sensitivity occurred with partial deletion of 4 (4^+/-), whereas a larger decrease in the component with higher ACh sensitivity was elicited by partial deletion of β2 (β2^+/-). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [³H]epibatidine binding sites in both regions contained only 4 and β2 subunits. Subsequently, 4 and β2 subunit content in the cortex and thalamus of 4 and β2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of 4 decreased and partial deletion of β2 increased the relative proportion of the 4 subunit in assembled receptors. Although these methods do not allow exact identification of stoichiometry of the subtypes present in wild-type cortex and thalamus, they do demonstrate that cortical and thalamic nAChRs of the 4^+/- and β2^+/- genotypes differ in relative expression of 4 and β2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that 4β2-nAChR with different stoichiometry are expressed in native tissue.

Address correspondence to: Dr. Michael Marks, Institute for Behavioral Genetics, 447 UCB, University of Colorado, Boulder, 1480 30th St., Boulder, CO 80303. E-mail: marksm{at}colorado.edu