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Alpha4 and beta2 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 (86)Rb(+) efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by alpha4beta2(*)-nAChR, because deletion of either the alpha4 or beta2 subunit reduces response at least 90%. A relatively larger decrease in the component of (86)Rb(+) efflux with lower ACh sensitivity occurred with partial deletion of alpha4 (alpha4(+/-)), whereas a larger decrease in the component with higher ACh sensitivity was elicited by partial deletion of beta2 (beta2(+/-)). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [(3)H]epibatidine binding sites in both regions contained only alpha4 and beta2 subunits. Subsequently, alpha4 and beta2 subunit content in the cortex and thalamus of alpha4 and beta2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of alpha4 decreased and partial deletion of beta2 increased the relative proportion of the alpha4 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 alpha4(+/-) and beta2(+/-) genotypes differ in relative expression of alpha4 and beta2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that alpha4beta2-nAChR with different stoichiometry are expressed in native tissue.

mRNAs for the neuronal nicotinic acetylcholine receptor (nAChR) alpha6 and beta3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that alpha6- or beta3-dependent nAChRs bind alpha-conotoxin MII (alpha-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of beta3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that beta3-null mutation selectively reduced striatal alpha6* nAChR expression by 76% versus beta3(+/+) control. Parallel experiments showed a smaller reduction in both midbrain alpha3* and alpha6* nAChRs (34 and 42% versus beta3(+/+) control, respectively). Sedimentation coefficient determinations indicated that residual alpha6* nAChRs in beta3(-/-) striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified beta3* nAChRs demonstrated that almost all wild-type striatal beta3* nAChRs also contain alpha4, alpha6, and beta2 subunits, although a small population of non-beta3 alpha6* nAChRs is also expressed. beta3 subunit incorporation seemed to increase alpha4 participation in alpha6beta2* complexes. (125)I-Epibatidine competition binding studies showed that the alpha-CtxMII affinity of alpha6* nAChRs from the striata of beta3(-/-) mice was similar to those isolated from beta3(+/+) animals. Together, the results of these experiments show that the beta3 subunit is important for the correct assembly, stability and/or transport of alpha6* nAChRs in dopaminergic neurons and influences their subunit composition. However, beta3 subunit expression is not essential for the expression of alpha6*, high-affinity alpha-CtxMII binding nAChRs.