Partial Deletion of the Nicotinic Cholinergic Receptor α4 or β2 Subunit Genes Changes the Acetylcholine Sensitivity of Receptor-Mediated⁸⁶Rb⁺Efflux in Cortex and Thalamus and Alters Relative Expression of α4 and β2 Subunits

Abstract

ABSTRACT␣4 and ␤2 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 ϩ efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by ␣4␤2*-nAChR, because deletion of either the ␣4 or ␤2 subunit reduces response at least 90%. A relatively larger decrease in the component of 86 Rb ϩ efflux with lower ACh sensitivity occurred with partial deletion of ␣4 (␣4 ϩ/Ϫ ), whereas a larger decrease in the component with higher ACh sensitivity was elicited by partial deletion of ␤2 (␤2 ϩ/Ϫ ). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [ 3 H]epibatidine binding sites in both regions contained only ␣4 and ␤2 subunits. Subsequently, ␣4 and ␤2 subunit content in the cortex and thalamus of ␣4 and ␤2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of ␣4 decreased and partial deletion of ␤2 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 ϩ/Ϫ and ␤2 ϩ/Ϫ genotypes differ in relative expression of ␣4 and ␤2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that ␣4␤2-nAChR with different stoichiometry are expressed in native tissue.

Structural and functional diversity of native brain neuronal nicotinic receptors

“…subunits, and that this correlates with differences in the functional properties of native nAChRs [50]. Overall, these findings support the conclusion that α4β2 nAChRs with different stoichiometries are expressed in native tissue.…”

“… subunit mRNA is not expressed in the IPn [66] and so nAChRs containing  cannot be synthesised in the IPn. 50 . This underlines the fact that, as has been suggested in the case of the α6β2β3* receptors in the visual and mesostriatal pathways, the  subunit may play a targeting role in the Hb [13,37].…”

α-Conotoxin AuIB Isomers Exhibit Distinct Inhibitory Mechanisms and Differential Sensitivity to Stoichiometry of α3β4 Nicotinic Acetylcholine Receptors

“…The existence of distinct stoichiometries for ␣4␤2 nAChRs heterologously expressed in Xenopus oocytes (22,23) and HEK cells (24) has been established and indications of their presence in vivo have also been reported recently (25). The distinct stoichiometries of ␣4␤2 nAChRs have been shown to have different sensitivities to agonists, antagonists, Zn 2ϩ modulation, as well as Ca 2ϩ permeability and induction by chronic nicotine exposure (22, 24, 26 -28).…”

Rare Human Nicotinic Acetylcholine Receptor α4 Subunit (CHRNA4) Variants Affect Expression and Function of High-Affinity Nicotinic Acetylcholine Receptors

“…Experiments that alter the relative expression of individual subunits (Zwart and Vijverberg 1998;Nelson et al, 2003;Moroni and Bermudez, 2006;Gotti et al, 2008) or use concatenated a4 and b2 subunits to force expression of defined subunit arrangements , have demonstrated that functional parameters of a4b2 nAChRs are largely dependent on the position of individual subunits in the pentamer. Overall, a4 2 b2 3 nAChRs are more sensitive to agonists than a4 3 b2 2 nAChRs , and both stoichiometries of a4b2 can be observed in the Fig.…”