Partial Deletion of the Nicotinic Cholinergic Receptor α4 or β2 Subunit Genes Changes the Acetylcholine Sensitivity of Receptor-Mediated 86Rb+ Efflux in Cortex and Thalamus and Alters Relative Expression of α4 and β2 Subunits

Authors:
  • MMMilena Moretti
    Milena Moretti
  • NMNatalie M. Meinerz
    Natalie M. Meinerz
  • FCFrancesco Clementi
    Francesco Clementi
  • AGAnnalisa Gaimarri
    Annalisa Gaimarri
  • ACAllan C. Collins
    Allan C. Collins
  • MMMichael J. Marks
    Michael J. Marks
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Abstract
α4 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 α4β2*-nAChR, because deletion of either the α4 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 α4 (α4+/-), 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 α4 and β2 subunits. Subsequently, α4 and β2 subunit content in the cortex and thalamus of α4 and β2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of α4 decreased and partial deletion of β2 increased the relative proportion of the α4 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 α4+/- and β2+/- genotypes differ in relative expression of α4 and β2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that α4β2-nAChR with different stoichiometry are expressed in native tissue.

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