Received for publication January 14, 2008.
Revised March 12, 2008.
Accepted for publication March 12, 2008.

Partial deletion of the nicotinic cholinergic receptor 4 and 2 subunit genes changes the acetylcholine sensitivity of receptor mediated ⁸⁶Rb⁺ efflux in cortex and thalamus and alters relative expression of 4 and 2 subunits

Abstract

4 and 2 nicotinic cholinergic receptor (nAChR) subunits can assemble in heterologuous 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 ACh concentration-response curves. Both phases are mediated primarily by 42*-nAChR, since deletion of either the 4 or 2 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 2 (2^+/-). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [³H]-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. While 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 2^+/- genotypes differ in relative expression of 4 and 2 subunits a result that corresponds to the relative functional changes observed following partial gene deletion. These results strongly suggest that 42-nAChR with different stoichiometry are expressed in native tissue.

Key words: Nicotinic cholinergic, Immunocytochemistry, Receptor binding studies, Knockout