A SUBTYPE OF NICOTINIC CHOLINERGIC RECEPTOR IN RAT-BRAIN IS COMPOSED OF ALPHA-4-SUBUNIT AND BETA-2-SUBUNIT AND IS UP-REGULATED BY CHRONIC NICOTINE TREATMENT

The subunit composition and pharmacological regulation of rat neuronal nicotinic cholinergic receptors were assessed. Specific immunoprecipi tation was determined in solubilized rat brain homogenates using [H-3] cytisine, a high affinity agonist at nicotinic receptors, in conjuncti on with polyclonal antisera generated against nonhomologous domains of the various subunits comprising this receptor class. In all brain reg ions tested, only antisera generated against the alpha-4 and beta-2 su bunits were able to immunoprecipitate specifically receptors labeled b y [H-3]cytisine. Thus, these sera were further characterized in order to validate and optimize their use in the immunoprecipitation protocol . Preincubation of solubilized receptors from rat forebrain with antis era generated against the alpha-2, alpha-3, alpha-5, beta-3, or beta-4 subunits did not decrease the amount of precipitable alpha-4 or beta- 2 subunit. On the other hand, when either anti-alpha-4 or anti-beta-2 serum was used to immunoprecipitate solubilized receptors from rat for ebrain, the supernatants contained little if any remaining receptors t hat could be specifically precipitated by either antibody. Because the se antisera do not cross-react, the data indicate that alpha-4 and bet a-2 subunits are associated with each other in at least one neuronal n icotinic receptor subtype that has high affinity for agonists. Moreove r, these results imply that all alpha-4 subunits that are labeled by [ H-3]cytisine are coupled to beta-2 subunits. We also present evidence that the alpha-4/beta-2 subtype characterized in this report is signif icantly increased in the cortex of rats chronically treated with nicot ine.