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CM Flores, SW Rogers, LA Pabreza, BB Wolfe and KJ Kellar
Department of Pharmacology, Georgetown University Medical Center, Washington, DC 20007.
The subunit composition and pharmacological regulation of rat neuronal nicotinic cholinergic receptors were assessed. Specific immunoprecipitation was determined in solubilized rat brain homogenates using [3H]cytisine, a high affinity agonist at nicotinic receptors, in conjunction with polyclonal antisera generated against nonhomologous domains of the various subunits comprising this receptor class. In all brain regions tested, only antisera generated against the alpha 4 and beta 2 subunits were able to immunoprecipitate specifically receptors labeled by [3H]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 antisera 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 forebrain, the supernatants contained little if any remaining receptors that could be specifically precipitated by either antibody. Because these antisera do not cross-react, the data indicate that alpha 4 and beta 2 subunits are associated with each other in at least one neuronal nicotinic receptor subtype that has high affinity for agonists. Moreover, these results imply that all alpha 4 subunits that are labeled by [3H]cystisine are coupled to beta 2 subunits. We also present evidence that the alpha 4/beta 2 subtype characterized in this report is significantly increased in the cortex of rats chronically treated with nicotine.
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