Four affinity chromatography stationary phases have beendeveloped based upon immobilized nicotinic acetylcholinereceptor (nAChR) subtypes, the
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4 nAChRs. The stationary phases were created usingmembranes from cell lines expressing the subtypes andan immobilized artificial membrane stationary phase. Theimmobilized nAChRs were characterized using frontalchromatography with the agonist epibatidine as the marker.The observed binding affinities for the agonists epibatidine, nicotine, and cytisine were consistent with reportedvalues, indicating that the nAChRs retained their abilityto bind agonists. The noncompetitive inhibitors (NCIs) ofthe nAChR (
R)- and (
S)-mecamylamine, phencylcidine,dextromethoprphan, and levomethorphan were also chromatographed on the columns using nonlinear chromatography techniques. The studies were carried out beforeand after exposure of the columns to epibatidine. The NCIretention times increased after exposure to epibtatidineas did the enantioselective separation of mecamylamineand methorphan. The results indicate that the immobilized nAChRs retained their ability to undergo agonist-induced conformational change from the resting to thedesensitized states. The columns provide a unique abilityto study the interactions of NCIs with both of theseconformational states.