Displacement and nonlinear chromatographic techniques in the investigation of interaction of noncompetitive inhibitors with an immobilized α3β4 nicotinic acetylcholine receptor liquid chromatographic stationary phase

A liquid chromatographic column containing immobilized alpha3beta4 nicotinic acetylcholine receptors (alpha3beta4-nAChRs) has been used to determine the equilibrium association constants (K-a), desorption rate constants (k(d)), and adsorption rate constants (k(a)) for the noncompetitive inhibitors: mecamylamine, ketamine, bupropion, and dextromethorphan. Displacement chromatography, with mecamylamine as the displacer, was used to verify that the four compounds bound to the same site on the immobilized alpha3beta4-nChRs. Nonlinear chromatographic techniques were then utilized to calculate the K-a, k(a), and k(d) values associated with the formation of the noncompetitive inhibitor-alpha3beta4-nAChR complexes. The k(a) values determined in this study ranged from 19.7 to 10.5 muM(-1)sec(-1), with a relative order of mecamylamine > dextromethorphan greater than or equal to ketamine > bupropion. The kd values determined in this study indicated that dextromethorphan-induced inhibition should produce a longer recovery time than the other three NCIs. This was consistent with results from a previous in vitro study. ne data from this study indicate that the immobilized alpha3beta4-nAChR column and nonlinear chromatography can be used in the study of NCIs at the alpha3beta4-nAChR.