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 α3β4 nicotinic acetylcholine receptors (α3β4-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 α3β4-nAChRs. 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−α3β4-nAChR complexes. The k_a values determined in this study ranged from 19.7 to 10.5 μM^-1sec^-1, with a relative order of mecamylamine > dextromethorphan ≥ ketamine > bupropion. The k_d 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. The data from this study indicate that the immobilized α3β4-nAChR column and nonlinear chromatography can be used in the study of NCIs at the α3β4-nAChR.