Immobilized nicotinic receptor stationary phase for on-line liquid chromatographic determination of drug-receptor affinities.

Nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels which mediate nicotinic cholinergic transmission in the nervous system. A nAChR subtype composed of alpha3 and beta4 subunits (alpha3/beta4 subtype), prepared from a stably transfected KXalpha3beta4R2 cell line, has been immobilized in the phospholipid monolayer of an immobilized artificial membrane (IAM) liquid chromatography (LC) stationary phase. Approximately 60 mg of protein was immobilized per gram IAM particles. The resulting phase was used for the rapid on-line chromatographic determination of drug binding affinities to nAChRs. Relative binding affinities were determined by frontal chromatography for (+/-)-epibatidine (Kd: 0.27 +/- 0.05 nM) > A85380 (Kd: 17.2 +/- 0.5 nM) > (-)-nicotine (Kd: 88 +/- 33 nM) > carbachol (Kd: 1280 +/- 30 nM) > atropine (Kd: 14,570 +/- 2600 nM). These results are consistent with the affinity rank order obtained from binding assays using membrane homogenates. The immobilized receptor LC stationary phase was stable and reproducible. This approach opens up the possibility of the production of a variety of immobilized receptor LC stationary phases which may be used for direct determination of drug-receptor binding interactions and for the rapid on-line screening of combinatorial pools for drug candidates.