Zhang Y, Xiao Y, Kellar K J, Wainer I W
Department of Pharmacology, Georgetown University School of Medicine, Washington, DC, 20007, USA.
Anal Biochem. 1998 Nov 1;264(1):22-5. doi: 10.1006/abio.1998.2828.
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.
烟碱型乙酰胆碱受体（nAChR）是配体门控离子通道，介导神经系统中的烟碱型胆碱能传递。一种由α3和β4亚基组成的nAChR亚型（α3/β4亚型），从稳定转染的KXα3β4R2细胞系制备而来，已固定在固定化人工膜（IAM）液相色谱（LC）固定相的磷脂单层中。每克IAM颗粒固定约60毫克蛋白质。所得固定相用于快速在线色谱测定药物与nAChR的结合亲和力。通过前沿色谱法测定相对结合亲和力，结果为（±）-依博加因（Kd：0.27±0.05 nM）＞A85380（Kd：17.2±0.5 nM）＞（-）-尼古丁（Kd：88±33 nM）＞卡巴胆碱（Kd：1280±30 nM）＞阿托品（Kd：14570±2600 nM）。这些结果与使用膜匀浆进行结合测定获得的亲和力排序一致。固定化受体LC固定相稳定且可重复。这种方法为生产各种固定化受体LC固定相开辟了可能性，这些固定相可用于直接测定药物-受体结合相互作用以及快速在线筛选药物候选物的组合库。
