Pereira E F, Reinhardt-Maelicke S, Schrattenholz A, Maelicke A, Albuquerque E X
Department of Pharmacology and Experimental Therapeutics, University of Maryland Medical School, Baltimore.
J Pharmacol Exp Ther. 1993 Jun;265(3):1474-91.
Electrophysiological and biochemical techniques were used to demonstrate that alpha-bungarotoxin-, methyllycaconitine-sensitive neuronal nicotinic acetylcholine receptors (nAChRs) can be activated via a novel agonist site(s). The residue proposed to be essential to this site is the amino acid Lys-125 of the receptor alpha subunit. In outside-out patches excised from cultured hippocampal neurons, physostigmine (PHY) and 1-methyl-PHY activated single channels whose main conductances were 46 and 23 pS. This action was insensitive to DL-2-amino-5-phosphonovaleric acid, atropine, tetrodotoxin and competitive nicotinic antagonists, but blocked by benzoquinonium or FK1, a nAChR-specific antibody raised against rat muscle nAChR alpha subunits that binds to the novel site. Indirect immunofluorescence staining demonstrated that FK1 binds to the hippocampal neurons, as would be expected based on the high degree of homology among nAChR alpha subunits from diverse sources in the region surrounding Lys-125. PHY prevented the binding of FK1, thus supporting that FK1 is a specific probe for the PHY site. High-affinity sites (KD approximately 35 nM) for 1-methyl-PHY were identified in hippocampal neurons. Similar to PHY, benzoquinonium (0.1-10 microM) and galanthamine (1-10 microM) activated nicotinic single channels. The agonists benzoquinonium and PHY were also open-channel blockers at the neuronal nAChRs, whereas galanthamine was predominantly a desensitizing agent. In mouse fibroblasts transfected with cDNAs of alpha 4 and beta 2 neuronal nAChR subunits, PHY also activated single channels that were blocked by FK1. In these cells, dihydro-beta-erythroidine blocked single channels activated by (+)-anatoxin-a and did not affect those opened by PHY. Thus, the present results suggest that the novel agonist site located on the receptor alpha subunit is a common feature of neuronal nAChRs.
采用电生理和生化技术证明，α-银环蛇毒素、甲基lycaconitine敏感的神经元烟碱型乙酰胆碱受体（nAChRs）可通过一个新的激动剂位点被激活。被认为对该位点至关重要的残基是受体α亚基的氨基酸Lys-125。在从培养的海马神经元上切下的外向型膜片中，毒扁豆碱（PHY）和1-甲基-PHY激活了主要电导分别为46和23 pS的单通道。这种作用对DL-2-氨基-5-膦酸戊酸、阿托品、河豚毒素和竞争性烟碱拮抗剂不敏感，但被苯甲喹铵或FK1阻断，FK1是一种针对大鼠肌肉nAChRα亚基产生的nAChR特异性抗体，可结合该新位点。间接免疫荧光染色表明，FK1与海马神经元结合，这与基于不同来源的nAChRα亚基在Lys-125周围区域的高度同源性所预期的情况一致。PHY可阻止FK1的结合，从而支持FK1是PHY位点的特异性探针这一观点。在海马神经元中鉴定出了1-甲基-PHY的高亲和力位点（KD约为35 nM）。与PHY类似，苯甲喹铵（0.1 - 10 μM）和加兰他敏（1 - 10 μM）激活了烟碱型单通道。激动剂苯甲喹铵和PHY也是神经元nAChRs的开放通道阻滞剂，而加兰他敏主要是一种脱敏剂。在转染了α4和β2神经元nAChR亚基cDNA的小鼠成纤维细胞中，PHY也激活了被FK1阻断的单通道。在这些细胞中，二氢-β-刺桐啶阻断了由（+）-anatoxin-a激活的单通道，而不影响由PHY打开的通道。因此，目前的结果表明，位于受体α亚基上的新激动剂位点是神经元nAChRs的一个共同特征。
