Zwart Ruud, Broad Lisa M, Xi Qian, Lee Martin, Moroni Mirko, Bermudez Isabel, Sher Emanuele
Eli Lilly and Company Ltd., Lilly Research Centre, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH, United Kingdom.
Eur J Pharmacol. 2006 Jun 6;539(1-2):10-7. doi: 10.1016/j.ejphar.2006.03.077. Epub 2006 Apr 5.
The neuronal nicotinic acetylcholine receptor alpha4 and beta2 subunits expressed in heterologous expression systems assemble into at least two distinct subunit stoichiometries of alpha4beta2 receptor. The (alpha4)2(beta2)3 stoichiometry is about 100-fold more sensitive to acetylcholine than the (alpha4)3(beta2)2 stoichiometry. In order to investigate if agonists in general distinguish high- and low-affinity alpha4beta2 nicotinic acetylcholine receptors, we have expressed human alpha4 and beta2 nicotinic acetylcholine receptor subunits in two different expression systems. The relative amounts of alpha4beta2 nicotinic acetylcholine receptors with high- and low-affinity for acetylcholine were manipulated by (a) injecting the subunit cDNAs at different alpha:beta ratios into Xenopus oocytes and (b) by culturing HEK-293 cells stably expressing alpha4beta2 nicotinic acetylcholine receptors overnight at different temperatures. The sensitivities of the alpha4beta2 nicotinic acetylcholine receptors to the agonists acetylcholine, 5-I A-85380, and TC-2559 were investigated using the voltage-clamp technique on Xenopus oocytes and using a fluorescent imaging plate reader to measure calcium responses from HEK-293 cells. Like acetylcholine, 5-I A-85380 produced biphasic concentration-response curves and the high-affinity component became larger when the cells were manipulated to produce a greater proportion of (alpha4)2(beta2)3 nicotinic acetylcholine receptors. Interestingly, under all circumstances, TC-2559 produced monophasic concentration-response curves. In oocytes injected with alpha4 and beta2 subunits in the 1:1 ratio the maximum effect of TC-2559 was 28% of that of acetylcholine. The EC50 for TC-2559 was not changed when oocytes were manipulated to express exclusively (alpha4)2(beta2)3 nicotinic acetylcholine receptors, however, the maximum effect of TC-2559 was dramatically enhanced. These results suggest that TC-2559 is a selective agonist of the (alpha4)2(beta2)3 nicotinic acetylcholine receptor stoichiometry.
在异源表达系统中表达的神经元烟碱型乙酰胆碱受体α4和β2亚基组装成至少两种不同亚基化学计量比的α4β2受体。（α4）2（β2）3化学计量比对乙酰胆碱的敏感性比（α4）3（β2）2化学计量比高约100倍。为了研究激动剂是否一般能区分高亲和力和低亲和力的α4β2烟碱型乙酰胆碱受体，我们在两种不同的表达系统中表达了人α4和β2烟碱型乙酰胆碱受体亚基。通过以下方式操纵对乙酰胆碱具有高亲和力和低亲和力的α4β2烟碱型乙酰胆碱受体的相对量：（a）以不同的α:β比例将亚基cDNA注射到非洲爪蟾卵母细胞中；（b）将稳定表达α4β2烟碱型乙酰胆碱受体的HEK-293细胞在不同温度下培养过夜。使用电压钳技术在非洲爪蟾卵母细胞上，并使用荧光成像板读数器测量HEK-293细胞的钙反应，研究α4β2烟碱型乙酰胆碱受体对激动剂乙酰胆碱、5-I A-85380和TC-2559的敏感性。与乙酰胆碱一样，5-I A-85380产生双相浓度-反应曲线，当细胞被操纵以产生更大比例的（α4）2（β2）3烟碱型乙酰胆碱受体时，高亲和力成分变得更大。有趣的是，在所有情况下，TC-2559产生单相浓度-反应曲线。在以1:1比例注射α4和β2亚基的卵母细胞中，TC-2559的最大效应是乙酰胆碱的28%。当卵母细胞被操纵以专门表达（α4）2（β2）3烟碱型乙酰胆碱受体时，TC-2559的半数有效浓度（EC50）没有变化，然而，TC-2559的最大效应显著增强。这些结果表明，TC-2559是（α4）2（β2）3烟碱型乙酰胆碱受体化学计量比的选择性激动剂。
