Gene targeting demonstrates that α4 nicotinic acetylcholine receptor subunits contribute to expression of diverse [3H]epibatidine binding sites and components of biphasic 86Rb+ efflux with high and low sensitivity to stimulation by acetylcholine
Gene targeting demonstrates that α4 nicotinic acetylcholine receptor subunits contribute to expression of diverse [3H]epibatidine binding sites and components of biphasic 86Rb+ efflux with high and low sensitivity to stimulation by acetylcholine
复制标题
DOI：
10.1016/j.neuropharm.2007.05.021
复制
发表时间：
2007-09-01
影响因子：
4.7
通讯作者：
Collins, Allan C.
中科院分区：
文献类型：
--
作者：
Marks, Michael J.;Meinerz, Natalie M.;Collins, Allan C.
[H-3]Epibatidine 以较高的亲和力（K-D 约为 0.02 nM）和较低的亲和力（K-D 约为 7 nM）与小鼠大脑中的 nAChR 亚型结合。可以通过选定的激动剂和拮抗剂的抑制进一步细分。这些子集受到 α 7、β 2 或 β 4 亚基定向删除的不同影响。大多数（但不是全部）较高和较低亲和力结合位点需要 β 2（Marks, M.J.、Whiteaker, P.、Collins, A.C., 2006。删除 α 7、β 2 或 β 4 烟碱受体亚基基因可鉴定对 [H-3]epibatidine 亲和力相对较低的高表达亚型。Mol, Pharmacol. 70, 947-959）。此处报告了功能性 α4 基因缺失的影响。 α 4 的删除实际上消除了金雀花碱敏感性、高亲和力的[H-3]epibatidine 结合，就像删除β 2 一样，证实这些位点是α 4 β 2*-nAChR。抗金雀花碱、高亲和力的 [H-3]epibatidine 结合位点多种多样，其中一些位点需要 α4 表达。较低亲和力[3 Hjepibatidine 结合位点也是异质的，可以细分为 α-银环蛇毒素敏感和耐药成分。删除α4并不会影响α-银环蛇毒素敏感成分，但显着减少了m-银环蛇毒素抗性成分。这种效果与 beta 2 缺失的效果相似，但不完全相同。这提供了第一个证据，表明大脑中低亲和力的皮巴替丁结合位点需要α4亚基的表达。使用Rb-86+外排测定法测量了α4基因靶向对受体功能的影响。 ACh 刺激的 Rb-86(+) 流出的浓度效应曲线是双相的（EC50 值 = 3.3 μM 和 300 μM）。在整个大脑和所检测的 14 个大脑区域的大部分区域中，针对 α4 在两个阶段均产生了显着的基因剂量依赖性减少。这些效应与删除 β 2 后的效应非常相似，因此，β 4 β 2*-nAChR 介导 ACh 刺激的 Rb-86(+) 流出的两个阶段的很大一部分。 (C) 2007 Elsevier Ltd. 保留所有权利。
[H-3]Epibatidine binds to nAChR subtypes in mouse brain with higher (K-D approximate to 0.02 nM) and lower affinity (K-D approximate to 7 nM). which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of alpha 7, beta 2 or beta 4 subunits. Most, but not all, higher and lower affinity binding sites require beta 2 (Marks, M.J., Whiteaker, P., Collins, A.C., 2006. Deletion of the alpha 7, beta 2 or beta 4 nicotinic receptor subunit genes identifies highly expressed subtypes with relatively low affinity for [H-3]epibatidine. Mol, Pharmacol. 70, 947-959). Effects of functional alpha 4 gene deletion are reported here. Deletion of alpha 4 virtually eliminated cytisine-sensitive, higher-affinity [H-3]epibatidine binding as did beta 2 deletion, confirming that these sites are alpha 4 beta 2*-nAChR. Cytisine-resistant, higher-affinity [H-3]epibatidine binding sites are diverse and some of these sites require alpha 4 expression. Lower affinity [3 Hjepibatidine binding sites are also heterogeneous and can be subdivided into alpha-bungarotoxin-sensitive and -resistant components. Deleting alpha 4 did not affect the alpha-bungarotoxin-sensitive component, but markedly reduced the m-bungarotoxin-resistant component. This effect was similar, but not quite identical, to the effect of beta 2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of alpha 4 subunits, The effects of alpha 4 gene targeting on receptor function were measured using a Rb-86+ efflux assay. Concentration-effect curves for ACh-stimulated Rb-86(+) efflux are biphasic (EC50 values = 3.3 mu M and 300 mu M). Targeting alpha 4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of beta 2, Thus, beta 4 beta 2*-nAChRs mediate a significant fraction of both phases of ACh stimulated Rb-86(+) efflux. (C) 2007 Elsevier Ltd. All rights reserved.