[¹²⁵I]AT-1012, a new high affinity radioligand for the α3β4 nicotinic acetylcholine receptors.
[¹²⁵I]AT-1012, a new high affinity radioligand for the α3β4 nicotinic acetylcholine receptors.
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[¤âµI]AT-1012，一种针对α3β4 烟碱乙酰胆碱受体的新型高亲和力放射性配体。
DOI：
10.1016/j.neuropharm.2013.09.023
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发表时间：
2014
影响因子：
4.7
通讯作者：
Zaveri,NurulainT
中科院分区：
文献类型：
--
作者：
Wu,Jinhua;Perry,DavidC;Bupp,JamesE;Jiang,Faming;Polgar,WillmaE;Toll,Lawrence;Zaveri,NurulainT
最近的遗传和药理学研究表明烟碱乙酰胆碱受体 (nAChR) 的 α3、β4 和 α5 亚基与尼古丁和其他滥用药物以及尼古丁戒断有关。 α3β4* nAChR 亚型已被证明与 α5 或 β3 nAChR 亚基共同组装，主要存在于自主神经节和选定的大脑区域中。研究 α3β4 nAChR 一直很困难，因为没有选择性的非肽配体可用于独立检查其药理学。我们最近报道了高亲和力、选择性小分子 α3β4 nAChR 配体 AT-1012 的 [125I] 放射性标记类似物的合成。我们在这里报告了这种放射性配体在受体结合中的体外表征以及针对 α3β4* nAChR 的体外放射自显影研究。 [125I]AT-1012 的结合特征在于转染至 HEK 细胞的大鼠 α3β4 和 α4β2 nAChR 以及 HEK 细胞中的人 α3β4α5 nAChR。 [125I]AT-1012 对大鼠 α3β4 nAChR 的结合亲和力为 1.4 nM，aBmax 为 10.3 pmol/mg 蛋白质，与使用 [3H]epibatidine 测定的未标记 AT-1012 相似。饱和等温线表明 [125I]AT-1012 与 α3β4 nAChR 上的单个位点结合。 [125I]AT-1012 在转染至 HEK 细胞的人 α3β4α5 nAChR 上也观察到类似的高结合亲和力。 [125I]AT-1012 不以高亲和力与 α4β2 nAChR 转染的 HEK 细胞的膜结合。 [3H]epibatidine 的结合研究进一步证实，AT-1012 对 α3β4 的结合选择性是 α4β2 nAChR 的 100 倍以上。使用 [125I]AT-1012 作为放射性配体测定已知 nAChR 化合物的 Ki 值与使用 [3H]epibatidine 获得的值相当。 [125I]AT-1012 还用于在体外使用放射自显影技术标记大鼠脑切片中的 α3β4 nAChR，结果显示大脑区域中放射性配体的高度局部化结合与 α3β4 nAChR 的谨慎定位一致。我们证明，[125I]AT-1012 是在存在其他 nAChR 亚型的情况下标记 α3β4 nAChR 的出色工具。
Recent genetic and pharmacological studies have implicated the α3, β4 and α5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [125I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [125I]AT-1012 was characterized at the rat α3β4 and α4β2 nAChR transfected into HEK cells, as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [125I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with aBmaxof 10.3 pmol/mg protein, similar to what was determined for unlabeled AT-1012 using [3H]epibatidine. Saturation isotherms suggested that [125I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [125I]AT-1012 at the human α3β4α5 nAChR transfected into HEK cells. [125I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells. Binding studies with [3H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for α3β4 over α4β2 nAChR.Kivalues determined for known nAChR compounds using [125I]AT-1012 as radioligand were comparable to those obtained with [3H]epibatidine. [125I]AT-1012 was also used to label α3β4 nAChR in rat brain slices in vitro using autoradiography, which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We demonstrate that [125I]AT-1012 is an excellent tool for labeling the α3β4 nAChR in the presence of other nAChR subtypes.