Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus
Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus
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DOI：
10.1073/pnas.0706773104
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发表时间：
2007-09-25
影响因子：
11.1
通讯作者：
Miledi, Ricardo
中科院分区：
文献类型：
--
作者：
Limon, Agenor;Reyes-Ruiz, Jorge Mauricio;Miledi, Ricardo
标记受体有助于神经递质受体定位的解剖学可视化，但这一过程会改变其功能特性。我们评价了谷氨酸受体3(GluR3)、α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)受体(WT GluR3)和两种将GFP标记到氨基末端(GFP-GluR3)或羧基末端(GluR3-GFP)的受体的分布和性质。虽然非洲爪哇卵母细胞由于内部结构(可能的受体产生、储存或再循环的部位)的定位，在植物半球的荧光较强，但受体插入质膜的过程与动物半球的极化有关。注射GluR3-GFP RNA的卵母细胞的荧光强度约为注射GFP-GluR3 RNA的卵母细胞的两倍。因此，GluR3-GFP卵母细胞产生的红藻氨酸电流比GFP-GluR3卵母细胞大，EC50值相近。在GluR3-GFP卵母细胞中，谷氨酸或AMPA与环噻嗪联合诱导的电流也更大。谷氨酸对红藻氨酸电流的幅度比不同，谷氨酸比WT或GFP-GluR3受体更有效地激活GluR3-GFP。这种模式与GluR3-GFP受体产生的谷氨酸诱导电流的较慢衰减有关。当GFP被标记到氨基末端时，这些变化没有观察到，这些受体的行为类似于WT。6-nitro-7-sulfamoylbenzo[f]quinoxaline-2，3-dione(NBQX)和6-氰基-7-硝基-2，3-二酮(CNQX)对标记受体的拮抗作用均不改变。我们的结论是，GFP是一个有用和方便的标记来可视化这些蛋白质。然而，在评估这些受体蛋白所起的作用时，必须考虑到不同的标签插入位置对受体特性的影响。
Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) alpha-amino-3-hydroxy5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-incluced currents than GFP-GluR3 oocytes, with similar EC50 values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate- to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins.