Expression of Glycine-Activated Diheteromeric NR1/NR3 Receptors in Human Embryonic Kidney 293 Cells Is NR1 Splice Variant-Dependent
Expression of Glycine-Activated Diheteromeric NR1/NR3 Receptors in Human Embryonic Kidney 293 Cells Is NR1 Splice Variant-Dependent
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DOI：
10.1124/jpet.109.158493
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发表时间：
2009-12-01
影响因子：
3.5
通讯作者：
Woodward, John J.
中科院分区：
文献类型：
--
作者：
Smothers, C. Thetford;Woodward, John J.
在卵母细胞中，甘氨酸激活由 N-甲基-D-天冬氨酸 (NMDA) NR1 和 NR3 亚基的二异体组合形成的受体。相反，哺乳动物细胞中的功能性受体需要同时表达 NR1 以及 NR3A 和 NR3B 亚基。在体内，NR3A 和 NR3B 亚基表现出差异表达模式，因此可能不会天然形成三异体受体。在这项研究中，我们检查了 NR1 剪接变体是否在允许哺乳动物细胞中功能性二异体受体组装方面发挥作用。在共表达 NR3A 或 NR3B 以及 NR1-1a 剪接变体的人胚胎肾 293 细胞中发现很少的电流。然而，在转染 NR3（A 或 B）和 NR1-2a、NR1-3a 或 NR1-4a 的细胞中产生了强大的甘氨酸激活电流，并且电流密度与 NR1 C 末端长度相关。 NR1-1a C 末端的截断适度增强了 NR1-1a/NR3A 电流，而控制运输或磷酸化的 C 末端残基突变仅观察到小幅增加。相反，当 NR1-1a 中影响甘氨酸进入的细胞外苯丙氨酸突变为丙氨酸时，观察到大电流。 NR1-1a 中破坏甘氨酸结合的单独突变不会单独在 NR1-1a/NR3A 受体中产生反应，但在甘氨酸和甘氨酸拮抗剂 7-氯犬尿酸共同应用期间产生超过 30 倍的电流增强。最后，用 NR1-4a 亚基以及 NR2 和 NR3 亚基转染细胞，导致 NR1/NR3 受体和常规 NMDA 受体电流的表达。这些结果表明 NR1 剪接变体在哺乳动物细胞中 NR1/NR3 受体的功能表达中发挥着重要作用。
In oocytes, glycine activates receptors formed by diheteromeric combinations of N-methyl-D-aspartate (NMDA) NR1 and NR3 subunits. In contrast, functional receptors in mammalian cells require the simultaneous expression of NR1 and both NR3A and NR3B subunits. In vivo, NR3A and NR3B subunits show differential expression patterns and thus may not naturally form triheteromeric receptors. In this study, we examined whether NR1 splice variants play a role in allowing assembly of functional diheteromeric receptors in mammalian cells. Little current was found in human embryonic kidney 293 cells coexpressing either NR3A or NR3B and the NR1-1a splice variant. However, robust glycine-activated currents were generated in cells transfected with NR3(A or B) and either NR1-2a, NR1-3a, or NR1-4a, and current density was correlated with NR1 C-terminal length. Truncation of the NR1-1a C terminus modestly enhanced NR1-1a/NR3A currents, whereas only small increases were observed with mutations of C-terminal residues that control trafficking or phosphorylation. In contrast, large currents were observed when an extracellular phenylalanine in NR1-1a that influences glycine access was mutated to alanine. A separate mutation in NR1-1a that disrupts glycine binding did not generate responses in NR1-1a/NR3A receptors alone, but it produced a greater than 30-fold potentiation of currents during coapplication of glycine and the glycine antagonist 7-chlorokynurenic acid. Finally, transfection of cells with the NR1-4a subunit along with NR2 and NR3 subunits resulted in the expression of both NR1/NR3 receptors and conventional NMDA receptor currents. These results indicate a prominent role for NR1 splice variants in the functional expression of NR1/NR3 receptors in mammalian cells.