Single nucleotide polymorphisms in the human norepinephrine transporter gene affect expression, trafficking, antidepressant interaction, and protein kinase C regulation
Single nucleotide polymorphisms in the human norepinephrine transporter gene affect expression, trafficking, antidepressant interaction, and protein kinase C regulation
复制标题
DOI：
10.1124/mol.105.011270
复制
发表时间：
2005-08-01
影响因子：
3.6
通讯作者：
Blakely, RD
中科院分区：
文献类型：
--
作者：
Hahn, MK;Mazei-Robison, MC;Blakely, RD
去甲肾上腺素（NE）在注意力、记忆、情绪、压力、心率和血压中的作用暗示了NE在精神和心血管疾病中的作用。去甲肾上腺素转运体介导释放的儿茶酚胺的再摄取，从而在中枢和外周神经系统信号强度的限制中发挥作用。影响转运体功能的人类NET （hNET）基因中的非同义单核苷酸多态性（snp）可导致疾病，例如在直立性不耐受中发现的无功能转运体A457P。在这里，我们研究了在包括心血管表型的人群中已确定但未表征的其他氨基酸变异。变体hNETs在COS-7细胞中表达，并通过细胞表面生物素化和Western blot分析，放射性标记底物的运输，拮抗剂相互作用以及通过phorbol酯β -phorbol-12-肉豆酸盐- 13-醋酸酯通过蛋白激酶C （PKC）连接途径进行调节来检测蛋白表达和运输。我们在研究的10个突变体中观察到6个功能扰动。一些变异在运输和运输方面存在缺陷，其中A369P的影响最为显著，它完全缺乏完全糖基化形式的转运蛋白，保留在细胞内，缺乏任何运输活性。此外，A369P和另一个转运变体N292T在共表达时阻碍了hNET的表面表达。F528C表现出转运增加，值得注意的是，它对PKC的下调不敏感，对三环抗抑郁药地西帕明的效力降低。这些发现揭示了可能损害人群中SNP携带者NE信号的功能缺陷，并确定了参与转运体生物合成、活性和调节的NET关键区域。
The role of norepinephrine ( NE) in attention, memory, affect, stress, heart rate, and blood pressure implicates NE in psychiatric and cardiovascular disease. The norepinephrine transporter (NET) mediates reuptake of released catecholamines, thus playing a role in the limitation of signaling strength in the central and peripheral nervous systems. Nonsynonymous single nucleotide polymorphisms (SNPs) in the human NET (hNET) gene that influence transporter function can contribute to disease, such as the nonfunctional transporter, A457P, identified in orthostatic intolerance. Here, we examine additional amino acid variants that have been identified but not characterized in populations that include cardiovascular phenotypes. Variant hNETs were expressed in COS-7 cells and were assayed for protein expression and trafficking using cell-surface biotinylation and Western blot analysis, transport of radiolabeled substrate, antagonist interaction, and regulation through protein kinase C (PKC)-linked pathways by the phorbol ester beta-phorbol-12-myristate- 13-acetate. We observed functional perturbations in 6 of the 10 mutants studied. Several variants were defective in trafficking and transport, with the most dramatic effect observed for A369P, which was completely devoid of the fully glycosylated form of transporter protein, was retained intracellularly, and lacked any transport activity. Furthermore, A369P and another trafficking variant, N292T, impeded surface expression of hNET when coexpressed. F528C demonstrated increased transport and, remarkably, exhibited both insensitivity to down-regulation by PKC and a decrease in potency for the tricyclic antidepressant desipramine. These findings reveal functional deficits that are likely to compromise NE signaling in SNP carriers in the population and identify key regions of NET contributing to transporter biosynthesis, activity, and regulation.