χ-conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter
χ-conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter
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DOI：
10.1074/jbc.m213101200
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发表时间：
2003-10-10
影响因子：
4.8
通讯作者：
Lewis, RJ
中科院分区：
文献类型：
--
作者：
Bryan-Lluka, LJ;Bönisch, H;Lewis, RJ
通过测定人去甲肾上腺素转运蛋白（hNET）点突变对MrIA抑制效力的影响，研究了嵌合肽MrIA与hNET的相互作用。通过定点突变产生突变体并在COS-7细胞中表达。MrIA抑制hNET摄取[H-3]去甲肾上腺素（Ki 1.89 μ M）的效力大于抑制[H-3]多巴胺（Ki 4.33 μ M）的效力，并且人多巴胺转运蛋白和5-羟色胺转运蛋白不受MrIA抑制（至7 μ M）。在hNET氨基酸残基与人多巴胺转运蛋白氨基酸残基交换的18个突变中，MrIA对3个突变（在预测的细胞外环3和4以及跨膜结构域（TMD）8中）的[H-3]去甲肾上腺素摄取的抑制效力增加，对1个突变（在TMD 6和细胞内环（IL）3中）的抑制效力降低。在TMD 2、4、5和11以及IL 1中的12个额外突变中，3个突变（TMD 2和IL 1中）具有降低的MrIA抑制效力。检测的所有其他突变均对MrIA效价无影响。结果与先前的去甲丙咪嗪和可卡因抑制突变hNET的去甲肾上腺素摄取的数据的比较表明，MrIA结合hNET发生在一个网站，这是不同的，但与三环类抗抑郁药和可卡因的结合位点重叠。
The interactions of chi-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [H-3] norepinephrine (K-i 1.89 muM) than [H-3] dopamine (K-i 4.33 muM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA ( to 7 muM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [H-3] norepinephrine uptake for three mutations ( in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine.