Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuT_Aa, and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuT_Aa, contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.

中文翻译：

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底物与甘氨酸转运蛋白GlyT2相互作用的分子决定因素。

SLC6家族中的转运蛋白在调节神经传递中起关键作用，并且是多种治疗方法的靶标。对SLC6转运蛋白的转运机制和药物相互作用的特异性的重要见解已从该家族细菌同系物LeuT _Aa的晶体结构中获得，近来，果蝇多巴胺转运蛋白和人类血清素转运蛋白也获得了重要的见解。然而，有争议的证据表明细菌亮氨酸转运蛋白LeuT _Aa底物包含两个底物结合位点，它们在运输机制中协同工作，第二底物的结合是底物从主要位点释放所必需的。另一种建议是可能存在低亲和力结合位点，以将底物流引导至主要位点。我们结合了底物相互作用的分子动力学模拟和GlyT2的同源性模型，并结合了放射性标记的氨基酸摄取分析以及野生型和突变型转运蛋白的电生理学分析，以提供证据表明GlyT2的底物选择性完全由GlyT2确定。第一底物结合位点，此外，如果存在第二位点，则它是低亲和力的非选择性氨基酸结合位点。