Laboratory for Membrane Protein Dynamics, Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
Cells. 2022 Nov 28;11(23):3811. doi: 10.3390/cells11233811.
The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na and Cl binding on [H]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (K) for [H]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.
多巴胺转运体（DAT）是神经递质：钠离子协同转运体（NSS）家族的一员，介导多巴胺从细胞外空间的钠离子驱动重摄取，从而终止多巴胺能神经传递。我们目前对 DAT 的结构理解来自 DAT 的分辨率（dDAT）。尽管对与各种抗抑郁药、精神兴奋剂及其内源性底物多巴胺复合的纯化 dDAT 进行了广泛的结构研究，但纯化全长 dDAT 的分子药理学尚未阐明。在这项研究中，我们使用闪烁接近测定法通过放射性配体结合来功能表征去污剂胶束中的纯化全长 dDAT。我们阐明了 Na 和 Cl 结合对 [H]nisoxetine 亲和力的影响，并利用这一点来评估底物和抑制剂对转运体的结合谱。此外，该技术允许我们直接确定 [H]多巴胺与 dDAT 的平衡结合亲和力（K）。为了与更天然的系统进行比较，在 COS-7 细胞中表达的 dDAT、人 DAT 和人去甲肾上腺素转运体上测定了指定单胺和抑制剂的亲和力。根据我们收集的数据，我们为纯化的全长 dDAT 建立了药理学特征，这将有助于随后使用 dDAT 作为哺乳动物 NSS 蛋白家族的模型蛋白进行生物物理研究。
