A Juxtamembrane Mutation in the N Terminus of the Dopamine Transporter Induces Preference for an Inward-Facing Conformation
A Juxtamembrane Mutation in the N Terminus of the Dopamine Transporter Induces Preference for an Inward-Facing Conformation
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DOI：
10.1124/mol.108.048744
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发表时间：
2009-03-01
影响因子：
3.6
通讯作者：
Gnegy, Margaret E.
中科院分区：
文献类型：
--
作者：
Guptaroy, Bipasha;Zhang, Minjia;Gnegy, Margaret E.
人类多巴胺转运蛋白（hDAT）调节突触多巴胺（DA）水平，是滥用和治疗药物的作用位点。在这里，我们研究了苏氨酸残基（hDAT中的Thr62）的作用，该残基在单胺转运体近膜n端区域的典型磷酸化位点（RETW）内高度保守。在稳定转染的人胚胎肾293T细胞中，与hDAT或T62A-hDAT相比，T62D-hDAT的表达降低。通过[H-3]多巴胺外排测量和电流测量，T62D-hDAT显示出[H-3]多巴胺摄取显著减少，但与hDAT或T62A-hDAT相比，显示出更高的基础多巴胺外排。T62D-hDAT的高组成外排妨碍了安非他明刺激的[H-3]多巴胺外排的测量，但当将多巴胺内部添加到电压箝位的T62D-hDAT细胞中时，安非他明诱导的外排与hDAT相当。研究发现，Zn2+可以挽救主要面向内的突变转运体中减少的DA摄取，微摩尔浓度的Zn2+可以显著增强T62D-hDAT中[H-3]多巴胺的摄取，并允许测量安非他明刺激的多巴胺外排。这些结果表明，T62D-hDAT在内向和外向构象之间的过渡中倾向于内向构象。然而，对于T62A-hDAT， [H-3]多巴胺摄取和[H-3]多巴胺外排都减少了50%，这与内向和外向构象之间的缓慢转变是一致的。这些发现表明Thr62在hDAT活性中的重要功能作用的机制是在结构背景下使用DAT的三维分子模型的动态模拟来研究的。
The human dopamine transporter (hDAT) regulates synaptic dopamine (DA) levels and is the site of action of abused and therapeutic drugs. Here we study the effect of a threonine residue (Thr62 in hDAT) that is highly conserved within a canonical phosphorylation site (RETW) in the juxtamembrane N-terminal region of monoamine transporters. In stably transfected human embryonic kidney 293T cells, expression of T62D-hDAT was reduced compared with hDAT or T62A-hDAT. T62D-hDAT displayed dramatically reduced [H-3] dopamine uptake but exhibited a higher basal dopamine efflux compared with hDAT or T62A-hDAT, as determined by measurements of [H-3] dopamine efflux and amperometry. The high constitutive efflux in T62D-hDAT precluded the measurement of amphetamine-stimulated [H-3] dopamine efflux, but when dopamine was added internally into voltage-clamped T62D-hDAT cells, amphetamine-induced efflux comparable with hDAT was detected by amperometry. In accordance with findings that Zn2+ can rescue reduced DA uptake in mutant transporters that are predominantly inward-facing, micromolar concentrations of Zn2+ markedly potentiated [H-3] dopamine uptake in T62D-hDAT and permitted the measurement of amphetamine-stimulated dopamine efflux. These results suggest that T62D-hDAT prefers an inward-facing conformation in the transition between inward- and outward-facing conformations. For T62A-hDAT, however, the measured 50% reduction in both [H-3] dopamine uptake and [H-3] dopamine efflux was consistent with a slowed transition between inward- and outward-facing conformations. The mechanism underlying the important functional role of Thr62 in hDAT activity suggested by these findings is examined in a structural context using dynamic simulations of a three-dimensional molecular model of DAT.