Abstract:
The monoamine transporters, including the serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET), are the therapeutic targets for the treatment of many neuropsychiatric disorders. Despite significant progress in characterizing the structures and transport mechanisms of these transporters, the regulation of their transport functions through dimerization or oligomerization remains to be understood. In the present study, we identified a conserved intramolecular ion-pair at the third extracellular loop (EL3) connecting TM5 and TM6 that plays a critical but divergent role in the modulation of dimerization and transport functions among the monoamine transporters. The disruption of the ion-pair interactions by mutations induced a significant spontaneous cross-linking of a cysteine mutant of SERT and an increase in cell surface expression but with an impaired specific transport activity. On the other hand, similar mutations of the corresponding ion-pair residues in both DAT and NET resulted in an opposite effect on their oxidation-induced dimerization, cell surface expression, and transport function. Reversible biotinylation experiments indicated that the ion-pair mutations slowed down the internalization of SERT but stimulated the internalization of DAT. In addition, cysteine accessibility measurements for monitoring SERT conformational changes indicated that substitution of the ion-pair residues resulted in profound effects on the rate constants for cysteine modification in both the extracellular and cytoplasmatic substrate permeation pathways. Furthermore, molecular dynamics simulations showed that the ion-pair mutations increased the interfacial interactions in a SERT dimer but decreased it in a DAT dimer. Taken together, we propose that the transport function is modulated by the equilibrium between monomers and dimers on the cell surface, which is regulated by a potential compensatory mechanism but with different molecular solutions among the monoamine transporters. The present study provided new insights into the structural elements regulating the transport function of the monoamine transporters through their dimerization.
摘要:
单胺转运蛋白,包括血清素转运蛋白(SERT),多巴胺转运蛋白(DAT),和去甲肾上腺素转运蛋白(NET),是治疗许多神经精神疾病的治疗靶点。尽管在表征这些转运蛋白的结构和运输机制方面取得了重大进展,通过二聚化或低聚化调节它们的转运功能仍有待理解。在本研究中,我们确定了在连接TM5和TM6的第三个胞外环(EL3)上保守的分子内离子对,它在调节单胺转运蛋白之间的二聚化和转运功能中起着关键但不同的作用.突变对离子对相互作用的破坏诱导了SERT的半胱氨酸突变体的显着自发交联和细胞表面表达的增加,但特异性转运活性受损。另一方面,DAT和NET中相应的离子对残基的相似突变对其氧化诱导的二聚化产生相反的影响,细胞表面表达,和运输功能。可逆的生物素化实验表明,离子对突变减慢了SERT的内化,但刺激了DAT的内化。此外,用于监测SERT构象变化的半胱氨酸可达性测量表明,离子对残基的取代会对细胞外和细胞质底物渗透途径中半胱氨酸修饰的速率常数产生深远的影响。此外,分子动力学模拟表明,离子对突变增加了SERT二聚体中的界面相互作用,但降低了DAT二聚体中的界面相互作用。一起来看,我们提出,细胞表面上的单体和二聚体之间的平衡调节运输功能,它受潜在的补偿机制调节,但在单胺转运蛋白之间具有不同的分子溶液。本研究为通过二聚化调节单胺转运蛋白运输功能的结构元素提供了新的见解。