VESICULAR TRANSPORTER SPECIFICITY

描述：将生物胺和乙酰胆碱包装成 神经元和神经内分泌细胞的专门分泌囊泡是 由特定的囊泡转运蛋白介导。 人类的两种亚型 囊泡单胺转运体（hVMAT 1和hVMAT 2）和囊泡Ach 最近已经克隆了人乙酰胆碱转运蛋白（hVAChT），并进行了功能测定， 每一个都被开发出来了。 因为这些转运蛋白在 结构和功能，底物和抑制剂分析 结合位点应提供深入了解囊泡的机制， 运输和进入影响发射器释放的方式 改变了转运蛋白的性质 该提案的主要目标是定义分子结构， 确定转运蛋白的特异性，并检验囊泡 转运蛋白可能是调节突触功能的潜在位点。 为了确定囊泡的跨膜结构域（TMD）， 参与底物转运孔的转运蛋白 以及决定转运蛋白特异性的分子结构， 嵌合转运蛋白和位点特异性突变体将用于测试 以下假设： 1)在这些转运蛋白上存在两个底物结合位点： 高亲和力细胞质识别位点和低亲和力放电位点 位于囊状腔的方向。 2)不同的氨基酸残基是 参与hVMAT 1和hVMAT 2的差异识别特性， 高亲和性和低亲和性未取代的芳族胺。 3)VMAT 1和 VMAT 2可能作为二聚体在功能上相互作用，并表达改变的 转运蛋白特异性，4）hVMAT 1和hVAChT的底物结合位点 存在于不同的TMD中。 为了验证分子结构的改变 确定囊泡转运蛋白特异性可以影响 调节神经分泌的神经递质， 进行预测：1）基于hVAChT的位点特异性突变 几个非致命的unc-17点突变的C.优雅会影响 囊泡ACh摄取动力学参数及稳态水平 在体外积累，和2）ACh对VAChT或 VAChT的表达水平会影响ACh的积累量 胆碱能神经元释放ACh的量 在文化中。

DESCRIPTION: The packaging of biogenic amines and acetylcholine into specialized secretory vesicles of neurons and neuroendocrine cells is mediated by specific vesicular transporters. Two isoforms of the human vesicular monoamine transporter (hVMAT1 and hVMAT2) and the vesicular Ach transporter (hVAChT) have recently been cloned and functional assays for each have been developed. Because these transporters are highly related in both structure and function, the analysis of their substrate and inhibitor binding sites should provide insight into the mechanisms of vesicular transport and into the means by which transmitter release is affected by altered transporter properties. The major goals of this proposal are to define the molecular structures that determine transporter specificity and to test the hypothesis that vesicular transporters may be a potential site for regulation of synaptic function. In order to define the transmembrane domains (TMD) of the vesicular transporter proteins which participate in the substrate translocation pore and the molecular structures that determine transporter specificity, chimeric transporter and site-specific mutants will be used to test the following hypotheses: 1) two substrate binding sites exist on these transporters: they include a high-affinity cytoplasmic recognition site and a low-affinity discharge site located towards the vesicular lumen. 2) distinct amino acid residues are involved in the differential recognition properties of hVMAT1 and hVMAT2 to high-affinity and low-affinity unsubstituted aromatic amines. 3) VMAT1 and VMAT2 can functionally interact, perhaps as a dimer, and express altered transporter specificity, 4) the substrate binding sites of hVMAT1 and hVAChT reside in different TMDs. To test the hypothesis that alterations in the molecular structures that determine vesicular transporter specificity can affect the levels of neurotransmitter available for regulated neurosecretion, the following predictions are made: 1) site-specific mutations in hVAChT that are based on several non-lethal unc-17 point mutations in C. elegans will affect the kinetic parameters of uptake and steady-state level of vesicular ACh accumulation in vitro, and 2) changes in the affinity of ACh for VAChT or the level of expression of VAChT will affect the amount of ACh accumulated in synaptic vesicles and the amount of ACh released from cholinergic neurons in culture.