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特化分泌小泡 由特定的囊泡转运蛋白介导。人类的两种异构体 囊泡单胺转运体(hVMAT1和hVMAT2)与囊泡型Ach 转运蛋白(HVAChT)最近被克隆并进行了功能分析 每一种都已经开发出来了。因为这些转运蛋白在 结构和功能，底物和抑制剂的分析 结合部位应该提供对囊泡机制的洞察。 传输并进入影响发射器释放的方式 改变了传送器的属性。 这项提案的主要目标是定义 确定转运蛋白的特异性并检验囊泡的假设 转运蛋白可能是调节突触功能的潜在部位。 为了确定囊泡的跨膜区(TMD) 参与底物转运孔的转运蛋白 以及决定转运蛋白专一性的分子结构， 嵌合转运体和定点突变将被用来测试 以下是假设： 1)在这些转运蛋白上存在两个底物结合位点：它们包括 高亲和力胞质识别部位和低亲和力放电部位 位于水泡腔的方向。2)不同的氨基酸残基是 参与hVMAT1和hVMAT2的差异识别特性 高亲和力和低亲和力的未取代芳香胺。3)VMAT1和 VMAT2可以在功能上相互作用，可能作为二聚体，并表达改变 转运体特异性，4)hVMAT1和hVAChT的底物结合部位 驻留在不同的TMD中。 来检验这样一个假设，即分子结构的变化 确定囊泡转运体的特异性可以影响 可用于调节神经分泌的神经递质如下 预测：1)hVAChT的位点特异性突变是基于 线虫中几个非致命性的UNC-17点突变将影响 囊泡ACh摄取的动力学参数和稳态水平 体外累积，以及2)ACh对Vacht或Vacht亲和力的变化 Vacht的表达水平会影响ACh的积累量 和胆碱能神经元释放的ACh的量 在文化上。

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.