VESICULAR TRANSPORTER SPECIFICITY

囊泡转运蛋白特异性

• 批准号: 6139547
• 负责人: JEFFREY D ERICKSON
• 金额: $ 20.17万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6139547
• 关键词: Caenorhabditis elegans; acetylcholine; animal genetic material tag; chimeric proteins; membrane transport proteins; neurotransmitter transport; point mutation; protein structure function; site directed mutagenesis; synaptic vesicles; tissue /cell culture

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.

描述：生物胺和乙酰胆碱的包装 神经元和神经内分泌细胞的专门分泌囊泡是 由特定囊泡转运蛋白介导。 人类的两个同工型 囊泡单胺转运蛋白（HVMAT1和HVMAT2）和囊泡ACH 转运蛋白（HVACHT）最近被克隆并进行功能测定 每个都已开发。 因为这些转运蛋白在 结构和功能，底物和抑制剂的分析 绑定位点应洞悉囊泡机制 运输并进入发射器释放受影响的方式 转运蛋白特性改变。 该提案的主要目标是定义分子结构 确定转运蛋白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。 为了定义囊泡的跨膜结构域（TMD） 参与底物易位孔的转运蛋白 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：它们包括 高亲和力的细胞质识别部位和低亲和力排放位点 位于囊泡腔。 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未取代的芳香胺。 3）VMAT1和 VMAT2可以在功能上相互作用，也许是二聚体，并表达已更改 转运蛋白特异性，4）HVMAT1和HVACHT的底物结合位点 居住在不同的TMD中。 检验以下假设：分子结构的改变 确定囊泡转运蛋白特异性可能会影响 可用于调节神经分泌的神经递质，以下 做出预测：1）基于HVACHT的位点特异性突变 秀丽隐杆线虫中的几个非致命UNC-17点突变将影响 摄取的动力学参数和稳态的囊泡ACH 在体外积累，以及2）ACH对VACHT或 VACHT的表达水平将影响ACH累积的量 在突触囊泡和胆碱能神经元释放的ACH量中 在文化中。