VESICULAR TRANSPORTER SPECIFICITY

囊泡转运蛋白特异性

基本信息

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
项目类别：
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

项目摘要

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：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蛋白（hVMAT1 和 hVMAT2）和囊泡 Ach 转运蛋白（hVAChT）最近已被克隆并进行了功能测定每一个都已被开发出来。因为这些转运蛋白在以下方面高度相关结构和功能、底物和抑制剂的分析结合位点应该提供对囊泡机制的深入了解运输和影响发射机释放的方式改变转运蛋白的特性。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位点。为了定义囊泡的跨膜域（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) 这些转运蛋白上存在两个底物结合位点：它们包括高亲和力细胞质识别位点和低亲和力放电位点位于朝向囊泡腔的位置。 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 可以功能性地相互作用，也许作为二聚体，并表达改变的转运蛋白特异性，4) hVMAT1 和 hVAChT 的底物结合位点驻留在不同的TMD中。检验以下假设：分子结构的改变确定囊泡转运蛋白的特异性会影响可用于调节神经分泌的神经递质，如下做出预测：1) hVAChT 中的位点特异性突变基于线虫中的几个非致命性 unc-17 点突变将影响囊泡乙酰胆碱摄取的动力学参数和稳态水平体外积累，以及 2) ACh 对 VAChT 的亲和力发生变化或 VAChT的表达水平会影响ACh积累的量突触小泡中的乙酰胆碱和胆碱能神经元释放的乙酰胆碱量在文化中。