VESICULAR NEUROTRANSMITTER TRANSPORT IN DROSOPHILA

描述(由申请人提供)： 包括组胺在内的单胺类神经递质调节多种生理功能 过程，包括人类的胃酸分泌。在果蝇中，组胺 介导光感受器突触传递。在这两种生物中，单胺 在胞吐过程中，递质从分泌小泡中释放出来。自.以来 递质合成发生在细胞质中，胞外释放需要 主动转运到水泡腔。特异囊泡单胺 转运体(VMATs)调节这一活动，并包括两个 不同的人类VMAT基因(肾上腺的VMAT1和神经元的VMAT2)，以及一个 果蝇的预测基因。在此之前，我们已经研究了对大鼠的调节 VMAT2，并已鉴定出编码在羧基中的二亮氨酸样信号 VMATs的末端，调节有效的内吞作用。此外，我们还有 确定了两个基序，它们调节转运蛋白对分泌囊泡的分选。 其中包括位于-4和-5位的两个酸性残基。 二亮氨酸基序，以及极端的一簇明显的酸性残基 VMAT2的羧基末端。为了启动对果蝇VMAT的研究，我们 最近克隆并测序了果蝇VMAT基因(DVMAT)。 序列分析预测至少有两个选择性剪接的dVMAT变异体 (A和B)包含不同的预测的羧基末端胞质结构域。 DVMAT-A包含与二亮氨酸基序惊人地相似的序列，并且是酸性的 调节大鼠VMAT2膜转运的簇。另一种变种是dVMAT-B， 缺乏这些信号。测试膜的可选剪接是否 运输信号代表了一种调节VMAT功能的新机制，我们 现在将在Vivo中确定这两个变体的本地化，并测试 潜在的二亮氨酸基序在dVMAT-A中的作用。要确定如何更改 在dVMAT本地化影响发射机在体内释放，我们也将筛选 对于dVMAT突变果蝇。在未来的实验中，我们将使用dVMAT突变体作为 表示显示不同模式的VMAT变体的背景为空 细胞内交易。使用视觉作为行为读数，我们将 确定dVMAT贩运的变化如何影响组胺存储和 放手。我还将使用果蝇中的VMAT突变体作为致敏背景 用于未来的基因筛查，以识别调节VMAT功能的分子。

DESCRIPTION (provided by applicant): Monoamine neurotransmitters including histamine regulate multiple physiological processes, including gastric acid secretion in humans. In Drosophila, histamine mediates photoreceptor synaptic transmission. In both organisms, monoamine transmitters are released from secretory vesicles during exocytosis. Since transmitter synthesis occurs in the cytoplasm, exocytotic release requires active transport into the vesicle lumen. Specific vesicular monoamine transporters (VMATs) mediate this activity and include the products of two distinct human VMAT genes (VMAT1 in adrenal and VMAT2 in neurons), and one predicted gene in Drosophila. Previously, we have studied the regulation of rat VMAT2, and have identified a dileucine-like signal encoded in the carboxy terminus of VMATs that mediates efficient endocytosis. In addition we have identified two motifs that regulate transporter sorting to secretory vesicles. These include two acidic residues at the -4 and -5 position relative to the dileucine motif, and a distinct cluster of acidic residues at the extreme carboxy terminus of VMAT2. To initiate studies of VMAT in Drosophila, we recently cloned and sequenced cDNAs encoding Drosophila VMAT (dVMAT). Sequence analysis predicts at least two alternatively spliced dVMAT varaints (A and B) that contain different predicted carboxy-terminal cytoplasmic domains. dVMAT-A contains sequences strikingly similar to the dileucine motif and acidic cluster that regulates membrane trafficking of rat VMAT2. The other variant, dVMAT-B, lacks these signals. To test whether alternative splicing of membrane trafficking signals represents a new mechanism for regulating VMAT function, we will now determine the localization of the two variants invivo, and test the function of the potential dileucine motif in dVMAT-A. To determine how changes in dVMAT localization affect transmitter release invivo, we will also screen for dVMAT mutant flies. In future experiments, we will use a dVMAT mutant as a null background to express VMAT variants showing different patterns of intracellular trafficking. Using vision as a behavioral readout, we will determine how changes in dVMAT trafficking affect histamine storage and release. I will also use VMAT mutants in Drosophila as a sensitized background for future genetic screens to identify molecules that regulate VMAT function.