CHARACTERIZATION OF VESICULAR MONOAMIE TRANSPORTERS

描述(调查者摘要)：封存 神经递质，如5-羟色胺、去甲肾上腺素、多巴胺和 组胺进入细胞内的囊泡中以便随后释放是一种 神经元和分泌细胞中的基本细胞事件。减少或 突触小泡单胺转运体的异常活动可能 在帕金森氏症中起着核心作用。利血平，临床用药 多年来控制高血压，导致体内的胺类物质消耗殆尽 通过抑制囊泡中的单胺转运体来储存囊泡 薄膜。脑内单胺类神经递质摄取的调节 储藏小泡在情感心理中可能起重要作用 通过改变5-羟色胺水平与抑郁症相关的疾病， 去甲肾上腺素、多巴胺或其他神经递质。这是一种战略 该提案是基于这样的基本原理，即确定抑制剂， 底物，以及单胺转运体和 VMAT相互作用的蛋白质将提供对 单胺滞留到囊泡中的生化作用机制 调节转运体活性的因素。这项工作将 实现四个具体目标：(1)确定VMAT2 底物结合位点(S)使用苯丙胺，多巴胺， 和神经毒素MPP+；(2)利血平结合部位的鉴定(S) 重组纯VMAT2在人工脂质体中的应用研究 放射性碘化利血平光标记；(3)特异性鉴定 [14C]二环己基碳二亚胺(DCD)-VMAT2中的反应性氨基酸，其是 参与质子运输；新的可切割的基于DCCD的光标记 探查DCD的分子环境--反应部位即可制备； (4)鉴定与VMAT相互作用的蛋白质 生物化学和分子生物学技术。这项研究将提供一种 基于逻辑蛋白质化学的结构和结构评价方法 囊泡单胺转运体的调节。这项工作将提供 深入了解单胺转运体的作用机制和 有助于我们理解药理和治疗方法 治疗帕金森氏症、心血管疾病、 过敏和神经系统紊乱。

DESCRIPTION (Investigator's Abstract): The sequestration of neurotransmitters, such as serotonin, norepinephrine, dopamine, and histamine into intracellular vesicles for subsequent release is a fundamental cellular event in neurons and secreting cells. Reduced or aberrant activity of the monoamine translocator of the synaptic vesicle may play a central role in Parkinson's Disease. Reserpine, used clinically for many years to control hypertension, causes the depletion of amines from the storage vesicles by inhibition of the monoamine translocator in the vesicle membrane. The regulation of uptake of monoamine neurotransmitters into storage vesicles may play an important role in affective psychological disorders related to depression by altering levels of serotonin, norepinephrine, dopamine, or other neurotransmitters. The strategy of this proposal is based on the rationale that identification of the inhibitor, substrate, and proton translocation sites on monoamine transporters and VMAT-interacting proteins will provide a basic understanding of the biochemical mechanism of action of monoamine sequestration into vesicles and the factors which regulate the activity of the translocator. This work will be accomplished in four Specific Aims: (1) identification of the VMAT2 substrate binding site(s) using novel derivatives of amphetamine, dopamine, and the neurotoxin MPP+; (2) identification of the reserpine binding site(s) on VMAT2 using reconstituted pure VMAT2 in artificial liposomes and novel radioiodinated reserpine photolabels; (3) identification of the specific [14C]dicyclohexylcarbodiimide (DCCD)-reactive amino acid in VMAT2 which is involved in proton transport; novel cleavable DCCD-based photolabels to probe the molecular environment of the DCCD-reactive site will be prepared; (4) identification of proteins which interact with VMAT using multiple biochemical and molecular biology techniques. This research will provide a logical protein chemistry-based approach for assessment of the structure and regulation of the vesicle monoamine transporter. This work will provide insight into the mechanism of action of the monoamine transporters and contribute to our understanding of how pharmacological and therapeutic strategies may be devised to treat Parkinsonism, cardiovascular disease, allergy, and disorders of the nervous system.