CHARACTERIZATION OF VESICULAR MONOAMIE TRANSPORTERS

描述（研究者摘要）： 神经递质，如血清素，去甲肾上腺素，多巴胺，和 组胺进入细胞内囊泡随后释放是一种 神经元和分泌细胞中的基本细胞事件。 减少或 突触囊泡的单胺转运蛋白的异常活性可能 在帕金森病中起着重要作用。 利血平，临床上用于 多年来，控制高血压，导致胺的消耗， 通过抑制囊泡中的单胺转运蛋白的储存囊泡 膜的 单胺类神经递质摄取的调节 储存囊泡可能在情感心理学中起重要作用 通过改变血清素水平而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 这个战略 该提案基于以下基本原理：抑制剂的鉴定， 底物和单胺转运蛋白上的质子转运位点， VMAT相互作用蛋白质将提供一个基本的了解， 单胺螯合进入囊泡的生化作用机制， 调节转运蛋白活性的因素。 这项工作将 实现四个具体目标：（1）识别VMAT 2 使用安非他明，多巴胺， （2）利血平结合位点的鉴定 使用在人工脂质体中重构的纯VMAT 2对VMAT 2的作用， 放射性碘利血平标记;（3）特异性标记物的鉴定 VMAT 2中的[14 C]二环己基碳二亚胺（DCCD）反应性氨基酸， 参与质子运输;新的可裂解的基于DCCD的光标记， 探针分子环境中的DCCD反应位点的制备; (4)使用多重免疫荧光法鉴定与VMAT相互作用的蛋白质 生物化学和分子生物学技术。 这项研究将提供一个 基于逻辑蛋白质化学的方法，用于评估结构和 调节囊泡单胺转运蛋白。 这项工作将提供 深入了解单胺转运蛋白的作用机制， 有助于我们理解药理学和治疗学 可以设计出治疗帕金森症，心血管疾病， 过敏和神经系统疾病。

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.