Characterization of Vesicular Monoamine Transporters

描述（由申请人提供）：该提案的策略基于单胺转运体（VMAT2）上抑制剂、底物、质子易位和功能相关磷酸化位点的鉴定，将提供对单胺隔离到囊泡中的作用机制和调节转运体活性的因素的基本理解。这项工作将在三个特定目标下完成：(1)确定利血平在VMAT2上的结合位点。新的利血平光亲和标记将被合成和表征，并且光标记肽将被识别以绘制利血平结合位点；(2)基材输送通道的识别。这一目标将涉及使用几种方法，包括放射性光活化底物类似物以共价衍生VMAT2上的底物结合位点；VMAT2在工程半胱氨酸残基上用半胱氨酸反应试剂甲基乙磺酸乙胺（MTSEA）和甲基乙基三甲基铵（MTSET）进行位点特异性衍生化；以及通道内潜在残基的定点诱变；(3)确定VMAT2两个高电荷区域的功能作用。这一目标将涉及使用生化和遗传（定点诱变）方法来确定VMAT2 n端磷酸化对转运蛋白功能和转运蛋白细胞内分布/寡聚状态的作用。通过直接或间接毒物（如MPP+、杀虫剂）和基因改变的神经元表达蛋白的作用，黑质多巴胺能神经元突触囊泡单胺转运蛋白活性降低或异常，可能在帕金森病中起核心作用。单胺类神经递质进入储存囊泡的摄取调节也可能通过改变血清素、去甲肾上腺素、多巴胺或其他神经递质的水平，在抑郁症相关的情感性心理障碍中发挥重要作用。这项工作将深入了解单胺转运体的作用机制，并有助于我们理解如何设计药物和治疗策略来治疗帕金森病或其他神经系统疾病。

DESCRIPTION (provided by applicant): The strategy of this proposal is based on the rationale that identification of the inhibitor, substrate, proton translocation, and functionally relevant phosphorylation sites on monoamine transporters (VMAT2) will provide a basic understanding of the mechanism of action of monoamine sequestration into vesicles and the factors which regulate transporter activity. This work will be accomplished in three Specific Aims: (1) Identification of the reserpine binding site(s) on VMAT2. Novel reserpine photoaffinity labels will be synthesized and characterized, and photo-labelled peptides will be identified in order to map the reserpine binding site; (2) Identification of the substrate transport channel. This aim will involve the use of several approaches, including radioactive photo-activatable substrate analogs to covalently derivatize the substrate binding site on VMAT2; site-specific derivatization of VMAT2 at engineered cysteine residues with the cysteine-reactive reagents, methanethiosulfonate ethyl amine (MTSEA), and MTS-ethyltrimethylammonium (MTSET); and site-directed mutagenesis of potential residues lining the channel; (3) Determination of the functional role of two highly charged regions of VMAT2. This aim will involve the use of biochemical and genetic (site-directed mutagenesis) approaches to determine the role of phosphorylation of the N-terminus of VMAT2 on transporter function and the intracellular distribution/oligomeric state of the transporter. Reduced or aberrant activity of the monoamine transporter of the synaptic vesicles in dopaminergic neurons of the substantia nigra through either direct or indirect actions of toxicants (e.g., MPP+, insecticides) and genetically altered neuronally expressed proteins may play a central role in Parkinson's Disease. The regulation of uptake of monoamine neurotransmitters into storage vesicles may also play an important role in affective psychological disorders related to depression by altering levels of serotonin, norepinephrine, dopamine, or other neurotransmitters. 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 or other disorders of the nervous system.