AMINE TRANSPORTERS--TRAFFICKING AND REGULATION

描述：(申请人摘要) 滥用和治疗药物(如可卡因、安非他明、氟西汀) 它们通过作用于质膜胺转运体而发挥作用。这是 胺转运体决定突触递质功能的证据 水平(以及突触前和突触后受体的反应)。近期 数据表明，转运蛋白的功能是受调控的。考虑到 胺类突触在药物滥用和神经精神疾病中的调节 将会产生重大后果。需要检验的一般假设 IS：转运体通过反应调节突触递质水平 与负责这些发射器水平的过程类似。这些 实验将确定调节转运蛋白的因素 贩运、表达和调制。可证伪的预测将是 来源：假设1：转运蛋白的表达是通过多种途径进行的 与那些参与递质分泌的基因完全相同。本地化和 转运体的表达将被评估。反义、毒素和 然后将使用药物策略来灭活分泌。 在特定阶段组成(例如，细胞骨架、对接蛋白)。 假设2：转运蛋白功能的调制发生在对 影响突触递质水平或受其影响的分子。 将对运输者表达和贩运进行评估，以应对 影响神经递质释放的信号(例如，钙)或该信号 细胞外递质水平(例如，突触前受体)。这个 作用机制(例如，蛋白质合成、转位)将是 对每个调制器进行评估。假设3：初生代的亮氨酸基序 转运蛋白的氨基酸序列控制着靶向受调控的 分泌途径。将产生突变的转运蛋白；它们的靶向 并将评估受监管的能力。野生型与野生型的互作 带有分泌蛋白的突变转运蛋白将被确定。 预测将通过人类5-羟色胺的表达来检验 神经分泌(PC12)细胞中的转运蛋白。结果衡量标准包括吸纳 放射性标记底物、拮抗剂结合、亚细胞分级 以及免疫印迹和免疫显微镜检查。这些实验将1) 阐明转运蛋白在突触信号中的作用；2)确定 递质分泌与再摄取过程的相互作用；3) 确定转运蛋白表达和调控所需的成分；以及 4)提供了另一种战略，用于在 突触在药物滥用和精神疾病的治疗中可能有用 生病了。

DESCRIPTION: (Applicant's Abstract) Drugs of abuse and therapy (e.g., cocaine, amphetamine, fluoxetine) exert their effects by acting on plasma membrane amine transporters. This is evidence that amine transporters function to determine synaptic transmitter levels (and thus the response of pre- and post-synaptic receptors). Recent data suggest that transporter function is regulated. Given the role of amine synapses in drug abuse and neuropsychiatric disease, such-regulation would have significant consequences. The general hypothesis to be tested is: Transporters regulate synaptic transmitter levels by responding similarly to the processes responsible for those transmitter levels. These experiments will determine the factors that regulate transporter trafficking, expression and modulation. Falsifiable predictions will be made from: Hypothesis 1: Expression of transporters occurs by pathways identical to those involved in transmitter secretion. Localization and expression of the transporter will be assessed. Antisense, toxin, and pharmacological strategies will then be used to inactivate secretion components (e.g., cytoskeleton, docking proteins) at specific stages. Hypothesis 2: Modulation of transporter function occurs in response to molecules that affect, or are affected by, synaptic transmitter levels. Transporter expression and trafficking will be assessed in response to signals that affect neurotransmitter release (e.g., Ca2+) or that signal extracellular transmitter levels (e.g., presynaptic receptors). The mechanism of action (e.g., protein synthesis, translocation) will be assessed for each modulator. Hypothesis 3: Leucine motifs in the primary amino acid sequence of the transporter govern targeting to the regulated secretion pathway. Mutant transporters will be produced; their targeting and ability to be regulated will be assessed. Interaction of wild-type and mutant transporters with secretion proteins will be determined. Predictions will be tested using expression of the human serotonin transporter in neurosecretory (PC12) cells. Outcome measures include uptake of radiolabeled substrates, antagonist binding, subcellular fractionation and western blots, and immunomicroscopy. These experiments will 1) elucidate the role of transporters in synaptic signaling; 2) determine the interaction between transmitter secretion and the reuptake process; 3) identify components necessary for transporter expression and regulation; and 4) provide an alternative strategy for regulating levels of amines at the synapse that could be useful in treatments for drug abuse and mental illness.