MOLECULAR STUDIES OF CATECHOLAMINE TRANSPORTERS

描述：(申请人摘要) 在之前的资助期间，实验室使用了第一个编码 一种对可卡因敏感的生物胺转运体--人去甲肾上腺素 转运蛋白(Net)，作为分离和鉴定 编码多巴胺转运体(DAT)的cDNA被认为是 快感和成瘾效应最重要的作用部位 可卡因。尽管许多神经生物学问题需要研究 在可以解释精神运动兴奋剂成瘾的生物学之前， 在这份续期申请中提出的研究将继续 基于这样一种观点，即重要的见解将来自于 运输者本身的生物学。这份提案概述了 进一步探索基本结构-功能关系， DAT和Net区的电学性质及细胞生物学调节 这提供了一些在上一次 授权期。使用嵌合体、精选的点突变和 半胱氨酸扫描和修饰(SCAM)方法正在与 转运、电生理途径和结合的动力学分析 进一步探索底物结构和拮抗剂结合的研究 以及离子的渗透途径。最近的工作来自于此 实验室已经证明，DAT对多巴胺的摄取是一种生电和 与电压相关的过程。此外，Na/CI-依赖的成员 神经递质转运体家族，包括DAT，可以调节 非化学计量比关联的宏观离子流 底物运动。这些电流的机制和后果，在 特别是它们激活电压门控钙通道的潜力和 细胞内信号通路，将被研究。建议数 实验还旨在研究细胞内信号机制如何 调节细胞表面表达的载体的数量，重点是 尤其是PKC的激活对细胞的内化和 Net和DAT蛋白的封存。我们的总体目标是与 蛋白质结构特征与新的功能特性和调控 机制，并确定DA和NE运营商对 神经功能。

DESCRIPTION: (Applicant's Abstract) During the previous grant period the laboratory used the first cDNA encoding a cocaine-sensitive biogenic amine transporter, the human norepinephrine transporter (NET), as the basis for the isolation and characterization of a cDNA encoding the dopamine transporter (DAT) which has been proposed to be the most significant site of action for the euphoric and addictive effects of cocaine. Although many neurobiological issues need to be investigated before the biology of psychomotor stimulant addiction can be explained, the studies proposed in this continuing renewal application will continue to build on the view that significant insights will come from the fundamental biology of the transporters themselves. This proposal outlines plans for the further exploration of basic structure-function relationships, electrogenic properties and cell biological regulation of DAT and NET, areas that provided some of the most interesting observations during the previous grant period. Continuing studies using chimeras, selected point mutants and cysteine-scanning and modification (SCAM) approaches are being coupled with kinetic analyses of transport, electrophysiological approaches and binding studies to further probe the structure of substrate and antagonist binding sites, as well as the permeation pathway for ions. Recent work from this laboratory has shown that dopamine uptake by DAT is an electrogenic and voltage-dependent process. Furthermore, members of Na+/CI- -dependent family of neurotransmitter transporters, including DAT, can mediate macroscopic ionic currents which are not stoichiometrically linked to substrate movement. The mechanism and consequences of these currents, in particular their potential to activate voltage-gated calcium channels and intracellular signaling pathways, will be examined. The proposed experiments also aim to examine how intracellular signaling mechanisms regulate the number of carriers expressed at the cell surface, focusing on a particularly striking effect of PKC activation on the internalization and sequestration of NET and DAT proteins. Our overall goal is to relate protein structural features to novel functional properties and regulatory mechanisms, and to establish the contributions of the DA and NE carriers to neuronal function.