Covalent labeling endogenous G-protein coupled receptors in living cells

阿片受体广泛分布于中枢和外周神经 系统和激活时有多种动作，包括：按下 呼吸，奖赏通路的激活，正常 胃肠动力和止痛。目前很难研究这一点。 广泛分布于野生动物中。抗阿片类药物抗体 受体通常非常不可靠，表位通常在细胞内 使得在活组织中检测是不可能的。使用转基因或 Knockin动物识别受体需要广泛的特性，可以 受到不同表达水平的限制，并仅限于在小鼠身上进行的研究。这个 这项提议的目标是使用配体导向的方法来共价标记 活组织中的内源性阿片受体。导向化合物，纳曲胺， 是一种非选择性阿片类拮抗剂，对所有亚型阿片类药物都有很高的亲和力 感受器。纳曲胺将通过一系列连接物-反应性酰基-偶联 咪唑和荧光配体。一旦与受体结合，反应性酰基 咪唑与受体上的赖氨酸反应，同时释放 纳曲胺。因此，纳曲克沙明可以自由地从受体上解离。 荧光配体以共价方式与受体结合。初步结果 表明该化合物与类阿片类药物相互作用非常有效。 受体。因此，内源性受体被标记并且标记不依赖于 关于物种。 这种化合物和未来的改进将用于CNS的领域，其中 阿片受体只在一小部分神经元中表达。这 建议将重点放在小鼠和大鼠的VTA和黑质。阿片类药物 活体脑片中表达受体的神经元将被鉴定和全细胞 我们会录下来的。通过以这些细胞为靶点，完全理解 在奖励系统的这一关键领域，阿片类药物的行动将是可能的。结果是 这项基于细胞的研究将解决围绕着 阿片类药物成瘾特性的一个方面的基础机制。

Opioid receptors are widely distributed in the central and peripheral nervous system and upon activation have manifold actions including; depression of respiration, activation of the reward pathway, disruption of normal gastrointestinal motility and analgesia. It is currently difficult to examine this widespread distribution in wild type animals. Antibodies against the opioid receptors are often very unreliable and the epitopes are most often intracellular such that detection in living tissue is not possible. The use of transgenic or knockin animals to identify receptors requires extensive characterization, can be limited by varied expression levels and has been limited to studies in mice. The goal of this proposal is to use a ligand directed approach to covalently label endogenous opioid receptors in living tissue. The guide compound, naltrexamine, is a non-selective opioid antagonist that has high affinity for all subtype opioid receptors. Naltrexamine will be coupled through a series of linkers, a reactive acyl imidazole and fluorescent ligand. Once bound to the receptor, the reactive acyl imidazole reacts with a lysine on the receptor and simultaneously releases the naltrexamine. Thus natrexamine is free to dissociate from the receptor, however the fluorescent ligand is left covalently bound to the receptor. Preliminary results indicate that this compound interacts very effectively with the mu-opioid receptor. Thus endogenous receptor are labeled and the labeling is not dependent on species. This compound and future refinements will be used areas of the CNS where opioid receptors are expressed in only a small population of neurons. This proposal will center on the VTA and Substantia Nigra in mouse and rat. Opioid receptor expressing neurons in living brain slices will be identified and whole cell recordings will be made. By targeting these cells a complete understanding of opioid action in this key area of the reward system will be possible. The results of this cell based study will address a long-standing question surrounding the mechanisms that underlie one aspect of the addictive properties of opioids.