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 系统和激活时具有多种作用，包括： 呼吸，奖赏途径的激活，正常的 胃肠蠕动和镇痛。目前很难对此进行考察 在野生动物中广泛分布。阿片类药物抗体 受体通常非常不可靠， 使得不可能在活组织中进行检测。使用转基因或 敲入动物以识别受体需要广泛的表征， 受到不同表达水平的限制，并且仅限于在小鼠中的研究。的 该提案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引导化合物，纳洛酮胺， 是对所有亚型阿片样物质具有高亲和力非选择性阿片样物质拮抗剂 受体。纳洛酮胺将通过一系列的连接体， 咪唑和荧光配体。一旦与受体结合， 咪唑与受体上的赖氨酸反应，同时释放 纳洛酮胺因此，纳曲沙明可以自由地从受体上解离， 荧光配体与受体共价结合。初步结果 表明该化合物与μ-阿片样物质非常有效地相互作用 受体的因此，内源性受体被标记，并且标记不是依赖性的 关于物种。 这种化合物和未来的改进将用于中枢神经系统的领域， 阿片样物质受体仅在一小部分神经元中表达。这 建议将集中在小鼠和大鼠的VTA和黑质。阿片 将鉴定活脑切片中表达受体的神经元， 将进行录音。通过靶向这些细胞， 阿片类药物在奖励系统的这一关键领域的行动将是可能的。的结果 这项基于细胞的研究将解决一个长期存在的问题， 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.