An integrative sturcture/functional analysis of mu-opioid receptor variants

描述（由申请人提供）：神经元阿片信号系统在中枢神经系统（CNS）生物学中的存在和重要性在基础研究和临床实践中得到了很好的确立和记录。一系列受体和内源性配体构成了该系统的分子基础，大量文献记录了它们在中枢神经系统细胞和系统生物学的正常控制中的作用。此外，已经确定中枢神经系统阿片受体是治疗剂和滥用药物的主要靶点，这导致了对受体结构，功能和药理特征的理解的重大兴趣。mu阿片受体（MOR）是特别感兴趣的，因为它在疼痛的治疗控制和麻醉成瘾中的核心作用。人类MOR基因（OPRM1）的许多多态性已经被确定，新兴的研究表明，MOR变异可能在人类对作用于MOR的药物敏感性的个体差异中发挥作用。特别令人感兴趣的是OPRM1 A118G变体，它在MOR的n端结构域（Asn40Asp）产生单个氨基酸替换，并编码40Asp突变受体（n40d变体）。了解这种突变的功能后果的核心是鉴定突变引起的MOR信号的功能变化。受体信号传导的一个关键方面是受体动力学，它决定了受体的可用性、功能和受体激活的下游后果。分子成像的最新进展为研究受体动力学提供了一种新的方法，荧光相关光谱（FCS）与共聚焦激光扫描显微镜（CLSM）相结合。FCS/CLSM能够实时无损地观察活细胞中的分子相互作用，具有最终的单分子灵敏度。我们建议利用这种新方法和平行生理分析来研究野生型和n40d变体的细胞动力学以及这些相互作用对神经元生理的功能后果。我们假设n40d变异将显示出受体动力学的改变，这是配体依赖的，并且受体动力学的改变将导致受体耦合到神经生理学的下游改变。本文提出了三个具体目标：(1)在分子和细胞水平上研究野生型人类MOR与其N40D变体在功能动力学上的潜在差异。(2)利用电生理和Ca2+成像技术确定MOR和n40d变体阿片受体在表达这些受体的活细胞中动态的下游功能后果的差异。(3)将MOR和n40d变体的研究扩展到天然神经元（培养的海马神经元），在神经元回路内的活细胞中研究受体动力学和功能偶联。

DESCRIPTION (provided by applicant): The existence and importance of the neuronal opioid signaling system in central nervous system (CNS) biology are well established and documented in basic research and clinical practice. A family of receptors and endogenous ligands form the molecular basis of this system and an extensive literature has documented their roles in the normal control of CNS cellular and system biology. Moreover, it has been well established that CNS opioid receptors are primary targets of therapeutic agents and abused drugs, which has lead to significant interest in an understanding of receptor structure, function and pharmacological profile. The mu opioid receptor (MOR) is of particular interest because of its central role in therapeutic control of pain and in narcotic addiction. A number of polymorphisms in the human gene (OPRM1) for MOR have been identified and emerging research suggests that MOR variants may play a role in the well-documented inter-individual variability in the sensitivity of humans to drugs that act at MOR. Of particular interest is the OPRM1 A118G variant, which generates a single amino-acid substitution in the N-terminal domain of MOR (Asn40Asp) and codes for 40Asp mutant receptors (N40D-variant). Central to an understanding of the functional consequences of this mutation is an identification of functional changes in MOR signaling due to the mutation. A critical aspect of receptor signaling is receptor dynamics, which determines receptor availability, function and the downstream consequences of receptor activation. Recent advances in molecular imaging now offer a new approach to study receptor dynamics, Fluorescence Correlation Spectroscopy (FCS) integrated with Confocal Laser Scanning Microscopy (CLSM). FCS/CLSM enables nondestructive observation of molecular interactions in living cells in real time with ultimate single-molecule sensitivity. We propose to utilize this new methodology and parallel physiological analyses to study the cellular dynamics of wild type and N40D-variant and the functional consequence of these interactions to neuronal physiology. We hypothesize that the N40D-variant will show altered receptor dynamics that are ligand dependent and that the altered receptor dynamics will result in downstream alterations in receptor coupling to neurophysiology. Three Specific Aims are proposed: (1) To investigate at the molecular and cellular level the potential differences in functional dynamics between the wild type human MOR and its N40D variant. (2) To identify differences in the downstream functional consequences of MOR and N40D-variant opioid receptor dynamics in live cells expressing these receptors using electrophysiological and Ca2+ imaging techniques. (3) To extend studies of MOR and N40D-variant to native neurons (cultured hippocampal neurons) where investigations of receptor dynamics and functional coupling can be pursued in live cells within neuronal circuits. PUBLIC HEALTH RELEVANCE: Accumulating evidence suggests that the normal occurrence of mutations in the human mu opioid receptor has consequences for opioid analgesia and drug addiction in humans expressing the variant form. Our understanding of how these mutations affect mu opioid receptor function is limited. To fill this gap in our knowledge we will use a newly developed approach to study the functional properties in both the normal mu opioid receptor and a variant that is commonly expressed in the population and has been implicated in altered sensitivity to drugs that act at the mu receptor.