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

描述(由申请人提供)：神经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证实和证明。一系列受体和内源性配体构成了这一系统的分子基础，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，中枢神经系统阿片受体是治疗药物和滥用药物的主要靶点，这使得人们对了解受体的结构、功能和药理学特征产生了浓厚的兴趣。MU阿片受体(MOR)因其在治疗控制疼痛和麻醉成瘾中的核心作用而引起人们的特别关注。人类MOR基因(OPRM1)中的一些多态已经被发现，新出现的研究表明，MOR变异可能在人类对MOR药物敏感性的个体间变异中发挥作用。特别令人感兴趣的是OPRM1 A118G变异体，它在MOR的N-末端区域产生一个氨基酸替代(Asn40Asp)，并编码40Asp突变受体(N40D-变异体)。理解这种突变的功能后果的核心是确定由于突变而导致的MOR信号的功能变化。受体信号传递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受体动力学的研究提供了一种新的方法，荧光相关光谱(FCS)与激光共聚焦扫描显微镜(CLSM)相结合。FCS/CLSM能够以终极的单分子灵敏度实时无损地观察活细胞中的分子相互作用。我们建议利用这种新的方法和平行的生理学分析来研究野生型和N40D变异体的细胞动力学以及这些相互作用对神经生理学的功能后果。我们假设N40D变异体将显示配体依赖的改变的受体动力学，并且改变的受体动力学将导致下游受体耦合到神经生理学的改变。我们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MOR与其N40D变异体在功能动力学方面的潜在差异。(2)利用电生理和钙成像技术，在表达这些受体的活细胞中识别MOR和N40D变异型阿片受体动力学的下游功能后果的差异。(3)将MOR和N40D变异的研究扩展到天然神经元(培养的海马神经元)，在神经元回路中的活细胞中可以进行受体动力学和功能耦合的研究。 与公共卫生相关：越来越多的证据表明，人类MU阿片受体突变的正常发生会对表现出变异形式的人类产生阿片类止痛和药物成瘾的后果。我们对这些突变如何影响u阿片受体功能的了解有限。为了填补这一知识空白，我们将使用一种新开发的方法来研究正常的u阿片受体和一种变异体的功能特性，该变异体在人群中普遍表达，并与作用于u受体的药物的敏感性改变有关。

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.