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

mu-阿片受体变体的综合结构/功能分析

• 批准号: 8033237
• 负责人: DONNA L GRUOL
• 金额: $ 19.05万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8033237
• 关键词: Absence of pain sensation; Affect; Agonist; Amino Acid Substitution; Basic Science; Binding; Cell Line; Cells; Cellular biology; Characteristics; Clinical Sensitivity; Cloning; Code; Coupling; Cultured Cells; Data; Development; Drug Addiction; Drug Receptors; Drug effect disorder; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Family; Fentanyl; Fluorescence; Fluorescence Microscopy; Fura-2; Genes; Genetic Polymorphism; Goals; Grant; Hippocampus (Brain); Human; Image; Imaging Techniques; Imaging technology; Individual; Individual Differences; Investigation; Ion Channel; Knowledge; Label; Laboratories; Laser Scanning Confocal Microscopy; Lead; Life; Ligands; Literature; Mediating; Medicine; Methadone; Methodology; Modeling; Molecular; Mus; Mutation; N-terminal; Naltrexone; Narcotic Addiction; Neuraxis; Neurons; Opioid; Opioid Peptide; Opioid Receptor; PC12 Cells; Pain management; Pharmaceutical Preparations; Physiological; Physiology; Play; Population; Positioning Attribute; Property; Protein Isoforms; Proteins; Protocols documentation; Receptor Activation; Receptor Signaling; Research; Research Design; Role; Signal Transduction; Spectrum Analysis; System; Systems Biology; Testing; Therapeutic; Therapeutic Agents; Time; Translating; Variant; base; clinical practice; drug of abuse; drug testing; endogenous opioids; fluorophore; interest; molecular imaging; mu opioid receptors; mutant; neurophysiology; new technology; novel strategies; public health relevance; receptor; receptor coupling; receptor function; receptor structure function; response; single molecule

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.

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已在基础研究和临床实践中得到充分证实和记录。一个家族的受体和内源性配体形成了这个系统的分子基础，大量的文献已经证明了它们在CNS细胞和系统生物学的正常控制中的作用。此外，已经确定CNS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、功能和药理学特征的理解的极大兴趣。μ阿片受体（莫尔）由于其在疼痛的治疗控制和麻醉剂成瘾中的中心作用而受到特别关注。已经鉴定了莫尔的人类基因（OPRM 1）中的许多多态性，并且新兴的研究表明，莫尔变体可能在人类对以莫尔起作用的药物的敏感性的充分记录的个体间变异性中起作用。特别令人感兴趣的是OPRM 1 A118 G变体，它在莫尔的N末端结构域中产生单个氨基酸取代（Asn 40 Asp）并编码40 Asp突变受体（N40 D变体）。理解这种突变的功能后果的核心是鉴定由于突变引起的莫尔信号传导的功能变化。受体信号传导的一个关键方面是受体动力学，其决定受体可用性、功能和受体活化的下游后果。分子成像的最新进展现在提供了一种新的方法来研究受体动力学，荧光相关光谱（FCS）与共聚焦激光扫描显微镜（CLSM）集成。FCS/CLSM能够真实的实时无损观察活细胞中的分子相互作用，具有极高的单分子灵敏度。我们建议利用这种新的方法和并行的生理分析来研究野生型和N40 D-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N40 D变体将显示配体依赖性的受体动力学改变，并且受体动力学改变将导致受体与神经生理学偶联的下游改变。本研究的目的有三：（1）从分子和细胞水平研究人莫尔与其N40 D变异体在功能动力学上的差异。(2)使用电生理学和Ca 2+成像技术确定表达这些受体的活细胞中莫尔和N40 D变体阿片受体动力学的下游功能后果的差异。(3)将莫尔和N40 D变体的研究扩展到天然神经元（培养的海马神经元），其中可以在神经元回路内的活细胞中进行受体动力学和功能偶联的研究。 公共卫生关系：越来越多的证据表明，人类μ阿片受体突变的正常发生对表达该变体形式的人类的阿片镇痛和药物成瘾具有影响。我们对这些突变如何影响μ阿片受体功能的理解是有限的。为了填补我们知识中的这一空白，我们将使用一种新开发的方法来研究正常μ阿片受体和一种变体的功能特性，该变体通常在人群中表达，并与对作用于μ受体的药物的敏感性改变有关。