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

基本信息

批准号：
7773440
负责人：
DONNA L GRUOL
金额：
$ 24.59万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7773440
关键词：
Absence of pain sensation Affect Agonist Amino Acid Substitution Basic Science Binding Cell Line Cells Characteristics Clinical Sensitivity Cloning Code Coupling Cultured Cells Data Development Drug Addiction Drug effect disorder Endorphins Enkephalin, Ala(2)-MePhe(4)-Gly(5)-Family Fentanyl Fluorescence Fluorescence Microscopy Fluorescence Spectroscopy 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 Morphine 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

项目摘要

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）：中枢神经系统（CNS）生物学中神经元阿片信号系统的存在和重要性已在基础研究和临床实践中得到很好的确立和记录。一个受体和内源性配体构成了该系统的分子基础，广泛的文献记录了它们在CNS细胞和系统生物学的正常控制中的作用。此外，已经很好地确定，中枢神经系统阿片类药物受体是治疗剂和滥用药物的主要靶标，这引起了人们对对受体结构，功能和药理特征的理解的重大兴趣。 MU阿片受体（MOR）特别令人感兴趣，因为它在疼痛的治疗控制和麻醉性成瘾中具有核心作用。已经确定了人类基因中的许多多态性（OPRM1），并且新兴的研究表明，MOR变体可能在人类对人类对在MOR的药物的敏感性的有据可查的个人间变异性发挥作用。尤其令人感兴趣的是OPRM1 A118G变体，该变体在MOR（ASN40ASP）的N末端结构域（ASN40ASP）中产生单个氨基酸取代，并编码40ASP突变受体（N40D-VARIANT）。理解该突变功能后果的核心是鉴定由于突变引起的MOR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，它决定了受体激活的受体可用性，功能和下游后果。现在，分子成像的最新进展为研究受体动力学，与共聚焦激光扫描显微镜（CLSM）集成的新方法，荧光相关光谱（FCS）。 FCS/CLSM可以实时对活细胞中分子相互作用进行无损观察，并具有最终的单分子敏感性。我们建议利用这种新方法和并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与神经元生理学的功能后果。我们假设N40D变体将显示出依赖配体的受体动力学改变，并且受体动力学的改变将导致受体偶联与神经生理学的下游改变。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变体之间功能动力学的潜在差异。（2）使用电生理学和CA2+成像技术表达这些受体的活细胞中MOR和N40D变异的阿片受体动力学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），其中可以在神经元电路内的活细胞中进行对受体动力学和功能偶联的研究。公共卫生相关性：积累的证据表明，人类Mu阿片类受体突变的正常发生对表示变异形式的人类中的阿片类镇痛和药物成瘾有后果。我们对这些突变如何影响阿片类药物受体功能的理解受到限制。为了填补我们的知识，我们将使用新开发的方法来研究正常MU阿片受体的功能特性和一种通常在人群中表达的变体，并且与对MU受体作用的药物的敏感性的改变有关。