Mechanism of cryptochrome-mediated photo transduction

隐花色素介导的光转导机制

• 批准号: 8502106
• 负责人: Todd C Holmes
• 金额: $ 26.07万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8502106
• 关键词: Action Potentials; Acute; Aloral; Binding; Biological; Biological Process; Biology; Brain; Butterflies; Cells; Chemicals; Couples; Coupling; Crying; Data; Drosophila genus; Ectopic Expression; Electron Transport; Event; Flavin-Adenine Dinucleotide; Flavins; Genetic; In Situ; Insecta; Ion Channel; Laboratories; Lateral; Light; Mammalian Cell; Measures; Mediating; Membrane; Membrane Potentials; Membrane Proteins; Molecular; Molecular Genetics; Neurons; Opsin; Oxidation-Reduction; Photophobia; Photoreceptors; Phototransduction; Physiological Processes; Potassium; Potassium Channel; Property; Proteins; Publishing; Resolution; Retinal; Signal Transduction; Speed; Structure; Technology; Testing; Time; Tryptophan; Vitamin B Complex; Voltage-Gated Potassium Channel; Work; Xenopus oocyte; base; chromophore; cryptochrome; fly; in vivo; intermolecular interaction; millisecond; mutant; novel; optogenetics; photoactivation; public health relevance; receptor; research study; response; sensor; spatial relationship; technology development; voltage; voltage clamp

DESCRIPTION (provided by applicant): Our laboratory recently discovered that blue light photoactivation of insect Cryptochrome (Cry) cause rapid membrane depolarization and up to 300% increased action potential firing rate over baseline dark spontaneous firing in central brain neurons (Sheeba et al., 2007; Fogle et al., 2011). The electrophysiological light response is robust in the absence of all opsin-based classical photoreceptor inputs (Fogle et al., 2011). Genetically targeted expression of Cry in normally light-insensitive olfactory neurons confers electrophysiological light responsiveness, indicating that Cry expression may be used for optogenetic applications (Fogle et al., 2011). A combination of molecular-genetic and pharmacological experiments indicate that Cry's light sensitivity is mediated through light-activated changes in the redox state of the flavin adenine dinucleotide (FAD) chromophore bound to dCry which then couples to a redox sensor in cytoplasmic potassium channel subunits and modulate potassium channel activity. We propose to extend these findings by determining the precise molecular mechanism of how light activated Cry undergoes an intramolecular transfer of redox state from the flavin chromophore to the protein surface of Cry by testing mutants which lack a well conserved tri-tryptophan motif characterized in other Cry proteins as conducting redox signals. We will then test the hypothesis that redox transfer takes place to target proteins in the membrane. Based on strong preliminary data that membrane coupling of Cry's light activated redox state occurs through voltage gated potassium channels, we will test the hypothesis that dCry then interacts with membrane redox-sensitive effector Hyperkinetic beta subunit (Hk) of voltage-gated potassium (Kv) channels. Our preliminary data indicates that light activation of Cry rapidly modulates cellular potassium currents and depolarizes the membrane potential. We have begun testing this hypothesis and find that the lLNv electrophysiological light response in almost completely abolished in Hk null mutant flies, suggesting that Hk is the primary membrane target for the novel dCry-based phototransduction mechanism. We will determine whether rapid translocation of dCry to the neuronal membrane increases the speed and the amplitude of the electrophysiological light response, as tested using a chemical biology-based inducible strategy. These experiments provide a unique opportunity to unravel a novel non-opsin phototransduction mechanism based on redox sensing. We have also the first opportunity to examine real-time actions of Cry in vivo and the possibility of determining a biological function for the highly conserved redox sensor in KvBeta subunits. As Cry's chromophore, FAD, is the ubiquitously expressed, our work may provide the basis of a new "Vitamin B-based" optogenetic technology applicable to cells that do not synthesize adequate levels of retinal.

描述（由申请人提供）：我们的实验室最近发现，昆虫加密型（CRY）的蓝光光学激活导致膜的快速去极化，并在中央脑神经元中基线深色自发性发射高达300％的动作电势发射速率（Sheeba等人，2007年，2007年； Fogle等； Fogle等； Fogle等； Fogle等，2011）。在没有所有基于OPSIN的经典光感受器输入的情况下，电生理光反应是鲁棒的（Fogle等，2011）。在正常光敏的嗅觉神经元中哭泣的遗传靶向表达赋予电生理光的响应能力，表明CRY表达可用于光遗传学应用（Fogle等，2011）。分子遗传学和药理学实验的组合表明，CRY的光敏度是通过在黄素腺嘌呤二核苷酸（FAD）染色体的氧化还原状态的光激活状态介导的，与DCRY结合，然后在细胞质量钾通道亚undumunits和调节盆腔通道中与Redox传感器结合，然后将其与Redox传感器结合。我们建议通过确定光活化哭泣的精确分子机制来扩展这些发现，从而通过测试缺乏保守良好的三色tryptophan基序在其他Cry蛋白中表征的氧化突变体的氧化还原状态从黄素发色团到哭泣的蛋白质表面的分子转移。然后，我们将检验以下假设：氧化还原转移发生到膜中的靶蛋白。基于强大的初步数据，CRY光激活的氧化还原状态的膜耦合通过电压门控钾通道发生，我们将检验以下假设：DCRY随后DCRY与膜氧化还原效应效应效应β亚基（HK）相互作用。我们的初步数据表明，哭泣的光激活迅速调节细胞钾电流并将膜电位去极化。我们已经开始检验这一假设，发现在HK Null突变蝇中几乎完全消除了LLNV电生理光反应，这表明HK是基于新型DCRY的基于DCRY的光转导机制的主要膜靶标。我们将确定使用基于化学生物学的诱导策略测试的电生理光反应的快速易位是否会增加电生理光反应的速度和幅度。这些实验提供了一个独特的机会，可以基于氧化还原传感来揭示新型的非蛋白质光转导机制。我们也有第一个检查体内哭泣的实时作用的机会，以及确定KVBETA亚基中高度保守的氧化还原传感器的生物学功能的可能性。正如Cry的发色团FAD是普遍表达的那样，我们的工作可能提供了一种新的“基于维生素B的”光遗传学技术的基础，适用于不合成足够水平的视网膜水平的细胞。