Mechanism of cryptochrome-mediated photo transduction

隐花色素介导的光转导机制

• 批准号: 8706189
• 负责人: Todd C Holmes
• 金额: $ 27.64万
• 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/8706189
• 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，昆虫隐花色素（Cry）的蓝光光激活会导致膜快速去极化，并且与中枢神经元的基线暗自发放电相比，动作电位放电率增加高达 300%（Sheeba 等人，2007 年；Fogle 等人，2011 年）。在没有所有基于视蛋白的经典光感受器输入的情况下，电生理光响应是稳健的（Fogle 等人，2011）。通常对光不敏感的嗅觉神经元中 Cry 的基因靶向表达赋予电生理光响应性，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相结合表明，Cry 的光敏感性是通过与 dCry 结合的黄素腺嘌呤二核苷酸 (FAD) 生色团的氧化还原状态的光激活变化来介导的，然后与细胞质钾通道亚基中的氧化还原传感器偶联并调节钾通道活性。我们建议通过测试缺乏在其他 Cry 蛋白中传导氧化还原信号的良好保守三色氨酸基序的突变体，确定光激活的 Cry 如何经历从黄素发色团到 Cry 蛋白表面的氧化还原态分子内转移的精确分子机制，从而扩展这些发现。然后我们将测试氧化还原转移发生到膜中的目标蛋白质的假设。基于 Cry 光激活氧化还原态的膜耦合通过电压门控钾通道发生的强有力的初步数据，我们将测试 dCry 随后与电压门控钾 (Kv) 通道的膜氧化还原敏感效应器 Hyperkinetic beta 亚基 (Hk) 相互作用的假设。我们的初步数据表明，Cry 的光激活可快速调节细胞钾电流并使膜电位去极化。我们已经开始测试这一假设，发现 Hk 无效突变果蝇中的 lLNv 电生理光反应几乎完全消失，这表明 Hk 是基于 dCry 的新型光转导机制的主要膜靶点。我们将确定 dCry 快速易位到神经元膜是否会增加电生理光反应的速度和幅度，正如使用基于化学生物学的诱导策略所测试的那样。这些实验为揭示基于氧化还原传感的新型非视蛋白光转导机制提供了独特的机会。我们还首次有机会检查 Cry 在体内的实时作用，以及确定 KvBeta 亚基中高度保守的氧化还原传感器的生物功能的可能性。由于 Cry 的发色团 FAD 是普遍表达的，我们的工作可能为一种新的“基于维生素 B”的光遗传学技术提供基础，该技术适用于不能合成足够水平视网膜的细胞。