Mechanism of cryptochrome-mediated photo transduction

描述(申请人提供)：我们的实验室最近发现，蓝光激活昆虫隐色素(Cry)可导致细胞膜快速去极化，并使中央大脑神经元的动作电位放电频率比基线暗自发放电增加300%(Sheeba等人，2007年；福格尔等人，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，电生理光反应是强劲的(Fogle等人，2011年)。在正常光不敏感的嗅神经元中，Cry的基因靶向表达赋予了电生理光反应，表明Cry的表达可能被用于光遗传学应用(Fogle等人，2011年)。分子遗传学和药理学实验相结合的结果表明，Cry的光敏感性是通过与dCry结合的黄素腺嘌呤二核苷酸(FAD)发色团的光激活改变来介导的，然后DCry与细胞质钾通道亚基中的氧化还原传感器偶联，调节钾通道的活性。我们建议通过测试缺乏与其他Cry蛋白相同的保守的三色氨酸基序的突变体作为氧化还原信号的传导来扩展这些发现，确定光激活的Cry如何经历氧化还原状态从黄素发色团到Cry蛋白表面的分子内转移的确切分子机制。然后，我们将测试氧化还原转移发生在膜上的目标蛋白的假设。根据强大的初步数据，Cry的光活化氧化还原状态的膜偶联通过电压门控钾通道发生，我们将检验dCry随后与电压门控钾通道的膜氧化还原敏感效应超动力β亚基(HK)相互作用的假说。我们的初步数据表明，Cry的光激活迅速调节细胞的钾电流，并使膜电位去极化。我们已经开始验证这一假说，发现在HK缺失突变的果蝇中，lLNv的电生理光反应几乎完全消失，这表明HK是基于dCry的新的光传导机制的主要膜靶点。我们将确定dCry快速移位到神经细胞膜是否会增加电生理光反应的速度和幅度，这是使用基于化学生物学的诱导策略进行的测试。这些实验为揭示一种基于氧化还原传感的新型非视素光传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学功能的可能性。由于Cry的发色团FAD是普遍表达的，我们的工作可能会为一种新的基于维生素B的光基因技术提供基础，该技术适用于不能合成足够水平的视网膜的细胞。

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.