Structure/Function of Channelrhodopsins and Related Retinylidene Proteins

我的实验室专注于微生物视紫红质的结构、功能和机制，广泛的视觉 具有多种功能的色素样蛋白。在过去的十年里，一个亚家族，光门控离子通道， （通道视紫红质），由于其在变革性技术中的核心作用， 光遗传学我们最初发现它们在叶绿素植物莱茵衣藻中作为趋光性 通过对光产生阳离子电流而使细胞膜脱附的受体。随后 神经科学家发现，这些在神经元中表达的光门控阳离子通道视紫红质（CCR）产生 使光能够触发动作电位的去极化电流。神经元的靶向光激活 通过在神经回路中表达CCR已经被证明是一种强大的技术， 神经科学研究。然而，它们的光门控通道活性是最不了解的视紫红质之一， 在分子机制方面发挥作用。在过去五年中，我们的工作取得了一些进展， 数十年来对微生物视紫红质研究的知识和专业知识，指导我们目前的研究战略。 在2015年，我们发现了唯一的阴离子传导（生理Cl-）通道视紫红质（ACR）在 隐藻的一个遥远的门。作为光遗传学的一项突破，ACR能够实现高效的光诱导 因此是神经元放电的有效抑制剂。对我们的研究计划也有重大影响， 光遗传学中最常用的ACR（来自Guillardia theta的GtACR 1）的最新晶体结构揭示了 我们提出的处于封闭黑暗状态的预先存在的隧道是由3个明确限定的收缩部封闭的通道。 GtACR 1通道是在通道视紫红质中成像的唯一候选离子通路，并且提供了有价值的 资源，用于阐明光通道门控的奥秘。从我们的研究中学到的原则可能会 也增强了我们对其他微生物视紫红质的理解。我们目前的研究调查了 和通道视紫红质的分子机制：（i）正在进行的基因组挖掘，以扩大我们的知识， 也推进光遗传学，重点是ACR，但包括CCR（例如可能的K+和Ca++通道）。 最近，我们鉴定了两个新的ACR家族和长期寻找的由细胞因子激活的红移ACR（“RubyACR”）。 穿透组织的长波长，对光遗传学有价值，并开辟了阐明颜色调谐的途径 通道视紫红质的机制;（ii）解开通道功能中的电步骤与 通过基于结构的诱变、体内光电生理学和动力学光学 和体外振动光谱;和（iii）通过X射线晶体学测定原子结构， cryoEM，包括创新的方法来成像的瞬时开放通道构象。阐明 通道视紫红质的机制将推进基础科学，也有利于工程优化， 为新的光遗传学应用定制它们。

My laboratory focuses on the structure, function, and mechanisms of microbial rhodopsins, widespread visual pigment-like proteins with diverse functions. Over the past decade, a subfamily, light-gated ion channels (channelrhodopsins), have had exceptional impact because of their central role in the transformative technology of optogenetics. We originally found them in the chlorophyte alga Chlamydomonas reinhardtii as phototaxis receptors that depolarize the cell membrane by producing cation currents in response to light. Subsequently neuroscientists found that these light-gated cation channelrhodopsins (CCRs) expressed in neurons produce depolarizing currents that enable light to trigger action potentials. Targeted photoactivation of neurons enabled by expression of CCRs in neural circuits has proven to be a powerful technique transforming many aspects of neuroscience research. Nevertheless, their light-gated channel activity is one of the least understood rhodopsin functions in terms of molecular mechanisms. Several advances in our work over the past 5 years, coupled to our knowledge and expertise over decades of research on microbial rhodopsins, guide our current research strategy. In 2015 we discovered exclusively anion-conducting (physiologically Cl-) channelrhodopsins (ACRs) in the distant phylum of cryptophyte algae. A breakthrough for optogenetics, ACRs enable efficient light-induced hyperpolarization and therefore are potent inhibitors of neuron firing. Also seminal to our research plans, our recent crystal structure of the most used ACR in optogenetics (GtACR1 from Guillardia theta) revealed a preexisting tunnel in the closed dark state that we propose is the channel closed by 3 well-defined constrictions. The GtACR1 tunnel is the only candidate ion pathway imaged in a channelrhodopsin, and provides a valuable resource for elucidating the mystery of channel gating by light. Principles learned from our study will likely enhance our understanding also of other microbial rhodopsins. Our current research investigates the diversity and molecular mechanisms of channelrhodopsins by: (i) ongoing genome mining to expand our knowledge and also advance optogenetics, focused on ACRs, but including CCRs (e.g. possible K+ and Ca++ channels). Recently we identified two new ACR families and long-sought red-shifted ACRs (“RubyACRs”) activated by tissue-penetrating long wavelengths, valuable for optogenetics and opening the way to elucidating color tuning mechanisms of channelrhodopsins; (ii) unraveling the relationship of electrical steps in channel function to photochemical transitions by structure-based mutagenesis, photo-electrophysiology in vivo, and kinetic optical and vibrational spectroscopy in vitro; and (iii) determination of atomic structures by X-ray crystallography and cryoEM, including innovative approaches to image the transient open-channel conformation. Elucidating mechanisms of channelrhodopsins will advance basic science and also facilitate engineering to optimize and tailor them for new optogenetic applications.