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，广泛存在于 具有多种功能的色素样蛋白。在过去的十年里，一个亚家族，光门离子通道 (通道视紫红质)，由于它们在变革性技术中的核心作用，产生了特殊的影响 光遗传学的研究。我们最初在绿藻衣藻中发现它们是趋光性的。 通过对光产生阳离子电流来使细胞膜去极化的受体。随后 神经学家发现，在神经元中表达的这些光门控阳离子通道视紫红质(CCRs)会产生 使光能够触发动作电位的去极化电流。启用神经元的靶向光激活 通过在神经电路中表达CCR已被证明是一种强大的技术，可以在许多方面改变 神经科学研究。然而，它们的光门通道活动是最不了解的视紫红质之一。 在分子机制方面发挥作用。在过去的5年里，我们的工作取得了一些进展，加上我们的 几十年来对微生物视紫红质研究的知识和专业知识指导着我们目前的研究战略。 2015年，我们在大脑中发现了独一无二的阴离子传导(生理氯离子)通道视紫红质(ACRs)。 遥远的隐芽藻门。光遗传学的突破，ACR使高效的光诱导成为可能 超极化，因此是神经元放电的有效抑制物。对我们的研究计划也是至关重要的，我们的 光遗传学中最常用的ACR(GtACR1来自Guillardia theta)的最新晶体结构揭示了 我们提出的处于封闭暗状态的预先存在的隧道是由3个明确定义的收缩关闭的通道。 GtACR1隧道是视紫红质通道中唯一的候选离子通路，它提供了一个有价值的 用于阐明光门通道之谜的资源。从我们的研究中学到的原则很可能 也增进了我们对其他微生物视紫红质的了解。我们目前的研究调查的是 和视紫红质通道的分子机制：(I)正在进行的基因组挖掘以扩大我们的知识和 先进的光遗传学，侧重于ACR，但包括CCR(例如，可能的K+和Ca++通道)。 最近我们发现了两个新的ACR家族和长期寻求的红移ACR(“RubyACRs”)激活 组织穿透长波长，对光遗传学有价值，并为阐明颜色调谐开辟了道路 通道视紫红质的机制；(Ii)解开通道功能中的电步骤与 基于结构的诱变的光化学转变、活体光电生理学和动力学光学 以及(Iii)用X射线结晶学和电子能谱测定原子结构。 包括成像瞬时明渠构象的创新方法。澄清 通道视紫红质的机制将促进基础科学的发展，也有助于工程优化和 为新的光遗传应用量身定做它们。

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.