Structure/Function of Channelrhodopsins and Related Retinylidene Proteins

视紫红质通道蛋白和相关视黄基蛋白的结构/功能

• 批准号: 10166003
• 负责人: JOHN LEE SPUDICH
• 金额: $ 62.74万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10166003
• 关键词: Action Potentials; Algae; Anions; Basic Science; Biomedical Research; Cardiac Myocytes; Cations; Cell membrane; Cells; Chlamydomonas reinhardtii; Color; Coupled; Cryoelectron Microscopy; Crystallization; Disease; Distant; Electrophysiology (science); Engineering; Epilepsy; Family; Genetic Techniques; Genome; Heart Diseases; Image; In Vitro; Investigational Therapies; Ion Channel Gating; Ions; Kinetics; Knowledge; Laboratories; Light; Maps; Microbial Rhodopsins; Mining; Molecular; Molecular Conformation; Mutagenesis; Neurons; Neurosciences Research; Optics; Parkinson Disease; Pathway interactions; Physiological; Proteins; Research; Resources; Retinal Pigments; Rhodopsin; Role; Seminal; Spectrum Analysis; Structure; Tachycardia; Techniques; Technology; Therapeutic Clinical Trial; Tissues; Visually Impaired Persons; Work; X-Ray Crystallography; base; brain circuitry; constriction; heart rhythm; in vivo; inhibitor/antagonist; innovation; light gated; microbial; nervous system disorder; neural circuit; optogenetics; painful neuropathy; photoactivation; programs; receptor; response; sight restoration; tool; vibration

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.

我的实验室主要研究微生物视紫红质的结构、功能和机制