Synthesis, Characterization, and Testing of Red-Shifted Glutamate Photoswitches

红移谷氨酸光电开关的合成、表征和测试

• 批准号: 8857474
• 负责人: Michael A Kienzler
• 金额: $ 5.8万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8857474
• 关键词: Action Potentials; Agonist; Amides; Amines; Animal Model; Behavior; Binding; Biological; Blindness; Cells; Chemicals; Cysteine; Dyes; Electrons; Engineering; Glutamate Receptor; Glutamates; Goals; Health; Hippocampus (Brain); Individual; Industry; Inherited; Ion Channel; Isomerism; Kainic Acid Receptors; Ligand Binding Domain; Ligands; Light; Literature; Macular degeneration; Maleimides; Metabotropic Glutamate Receptors; Methods; Mus; Nervous system structure; Neurons; Neurosciences Research; Organism; Penetration; Photochemistry; Property; Public Health; Rattus; Retina; Retinal Ganglion Cells; Retinitis Pigmentosa; Series; Shapes; Staging; System; Testing; Time; Tissues; Translating; Ultraviolet Rays; Vertebrate Photoreceptors; Visible Radiation; Vision; analog; azobenzene; base; blind; irradiation; metabotropic glutamate receptor 2; mouse model; multi-electrode arrays; mutant; neurotransmitter release; optical switch; optogenetics; patch clamp; protein function; research study; response; restoration; success; tool

DESCRIPTION (provided by applicant): Promising new tools which enable an individual protein's function to be controlled with light have been developed and applied in the nascent field of optogenetics. Many of these tools are based on light-modulated ion channels which provide unrivaled temporal and spatial control. Recently, a two-component chemical and biological approach based on a Photoswitchable Tethered Ligand (PTL) and a modified ionotropic glutamate receptor 6 (LiGluR) was developed. The PTL Maleimide-Azobenzene-Glutamate (MAG) takes advantage of the remarkable photochemistry of azobenzene, changes its shape (between the cis and trans isomers) depending on the irradiation wavelength used. When MAG covalently binds via the maleimide moiety to an engineered cysteine residue near the ligand binding domain of LiGluR, it allows for the precise control of channel gating. When expressed in neurons, this light-induced current allows for the induction of action potentials with light. Despite the success of LiGluR, the photochemical properties of this optical switch offer many opportunities for improvement. The most significant of which is the requirement of irradiation with UV light (380 nm), which is problematic because it is both toxic to cells and strongly scattered by many tissues, making penetration in larger organisms nearly impossible. The initial goal of this proposal is to synthesize and characterize the photophysical properties of a red- shifted (visible light modulated) MAG analog. Due to extensive use of azobenzene compounds in the dye industry, the literature describes a variety of methods which can be used for the synthesis of red-shifted MAG. The least complicated method involved synthesizing a "push-pull" type azobenzene core by installing an electron donating amine functionality ("push") at one end of the core counterbalanced by an electron withdrawing amide bond ("pull") at the other. The synthesis of such a red-shifted MAG has been completed and preliminary traces from whole-cell patch clamp experiments in HEK293 cells expressing LiGluR have established that the new MAGs function as state dependant tethered agonists. Further synthesis of a related MAG for testing on light-activated metabotropic glutamate receptors (LimGluR2 and 3) is planned. The next stage will be whole-cell patch clamping to test the red- shifted MAG for ability to elicit action potential firing in dissociated rat hippocampal neurons expressing LiGluR and inhibition of action potential firing and neurotransmitter release with LimGluR2. In addition to further developing a useful tool for basic neuroscience research, the creation of visible light activated versions of LiGluR and LimGluR has applications in the retina. Indeed, these visible-light activated tools will be tested for the ability to restore vision in mouse models of inherited blindness by expressing them in retinal ganglion cells and ON-bipolar cells.

描述（由申请人提供）：已经开发出有前途的新工具，这些工具使单个蛋白质的功能能够用光来控制，并应用于光遗传学的新生领域。其中许多工具都基于光调制离子通道，可提供无与伦比的时间和空间控制。最近，开发了一种基于光可转换拴系配体（PTL）和修饰的离子型谷氨酸受体6（LiGluR）的双组分化学和生物学方法。PTL马来酰亚胺-偶氮苯-谷氨酸盐（MAG）利用偶氮苯的显著光化学作用，根据所用的照射波长改变其形状（在顺式和反式异构体之间）。当MAG通过马来酰亚胺部分共价结合到LiGluR的配体结合结构域附近的工程化半胱氨酸残基时，其允许精确控制通道门控。当在神经元中表达时，这种光诱导电流允许用光诱导动作电位。尽管LiGluR取得了成功，但这种光开关的光化学性质提供了许多改进的机会。其中最重要的是需要用UV光（380 nm）照射，这是有问题的，因为它对细胞有毒并且被许多组织强烈散射，使得在较大的生物体中穿透几乎是不可能的。 该提议的最初目标是合成和表征红移（可见光调制）MAG类似物的光物理性质。由于偶氮苯化合物在染料工业中的广泛使用，文献描述了多种可用于合成红移MAG的方法。最不复杂的方法涉及通过在核的一端安装供电子胺官能团（“推”）并通过在另一端的吸电子酰胺键（“拉”）平衡来合成“推拉”型偶氮苯核。这种红移MAG的合成已经完成，并且来自表达LiGluR的HEK 293细胞中的全细胞膜片钳实验的初步迹线已经确定新MAG作为状态依赖性栓系激动剂起作用。计划进一步合成用于光激活代谢型谷氨酸受体（LimGluR 2和3）测试的相关MAG。下一阶段将是全细胞膜片钳以测试红移MAG在表达LiGluR的解离大鼠海马神经元中引发动作电位放电的能力以及用LimGluR 2抑制动作电位放电和神经递质释放的能力。除了进一步开发基础神经科学研究的有用工具外，LiGluR和LimGluR的可见光激活版本的创建在视网膜中具有应用。事实上，这些可见光激活的工具将通过在视网膜神经节细胞和ON双极细胞中表达它们来测试在遗传性失明的小鼠模型中恢复视力的能力。