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

描述(申请人提供)：有希望的新工具，使单个蛋白质的功能可以用光控制已经开发和应用于光遗传学的新领域。这些工具中的许多都是基于光调制离子通道，提供无与伦比的时间和空间控制。最近，一种基于光开关连接配体(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被开发出来。PTL马来酰亚胺-偶氮苯-谷氨酸(MAG)利用偶氮苯显著的光化学性质，根据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与LiGluR配体结合域附近的工程半胱氨酸残基共价结合时，它允许精确控制通道门控。当在神经元中表达时，这种光感应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特性提供了许多改进的机会。其中最重要的是要求用紫外光(380纳米)照射，这是有问题的，因为它既对细胞有毒，又被许多组织强烈散射，使其几乎不可能穿透较大的生物体。这项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应用，文献描述了各种可用于合成红移MAG的方法。最简单的方法是通过在核的一端安装一个电子供给胺官能团(“推”)，在另一端用一个吸电子的酰胺键(“拉”)来平衡，来合成“推-拉”型偶氮苯核。这种红移的MAG的合成已经完成，表达LiGluR的HEK293细胞的全细胞膜片钳实验初步证实，新的MAG具有状态依赖的拴系激动剂的功能。计划进一步合成一种相关的MAG，用于测试光激活的代谢型谷氨酸受体(LimGluR2和3)。下一阶段将是全细胞膜片钳，以测试红移的MAG在表达LiGluR的分离的大鼠海马神经元中诱发动作电位放电的能力，以及用LimGluR2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，创造可见光激活版本的LiGluR和LimGluR在视网膜中也有应用。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双极细胞中表达来测试在遗传性失明小鼠模型中恢复视力的能力。

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.