Genetic code expansion and the study of CACNG proteins

遗传密码扩展和CACNG蛋白的研究

• 批准号: 10214790
• 负责人: David Malcom MacLean
• 金额: $ 15.4万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214790
• 关键词: AMPA Receptors; Agonist; Amber; Amino Acids; Amino Acyl-tRNA Synthetases; Area; Biological; C-terminal; Cells; Codon Nucleotides; Communication; Complex; Data; Deposition; Development; Electrophysiology (science); Extravasation; Family; Family member; Flow Cytometry; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Future; GTP-Binding Proteins; Genetic Code; Genome; Glutamate Receptor; Goals; Integral Membrane Protein; Investigation; Kinetics; Libraries; Location; Maps; Mass Spectrum Analysis; Measures; Methods; Molecular; Mutation; Optics; Pharmaceutical Preparations; Plasmids; Polyamines; Positioning Attribute; Probability; Property; Protein Family; Proteins; Resources; Site; Structure; Surface; Synapses; Synaptic Transmission; Techniques; Testing; Transfer RNA; UV induced; Variant; Western Blotting; Work; base; biophysical properties; brain cell; cDNA Library; crosslink; desensitization; gain of function; genetic regulatory protein; innovation; insight; kainate; nervous system disorder; novel; operation; patch clamp; programs; protein function; receptor; screening; stable cell line; tool; trafficking; unnatural amino acids

PROJECT SUMMARY - ABSTRACT AMPA receptors are critical components of excitatory synaptic transmission. The current view is that AMPA receptors are nearly always accompanied by one or more types of auxiliary proteins. There are several families of these including the CMPK, GSGL, CNIH and CACNG of which the CACNG is the best studied. The CACNG proteins not only enhance the surface trafficking of AMPA receptors but also act as gain of function proteins, increasing agonist efficacy and potency, open probability, and reducing desensitization. The various CACNG proteins exert these effects to differing extents and thus detailed structure-function analysis of AMPA-auxiliary complexes deepen our understanding of which portions of the channel control these biophysical properties. Moreover, the diversity of CACNG proteins is a molecular handle by which distinct drugs, selective for specific CACNG proteins, can be developed. To further these goals, we will leverage the power of genetic code expansion, which enables non-canonical or unnatural amino acids with advantageous properties to be introduced in specific locations. Within the funding period, we will compile cDNA libraries for 4 CACNG family members which can be used to screen for non-canonical amino acid incorporation. We will also test these positons for leakage and for Bpa incorporation using an innovative fluorescence and FRET based-approach. Finally, we will confirm function using electrophysiology. Together, these resources and data will catalyze the investigation of these IDG-program proteins using genetic code expansion. Further, our efforts may encourage the use of powerful genetic code expansion techniques to study of IDG-proteins by other labs.

项目摘要 - 摘要 AMPA受体是兴奋性突触传播的关键成分。当前的观点是AMPA 受体几乎总是伴有一种或多种类型的辅助蛋白。有几个家庭 其中包括CMPK，GSGL，CNIH和CACNG的CACNG是最好的。 cacng 蛋白质不仅可以增强AMPA受体的表面运输，而且还起到功能蛋白的增益， 提高激动剂的功效和效力，开放概率和降低脱敏。各种cacng 蛋白质将这些作用施加到不同的范围，从而对AMPA-Auxilariary的详细结构功能分析分析 复合物加深了我们对信道哪些部分控制这些生物物理特性的理解。 此外，CACNG蛋白的多样性是一种分子手柄 可以开发CACNG蛋白。为了实现这些目标，我们将利用遗传密码的力量 扩展，可以引入具有优势性能的非典型或非天然氨基酸 在特定位置。在资金期内，我们将为4个CACNG家庭成员编译cDNA库 可用于筛选非经典氨基酸掺入。我们还将测试这些声音的 使用创新的荧光和基于FRET的接口进行泄漏和BPA掺入。最后，我们会的 使用电生理学确认功能。这些资源和数据一起将催化对 这些IDG程序蛋白使用遗传代码扩展。此外，我们的努力可能鼓励使用 其他实验室研究IDG蛋白质的强大遗传代码扩展技术。