Genetic code expansion and the study of CACNG proteins

项目摘要-摘要 AMPA受体是兴奋性突触传递的重要组成部分。目前的观点是AMPA 受体几乎总是伴随着一种或多种辅助蛋白。有几个家庭 其中包括CMPK、GSGL、CNIH和CACNG，其中以CACNG研究最多。中国天然气集团公司 蛋白质不仅促进AMPA受体的表面转运，而且作为功能蛋白的获得， 增加激动剂的效力和效力，开放概率，减少脱敏。各式各样的CACNG 蛋白质在不同程度上发挥这些作用，从而详细分析AMPA辅助物的结构和功能 复合体加深了我们对通道的哪些部分控制这些生物物理特性的理解。 此外，CACNG蛋白的多样性是一个分子句柄，通过它可以区分不同的药物，对特定的药物具有选择性 CACNG蛋白，可以开发。为了实现这些目标，我们将利用遗传密码的力量 扩展，这使得能够引入具有有利性质的非正则或非天然氨基酸 在特定的地点。在资助期内，我们将为CACNG家族的4个成员编制cDNA文库 它可用于筛选非规范氨基酸的掺入。我们还将测试这些位置以确定 利用一种创新的基于荧光和FRET的方法，用于BPA的渗漏和掺入。最后，我们会 使用电生理学确认功能。总而言之，这些资源和数据将催化对 这些IDG-使用遗传密码扩展来编程蛋白质。此外，我们的努力可能会鼓励使用 强大的遗传密码扩展技术，用于其他实验室研究IDG-蛋白质。

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.