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.

PROJECT NARRATIVE Communication between brain cells often involves proteins known as AMPA receptors and numerous neurological disease states involve AMPA receptor dysfuntion. Interestingly, AMPA receptors are nearly always accompanied by an array of different auxiliary proteins. Using cutting edge molecular biological and optical techniques, we will develop a suite of tools for us and other labs to investigate the structure and function of these auxiliary proteins, potentially developing drugs for specific AMPA-auxiliary complexes.