PRE-CRYSTALLIZATION SCREENING OF A GABA-A RECEPTOR

The Gamma-aminobutyric acid type A (GABA-A) receptor is a pentameric ligand gated ion channel that mediates inhibitory neurotransmission. GABA binds to the extracellular domain of the receptor, triggering the opening of the chloride ion-selective pore. Channel mutations have been associated with Angelman syndrome, Prader-Willi syndrome, and autism. We are working to determine the 3-D structure of the homomeric GABA-A receptor using fluorescence detection size-exclusion chromatography (FSEC). Two forms of the receptor are used; a full length and a shortened GABA-A receptor. Both of these have an internal green fluorescent protein (GFP). We have transfected HEK293t cells to observe fluorescence, indicating expressed GABA-A chimeric receptors.  FSEC was used to monitor the elution and oligomerization of chimeric receptors.  Conditions such as time, and buffers were varied to assess the expression of the pentameric conformation. A time-course experiment was performed at 24, 48, 72, and 96 hours to see the progression of expression of the two GABA-A constructs in HEK293t cells and SF9 cells. Each sample was analyzed using FSEC. Both constructs had the most pronounced peaks at 96 hours in HEK293t cells, with the detergents undecyl-β-d-maltoside and LDAO respectively. Each receptor was then sub-cloned into SF9 cells and assayed using FSEC. Next in SF9 cells we concluded that 72 hours is the optimum time period for each receptor, and is suitable for large scale growth. Large amounts of cultures of each construct will be produced, harvested at 72 hours, and used to obtain membrane for purification and crystallization of the protein.