Precrystallization Screening of a GABA-rho (GABAc) receptor

The GABA neurotransmitter is the major inhibitory neurotransmitter in the vertebrate brain, and targets include the GABA receptors. One of the targets of GABA is the GABAC, or GABA-rho, receptor, a ligand gated ion channel and member of the Cys-loop family of receptors. The GABA-rho receptor is located on the axonal terminal of retinal bipolar cells, regulating vision signaling and has been implicated in several retinal disorders; such as retinal leakage. These inhibitory ion channels could be the targets for therapies in these vision disorders. One limitation to novel drug design is that the complete three-dimensional structure of the GABA-rho receptor remains elusive. Our goal is to determine the three-dimensional structure of the GABA-ρ1 receptor. To do this, we have generated a GABA-rho receptor with a C-terminal histidine tag for our studies. Furthermore, we have used metal affinity chromatography and size exclusion chromatography to purify the GABA-rho receptor, a pentameric protein.  Thus far, we are able to isolate microgram quantities of the receptor in the n-Dodecyl-beta-D-Maltopyranoside (C12M) solubilization buffer, which was found to be the optimal buffer for expression. Within the next several months, we will isolate milligram quantities of GABA-rho receptor in this buffer for setting up hanging drop vapor diffusion crystallization trials and x-ray diffraction data collection. Obtaining the GABA-rho receptor structure through crystallization will provide a template for the drug design of therapeutics, which could target these specific receptor subunits.