Structural Insights into GluCl Receptor

GluCl_cryst was expressed from baculovirus-infected Sf9 cells and purified by metal ion affinity chromatography. The Fab complex was isolated by size-exclusion chromatography. The GluCl_cryst-Fab complex was concentrated to 1-2 mg/mL and supplemented with synthetic lipids and ivermectin. Crystallization was performed by hanging drop vapor diffusion at 4°C with a precipitating solution containing 21-23% PEG 400, 50 mM sodium citrate pH 4.5 and 70 mM sodium chloride. Cryoprotection was achieved by soaking crystals in precipitant solution supplemented with 30% PEG 400. Additional complexes were obtained by soaking crystals in cryoprotectant containing L-glutamate, picrotoxin or sodium iodide. Diffraction data were indexed, integrated and scaled and the structure solved by molecular replacement using a GLIC-derived homology model of GluCl_cryst and a Fab homology model as search probes. The molecular replacement phases were used to initiate autobuilding and the resulting model was iteratively improved by cycles of manual adjustment and crystallographic refinement. Function of GluCl was examined by two-electrode voltage clamp experiments and by [³H]-L-glutamate saturation and competition binding assays.