Intravenous bolus injection of 479.6 MBq ± 25.6 [11C]Ro15 4513

Human Embryonic kidney 293 were transfected with Recombinant GABAA Receptors. Approximately 60 h after transfection, the cells were harvested by washing with ice-cold phosphate- buffered saline (130 mM NaCl, 20 mM Na2HPO4, and 4 mM KH2PO4, pH 7.4) and centrifuged at 560 x g. Two more washing steps were performed with phosphate buffer (10 mM KH2PO4, 100mMKCl, and 0.1mMK-EDTA,pH7.4). Cells were sonicated in the presence of 700 mkM phenylmethylsulfonyl fluoride and 1mM EDTA. The membranes collected by three centrifugation resuspension cycles (100,000 x g for 20 min) were then used for ligand binding. Membranes were resuspended in phosphate buffer using a Teflon homogenizer. They were incubated in a total volume of 360 mkl for 1 h on ice in the presence of [3H]Ro15-1788 (78.6 Ci/mmol; PerkinElmer Life Sciences) or [3H]flunitrazepam (71–84 Ci/mmol; PerkinElmer Life Sciences). The final protein concentration was 0.1–1 mg of protein/ml. Total binding was measured at 2 and 20 nM [3H]Ro15-1788 or [3H]flunitrazepam. Nonspecific binding was determined under the same condition but in the presence of 100 mkM unlabeled Ro15-1788 or flunitrazepam, respectively. To perform displacement binding assays, various concentrations of competing ligands (RO 15-4513) were added. The concentration of [3H]Ro15-1788 used was 3 times the Kd value.