Radioligand Binding

Experiments to measure binding of [³H]-epibatidine (PerkinElmer, 32.46 Ci/mmol) to the α4β2 receptor, as well as competition with other ligands, were performed with protein purified as for crystallization but in the absence of ligands. The concentration of binding sites was kept at 0.1 nM after a preliminary experiment to determine optimal receptor concentration. In addition to the protein, the binding assay conditions included 20 mM Tris pH 7.4, 150 mM NaCl, 1 mM DDM, and 1 mg/mL streptavidin-YiSi scintillation proximity assay beads (SPA; GE Healthcare Life Sciences). Non-specific signal was determined in the presence of 100 μM [¹H]-nicotine; all data shown are from background-subtracted measurements. For competition assays [³H]-epibatidine concentration was fixed at 1 nM. All data were analyzed using Prism 6 software (GraphPad) with variable Hill slope. K_i values were calculated based on the experimentally determined K_d of 96 pM for [³H]-epibatidine.