5.8. nAChR Competition Radioligand Assay

The competition binding assays with radio-iodinated α-bungarotoxin (¹²⁵I-α-Bgt) were performed as in [37]. Briefly, suspension of GH₄C₁ cells stably transfected with human α7 nAChR (0.4 nM αBgt binding sites) were incubated in 50 μL binding buffer (20 mM Tris-HCl, pH 8.0, containing 1 mg/mL bovine serum albumin) for 90 min with various amounts of toxins. Thereafter, 0.1–0.2 nM ¹²⁵I-α-Bgt (500 Ci/mmol) was added, and after an additional 5 min incubation, cell suspensions were applied to GF/C glass filters (Cytiva, Marlborough, MA, USA) pretreated with 0.3% polyethyleneimine. The samples were then washed (3 × 4 mL) with 20 mM cold Tris-HCl buffer, pH 8.0, containing 0.1 mg/mL bovine serum albumin and bound radioactivity was measured with a Wallac 1470 Wizard Gamma Counter (PerkinElmer, Waltham, MA, USA). Nonspecific ¹²⁵I-αBgt binding was determined in the presence of 200-fold excess of α-Ctx. The competition binding assays with AChBPs were performed as described in [38].