3h epibatidine

(±)-[³H]Epibatidine (specific activity of 56–60 Ci/mmol) was purchased from Perkin Elmer (Boston, MA, United States). Non-radioactive epibatidine was purchased from Sigma-Aldrich. Saturation experiments were performed by incubating aliquots of membranes from HEK293 cells expressing α2β4 or α2^Tyr252Hisβ4 nAChR with 0.01–5 nM concentrations of (±)-[³H]Epibatidine (Perkin Elmer) overnight at 4°C. Non-specific binding was determined in parallel by incubation in the presence of 100 nM unlabeled epibatidine. After incubation, the samples were filtered on GFC filters soaked in 0.5% polyethyleneimine and washed with 15 mL ice-cold phosphate buffered saline (PBS) and the filters were counted for radioactivity in a β counter.

Groups of 8 oocytes were homogenized in 1 ml of buffer A (50 mM NaCl, 50 mM sodium phosphate buffer, pH 7.5, 5 mM EDTA, 5 mM EGTA, 5 mM benzamide, 5 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride). Then a crude membrane fraction was pelleted by centrifugation for 15 minutes at 13,400 rpm. Membrane proteins were resuspended by pipetting and solubilized in 150 µl of buffer A containing 2% Triton X-100 for 1 hour at room temperature. Debris was removed by centrifugation at 13,400 g for 15 min. Next, mAb 295 coated wells were loaded with aliquots of detergent extracts with 2 nM ³H epibatidine (PerkinElmer Life Sciences, Emeryville, CA) in a total volume of 100 µL in phosphate-buffered saline (PBS) buffer containing 0.5% Triton X-100 and 10 mM NaN₃[12] . These plates were left overnight on a shaker at 4°C. Wells were then washed three times with 0.5% Triton X-100 in PBS. ³H epibatidine bound to AChRs bound to the wells through mAb 295 or mAb 210 was eluted with 30 µl of 0.1 M NaOH and quantitated by liquid scintillation counting [22] (link). Nonspecific binding was determined using non-injected oocytes.

[³H]Tropisetron was synthesized (Metis Laboratories Inc, NY, USA) from its desmethyl analogue (0.5 mg) in a tritiomethylation reaction by heating at 80 °C for 2 h with 50 mCi [³H]methyl iodide in tetrahydrofuran (THF) (0.4 ml). After evaporation of the THF the residue was subjected to reversed phase chromatography (Kromasil 100 C18, 7 μm, 250 × 10 mm, 2 mL min⁻¹) using 31% ACN in 0.1% TFA as eluent. The eluent was stripped off and the residue was dissolved in 12 mL absolute ethanol. The [³H]tropisetron had a specific activity of 73.5 Ci/mmol and radiochemical purity of >99%. [³H]granisetron (63.5 Ci/mmol) and [³H]epibatidine (55.8 Ci/mmol) were purchased from Perkin Elmer (Waltham, MA) and were >97% chemical purity.