3.6. nAChRs Expressed on Xenopus Oocytes

The plasmids of the rat (α3, β2) and human (α7) nAChR subunits were linearized by the corresponding enzymes for in vitro cRNA transcription using the mMessage mMachine kit (Ambion, Austin, TX, USA). The cRNA was purified by the MEGA Clear Kit (Ambion). The cRNA of the nAChR subunits was injected into the Xenopus oocytes (within 59.8 nL). Although the physiological and pharmacological properties of the α3β4 nAChR and α4β2 nAChR isoforms can be affected by the ratio of α to β subunits [40,41], a study in our group found that the stoichiometry of the α3 and β2 subunits has no effect on the α3β2 nAChR. Therefore, for the α3β2 nAChR, the cRNA was mixed in a ratio of 1:1. The injected oocytes were then incubated at 17 °C in an ND-96 buffer with the mixed antibiotic (10 mg/L penicillin, 10 mg/L streptomycin, and 100 mg/L gentamicin) for 1–5 days. Finally, the oocytes expressed rα3β2 and hα7 nAchRs which were harvested.