4.4. Electrophysiological Activity on Different nAChRs

The samples were dissolved in ND-96 solution (96.0 mM NaCl, 1.0 mM MgCl₂, 2.0 mM KCl, 1.8 mM CaCl₂, 5mM HEPES, pH 7.1–7.5) for use. The plasmids of mouse (α1, β1, δ and ε) and rat (α3, α4, α6/α3, α7, α9, α10, β2, β3 and β4) nAChR subunits were linearized by 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 different nAChR subunits were combined in a specific ratio and injected into Xenopus oocytes (within 59.6 nL). The cRNA-injected oocytes were incubated at 17 °C in ND-96 buffer with an antibiotic (10 mg/L penicillin, 10 mg/L streptomycin and 100 mg/L gentamicin) for 1–5 days. Then the oocyte was fixed in the oocyte chamber (a cylindrical well, 50 µL in volume) and ND-96 solution (1 µM atropine and 0.1 mg/mL bovine serum albumin) was gravity-perfused at a rate of 2 mL/min. The membrane currents of the Xenopus oocytes expressed different subtypes of nAChR and were recorded by a two-electrode voltage clamp. The membrane potential of the oocytes was clamped at −70 mV. Two seconds of 100 µM ACh and another 58 seconds of ND-96 were applied to the oocyte, which was repeated until a stable baseline was obtained three times. Next, the oocytes were incubated in either ND-96 or different concentrations of conotoxins in ND-96 for 5 min, followed by Ach stimulation (2 s 100 µM Ach and 58 s ND-96) repeatedly. The membrane currents were recorded by a two-electrode voltage clamp at room temperature. All the nAChR inhibitory activities were tested in this method.