Xenopus Oocyte Expression and Electrophysiology

X. laevis (Xenopus Express) and X. borealis (Nasco) oocytes were surgically removed from anaesthetised (Tricaine methanesulfonate, MS-222) female frogs and treated with collagenase (1 mg/ml; Sigma type I) for defolliculation. cRNAs were synthesised using an mMessage mMachine cRNA transcription kit (Ambion Inc., Austin, TX, USA) and injected into stage V–VI oocytes at 4–200 ng per cell. Oocytes were stored at 17 °C in ND96 solution (96 mM NaCl, 1.8 mM CaCl₂, 2 mM KCl, 2 mM MgCl₂, 5 mM HEPES, pH 7.4) supplemented with 2.5 mM pyruvic acid, 50 μg/mL gentamicin, and either 2.5% horse serum or 0.5 mM theophylline. Membrane currents were recorded 2–10 days after injection under voltage-clamp (Axoclamp 900A or Geneclamp 500B, Molecular Devices, CA, USA) using two standard glass microelectrodes of 0.5–2 MΩ resistance when filled with 3 M KCl solution. Data acquisition and analysis were performed using pCLAMP software (Version 9.2 or 10, Molecular Devices, Sunnyvale, CA, USA). All experiments were performed at room temperature (18–21 °C) in ND96 solution. Experiments containing peptides were performed in ND96 solution containing 0.1% fatty acid free-bovine serum albumin (Sigma). Recordings were performed as previously described for ASICs30 (link)31 (link), GABA_AR32 (link)33 (link), GlyR34 (link), K_V6 (link)8 (link)35 (link), and Na_V36 (link) channels.