Two-electrode voltage clamp

Defolliculated oocytes were purchased from Ecocyte or Xenopus1 and stored in Modified Barth’s Solution (88 mM NaCl, 1 mM KCI, 5 mM Tris-HCI, 1 mM MgSO₄,0.4 mM CaCI₂,0.33 mM Ca(NO₃)₂.2.4 mM NaHCO₃, pH 7.4) supplemented with 0.1 mg ml ¹ gentamycin for up to 1 week at 4 °C until use. For oocyte expression, plasmid DNA was linearized using Notl-HF (NEB, R3189) for 2 h at 37 °C. Linearized DNA was purified using a PCR purification Kit (Qiagen, 28104) and eluted in 30 ml RNase-free water. RNA synthesis was performed with 1–3 μg DNA using the mMessage mMachine T7 Transcription Kit including 15 min of DNase treatment (Ambion, AM1344). RNA was treated with the Zymo Clean & Concentrator Kit and aliquoted at a concentration of approximately 10 μg ml⁻¹ for injection. Oocytes were injected with 25 ng RNA using the Nanoject III (Drummond scientific) and incubated in Modified Barth’s Solution at 17 °C overnight. Two-electrode voltage recordings were carried out at room temperature with an Oocyte Clamp OC-725C amplifier (Warner Instruments) and digitized using a Digidata 1550B (Axon Instruments) interface and pClamp 11 software. Data were filtered at 1 kHz and sampled at 10 kHz. Recordings were performed using borosilicate glass pipettes with resistances of 7–10MΩ when filled with 3 M KCI. All chemicals were diluted in ND96 extracellular solution (96 mM NaCl, 2 mM KCI, 5 mM HEPES, 1 mM MgCl₂,2 mM CaCl₂ adjusted to pH 7.4 with NaOH). Insoluble chemicals were first reconstituted in DMSO, and then diluted in ND96 to 1% final DMSO. Stimulus-evoked currents were obtained using200 ms voltage ramps from −120 mV to 120 mV applied every 500 ms with an interstimulus holding potential of −40 mV. Dose-response relationships were calculated using peak currents measured at −115 mV.