2.4. Two‐electrode voltage clamp recordings on recombinant human GABAA receptors in Xenopus oocytes

Different subunits of the human GABA_AR, including α1 (NM000806), α2 (NM000807), α3 (NM000808), α4 (NM000809), α5 (NM000810), β2 (NM021911), and γ2 ({"type":"entrez-nucleotide","attrs":{"text":"BC059389","term_id":"37590802","term_text":"BC059389"}}BC059389) were cloned into pcDNA3.1(+) (Invitrogen). GABA_AR subunit mRNA for oocyte injections were prepared by RD‐Biotech (Besançon, France). Xenopus leavis oocytes (stage V–VI), dissected and defolliculated, were purchased from EcoCyte Bioscience (Castrop‐Rauxel, Germany). Microinjections of mRNA for the GABA_AR subunits (α1‐5:β2: γ2; ratio 1:1:5.5) were performed with the automated system Roboocyte (Multi Channel Systems MCS, Reutlingen, Germany) using a total volume ~50 nL mRNA dissolved in RNAse‐free water (Ambion). Injected oocytes were kept at 17°C for 3‐6 days in Barth's solution containing 88 mmol/L NaCl, 2.4 mmol/L NaHCO₃, 1 mmol/L KCl, 0.33 mmol/L Ca(NO₃)₂, 0.41 mmol/L CaCl₂, 0.82 mmol/L MgSO₄, 5 mmol/L Tris/HCl, supplemented with penicillin (100 UI/mL)/streptomycin (100 μg/mL) (Whittaker Cambrex) (adjusted to pH 7.4 with NaOH 1 N).

Two‐electrode voltage clamp (TEVC) recordings were performed with the automated Roboocyte system (Multi Channel Systems MCS) using standard recording protocols. Oocytes were impaled and voltage clamped at a holding potential of −60 mV. After impalement, the oocytes were rinsed with normal frog ringer buffer for 60 s, and allowed to stabilize for a further 60 s. Normal frog ringer solution contained 115 mmol/L NaCl, 2.5 mmol/L KCl, 1.8 mmol/L CaCl₂, 10 mmol/L HEPES (adjusted to pH 7.2 with NaOH 5 N). For each oocyte, GABA was applied twice for 20 s with a 6 min interval to evoke inward Cl⁻ currents and was used as the control GABA current. The concentration–response curve was started by a 6‐min pre‐incubation of Padsevonil (1 nmol/L to 30 μmol/L) followed by co‐application of GABA and Padsevonil for 20 s.

Drugs were applied via a liquid dispenser (Gilson GX271) coupled to a peristaltic pump (Gilson MINIPULS 3). The perfusion rate of oocytes in the 96‐well plates was set at approximately 3 mL/min. All solutions were prepared freshly before each experiment. During measurements, oocytes were perfused with normal frog ringer solution.

GABA‐evoked Cl^‐ currents were analyzed by the Roboocyte Software version 2.2 (Multi Channel Systems MCS). The magnitude of the potentiation of the currents by the drug was calculated based on the ratio I (GABA + drug)/IGABA where I (GABA + drug) is the current response evoked by co‐application of drug and GABA and IGABA represents the current amplitude evoked by GABA alone.

Dose–response curves were analyzed using GraphPad Prism software and EC₅₀ values calculated by nonlinear regression analysis using a sigmoidal dose–response equation, where Y = Bottom + (Top‐Bottom)/(1 + 10^((LogEC₅₀‐X)). Data were normalized to maximum potentiation values of the drug.