Geneclamp 500b

ERG was performed as previously described (37 (link)). Flies were anesthetized with CO₂ and immobilized on a glass slide. A recording electrode with 3M NaCl was placed on the surface of the left eye, and another reference electrode was inserted into the thorax. After 5 minutes of dark adaptation, flies were given a 1-second light stimulus (Digitimer). The response was amplified unipolarly by GeneClamp 500B (Axon Instruments), and the traces were recorded and analyzed by pCLAMP 10 Electrophysiology Data Acquisition & Analysis Software (ver.10.5).

Whole cell membrane currents of oocytes were measured using a two-electrode voltage clamp (Gene Clamp 500B, Axon Instruments/Molecular Devices Sunnyvale, CA, USA). Glass pipettes were pulled using a P-97 Flaming/Brown type puller (Sutter, Novato, CA, USA). The recording chamber was perfused continuously with frog Ringer (OR) solution (in mM: 82.5 NaCl, 2.5 KCl, 1 CaCl2, 1 MgCl2, 1 Na2HPO4, and 5 HEPES, pH 7.5). Membrane conductance was determined using voltage pulses, and the pulse-induced current amplitudes were divided by the amplitudes of the voltage steps. For calculation of % inhibition, leak currents were subtracted. Typically, leak currents after the interventions were smaller than at the beginning of the experiment, and this smaller value was used for the subtraction to avoid overestimation of the inhibitory effect. For an independent measure of the leak current, oocytes at the end of the experiment were exposed to 100 µM carbenoxolone, which is known to close Panx1 channels 100%. Oocytes expressing Panx1 were held at −60 mV, and pulses to +60 mV were applied to transiently open the channels by means of the voltage gate. Pulses 5 s in duration were applied at 0.1 Hz for current and conductance measurements.

Gating pore currents through mutant Na_V1.4 channels were studied using two-electrode voltage clamp in Xenopus oocytes. All procedures were carried out as described previously (Männikkö et al., 2018 (link)). Briefly, oocytes for Na_V1.4 expression were isolated from Xenopus laevis in accordance with the UK Animal (Scientific Procedures) Act 1986. The oocytes were injected with rat SCN4A encoding R669G mutant channel (analogous to human R675G mutation) and SCN1B messenger RNA transcribed in vitro (mMESSAGE mMACHINE kit, Thermo Fisher Scientific) at a 1:1 mass ratio using Nanoject (Drummond). GeneClamp 500B, Digidata 1200, and pCLAMP programs (Molecular Devices) were used to collect data. The bath solution contained 60-mM sodium methanesulfonate, 60-mM guanidine sulfate, 1.8-mM CaSO₄, and 10-mM Hepes (pH 7.4), and the oocytes were perfused with 1–2-μM tetrodotoxin (TTX) to block the main pore. To measure gating pore currents, the mean current during the last 100 ms of a 300-ms step to voltages ranging from −140 mV to +50 mV in 5-mV increments was plotted against the voltage. Holding voltage was -100 mV. Data were analyzed and presented using pCLAMP (Molecular Devices) and Origin (OriginLab) software. All data are presented as mean ± SEM.

Animal protocols used in this study were approved by the IACUC at Academia Sinica or University of California, San Francisco, and Xenopus oocytes were prepared as described previously.⁴⁷ (link) Briefly, stage V–VI Xenopus laevis oocytes were harvested, injected with 30 ng of each cRNA, and incubated at 16°C for 2–4 days before recording. hKir6.2 and hSUR1 were cloned into the pGEM vector. hKir6.2 and hSUR1 cDNAs were first linearized by restriction enzyme digestion, and cRNA was then synthesized by using the mMESSAGE mMACHINE SP6 Transcription kit (Thermo-Fisher, USA). Macroscopic currents were recorded from oocytes with two-electrode voltage clamp (GeneClamp 500B, Molecular Device, USA). Electrodes were filled with 3 M KCl and had a resistance between 0.4–1 MΩ. A small chamber with a fast perfusion system (AutoMate Scientific, USA) was used to change extracellular ND96 solution containing: 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES and pH 7.4, adjusted with NaOH (in some cases, the Na⁺ was replaced with equimolar K⁺ to keep the osmolarity constant).

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.