Electrophysiological_analysis_Slo1-gamma1

Description

Source data for Inside-out patch clamp using Xenopus leavis oocyte The human KCNMA1 (NM_001014797; WT and mutants) and human LRRC26 (NM_001013653; WT and the R295A mutant) cDNAs were inserted into the pGEMHE expression vector67. The cRNAs were transcribed using the mMESSAGE mMACHINE™ T7 Transcription Kit (ThermoFisher Scientific). Oocytes were surgically taken from female Xenopus laevis anesthetized in water containing 0.15% tricaine (Sigma-Aldrich, E10521) for 15-30 min. They were treated with collagenase (Sigma-Aldrich, C0130) for 6-7 h at room temperature to remove the follicular cell layer. Defolliculated oocytes of a similar size at stage V or VI were selected for cRNA injection. The cRNA was injected into each oocyte using Nanoject II (Drummond Scientific Company). Slo1 and LRRC26 cRNAs were coinjected at a ratio of 1:2. cRNA-injected oocytes were incubated for 2-7 days at 18 °C in MBSH buffer (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, and 0.82 mM MgSO4, pH 7.6) supplemented with 0.1% penicillin-streptomycin solution (Sigma-Aldrich, P4333) The microelectrodes were drawn from borosilicate glass capillaries (Sutter Instruments, B150-86-10) using a P-1000 micropipette puller (Sutter Instrument) to a resistance of 1.5-3.5 MΩ. An agar bridge containing 3M KCl was used as the reference electrode. The pipette and bath solutions were prepared as described previously21. The pipette solution contains 140 mM K-methanesulfonate, 10 mM HEPES, and 2 mM Mg Cl2. The bath solution contains 140 mM K-methanesulfonate, 10 mM HEPES. Both pipette and bath solutions were adjusted to pH 7.0 by KOH. For the recordings in the absence of CaCl2, the bath solution supplemented with 5 mM EGTA was used. For the recordings in the presence of CaCl2, the bath solution supplemented with 1 µM Ca Cl2 (not the free [Ca2+]) was used. Before experiments, the vitelline membrane of oocytes was removed manually. Devitellinized oocytes were placed in a 35 mm dish (Corning) filled with 3 ml of bath solutions. Current recordings were acquired with an Integrated Patch Clamp (IPA) Amplifier (Sutter Instrument), sampled at 50 kHz, and filtered at 10 kHz. All experiments were performed at room temperature. Residual capacitance and leak currents were subtracted by a P/8 protocol at holding potentials of -120 mV for Slo1 alone condtions or -160 mV for Slo1 with LRRC26 conditions

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