Electrophysiological_analysis_Slo1-gamma1

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

来源数据集：基于 Xenopus laevis 胚囊的 Inside-out Patch Clamp 实验数据。
人类 KCNMA1（NM_001014797；野生型和突变型）及人类 LRRC26（NM_001013653；野生型和 R295A 突变型）的 cDNA 被插入至 pGEMHE 表达载体中。cRNA 的转录采用 ThermoFisher Scientific 的 mMESSAGE mMACHINE™ T7 转录试剂盒完成。
从雌性 Xenopus laevis 胚囊中取出卵母细胞，在含有 0.15% tricaine（Sigma-Aldrich，E10521）的水中麻醉 15-30 分钟。在室温下，卵母细胞经胶原酶（Sigma-Aldrich，C0130）处理 6-7 小时以去除卵泡细胞层。在 V 或 VI 期选择大小相似的脱去卵泡的卵母细胞进行 cRNA 注射。使用 Nanoject II（Drummond Scientific Company）将 cRNA 注入每个卵母细胞。Slo1 和 LRRC26 cRNA 以 1:2 的比例共注射。注射 cRNA 的卵母细胞在 18 °C 的 MBSH 缓冲液（88 mM NaCl，1 mM KCl，2.4 mM NaHCO3，10 mM HEPES，0.3 mM Ca(NO3)2，0.41 mM CaCl2，和 0.82 mM MgSO4，pH 7.6）中补充 0.1% 青霉素-链霉素溶液（Sigma-Aldrich，P4333）的条件下孵育 2-7 天。
采用 Sutter Instruments 的 B150-86-10 硼硅酸盐玻璃毛细管，使用 P-1000 微吸管拉力器（Sutter Instrument）拉制微电极，电阻范围为 1.5-3.5 MΩ。使用含有 3M KCl 的琼脂桥作为参考电极。电极和浴液制备方法如前所述。电极溶液含有 140 mM K-methanesulfonate，10 mM HEPES 和 2 mM Mg Cl2。浴液含有 140 mM K-methanesulfonate 和 10 mM HEPES。两种溶液均通过 KOH 调节至 pH 7.0。在无 CaCl2 的记录中，使用补充 5 mM EGTA 的浴液。在有 CaCl2 的记录中，使用补充 1 µM Ca Cl2（非游离 [Ca2+]）的浴液。在实验前，手动移除卵母细胞的卵黄膜。去卵黄膜的卵母细胞置于装有 3 ml 浴液的 35 mm 培养皿（Corning）中。使用集成 Patch Clamp（IPA）放大器（Sutter Instrument）获取电流记录，采样频率为 50 kHz，滤波频率为 10 kHz。所有实验均在室温下进行。通过 P/8 协议在保持电势 -120 mV（仅 Slo1 条件）或 -160 mV（Slo1 与 LRRC26 条件）下减去残留电容和漏电流。