双电极电压钳（TEVC）操作规程Two-Electrode Voltage Clamp (TEVC) Protocol

1. 方法原理Principle

双电极电压钳（Two-Electrode Voltage Clamp, TEVC）是研究爪蟾卵母细胞异源表达蛋白的经典电生理方法。两根尖端直径约 1 μm 的玻璃微电极同时刺入同一卵母细胞：电压电极持续监测跨膜电位（V_m）；电流电极由钳位放大器实时注入补偿电流，使 V_m 精确维持在设定的钳制电位（V_cmd）。所记录的补偿电流即等于流过目标离子通道或转运体的宏观电流（I_m），可直接反映蛋白质功能。Two-Electrode Voltage Clamp (TEVC) is the standard electrophysiology method for characterising proteins heterologously expressed in Xenopus oocytes. Two glass microelectrodes (~1 µm tip) are impaled into the same oocyte: the voltage electrode continuously monitors membrane potential (V_m); the current electrode injects compensating current via a clamp amplifier to maintain V_m precisely at the command potential (V_cmd). The injected current equals the macroscopic transmembrane current (I_m) through the target channel or transporter.

与膜片钳相比，TEVC 无需形成高阻封接，操作简便；爪蟾卵母细胞直径约 1.2 mm，表面积大（信号电流可达 μA 级），适合研究电流密度较低的通道、转运体或受体。Unlike patch clamp, TEVC requires no high-resistance seal. The large oocyte surface (~1.2 mm diameter) generates macroscopic currents in the µA range, making TEVC ideal for low-density channels, transporters and receptors.

2. 实验设备Equipment

3. 微电极制备Electrode preparation

• 玻璃管：硼硅酸盐薄壁毛细管（外径 1.5 mm，内径 1.1 mm），使用前 180 ℃ 烘干 4 h 去除内壁水分。Glass: borosilicate thin-wall capillaries (1.5/1.1 mm OD/ID); bake at 180 ℃ for 4 h to remove moisture.
• 拉制参数：目标阻抗 0.5–2 MΩ（3M KCl 填充后于外液中测定）；阻抗过高（>5 MΩ）噪声大，过低（<0.3 MΩ）易损伤卵母细胞。Pull parameters: target resistance 0.5–2 MΩ (measured in external solution with 3M KCl fill); >5 MΩ is noisy, <0.3 MΩ damages the oocyte.
• 填充：两根电极（电压电极与电流电极）均后端灌入 3M KCl，轻弹去除气泡后接入 Ag/AgCl 丝。TEVC 电极不透析细胞内液，无需配制膜片钳式的胞内液。部分实验室使用 1–2M KAc 替代，以减少 Cl⁻ 沿电极尖端漏入浴槽。Filling: back-fill both electrodes (voltage and current) with 3M KCl; flick out bubbles; insert Ag/AgCl wire. TEVC electrodes do not dialyse the cell — no patch-clamp-style internal solution is needed. Some labs use 1–2M KAc instead to minimise Cl⁻ leakage at the electrode tip.
• 液接电位：入浴前在外液中调零；每次更换批次电极或外液成分须重新调零。Liquid junction potential: zero offset in external solution before impalement; re-zero when changing electrode batch or external solution composition.

4. 实验溶液Solutions

电极充填液（两根电极通用）Electrode fill (both electrodes)

TEVC 两根电极均为"穿刺式"——仅穿透卵母细胞膜建立电学通路，不透析细胞内液，因此无需配制膜片钳式的胞内液（含 Cs⁺、TEA、ATP/GTP 等）。3M KCl 液接电位约 −4 mV（与 ND96 接触），入浴调零后可忽略。TEVC electrodes are "sharp" — they penetrate the membrane to make electrical contact only and do not dialyse the cytoplasm. No patch-clamp-style internal solution (with Cs⁺, TEA, ATP/GTP) is needed. The liquid junction potential of 3M KCl vs. ND96 is approximately −4 mV and is nulled before impalement.

浴槽灌流液：标准 ND96Bath solution: standard ND96

NaOH 调 pH 至 7.4±0.05；渗透压约 200 mOsm/kg；4 ℃ 保存 ≤7 天。适用于大多数离子通道、配体门控受体与转运蛋白的记录。Adjust pH to 7.4±0.05 with NaOH; osmolarity ~200 mOsm/kg; store ≤7 days at 4 ℃. Suitable for most ion channels, ligand-gated receptors and transporter recordings.

浴槽灌流液：无 Ca²⁺ ND96（用于抑制 I_Cl(Ca)）Bath solution: Ca²⁺-free ND96 (to suppress endogenous I_Cl(Ca))

Ba²⁺ 不激活爪蟾卵母细胞内源性 Ca²⁺ 激活的 Cl⁻ 电流（I_Cl(Ca)）；适用于研究 Ca²⁺ 通透通道或 Ca²⁺ 转运体时，以避免内源性 I_Cl(Ca) 污染信号。pH 7.4；4 ℃ 保存 ≤7 天；Ba²⁺ 本身可轻度阻断某些 K⁺ 通道，注意对照。Ba²⁺ does not activate the endogenous Ca²⁺-activated Cl⁻ current (I_Cl(Ca)); use when studying Ca²⁺-permeable channels or Ca²⁺ transporters to prevent endogenous I_Cl(Ca) contamination. pH 7.4; store ≤7 days at 4 ℃. Note that Ba²⁺ can mildly block some K⁺ channels — include appropriate controls.

5. 卵母细胞准备Oocyte preparation

• 选用注射 cRNA 后 16–48 h 的 V–VI 期卵母细胞；形态：动物极深棕、植物极乳白、赤道界清晰、表面光滑无损。Use stage V–VI oocytes 16–48 h after cRNA injection; morphology: dark animal pole, pale vegetal pole, clear equatorial border, smooth surface.
• 将单个卵母细胞转入记录槽（外液灌流中），植物极朝上放置有助于减少内源性电流干扰。Transfer a single oocyte to the recording chamber under external solution perfusion; orient with vegetal pole up to minimise endogenous current contamination.
• 必须设阴性对照：非注射卵母细胞（uninjected）与注射水的卵母细胞（water-injected）在相同条件下同步记录，以扣除内源性背景电流。Always include negative controls: uninjected and water-injected oocytes recorded under identical conditions to subtract endogenous background currents.

6. 记录操作步骤Recording procedure

1. 入浴调零：将两根充填好的电极浸入外液，调零液接电位（offset）；记录此时电极阻抗（目标 0.5–2 MΩ）。Bath offset: immerse both filled electrodes in external solution; zero liquid junction potential; record electrode resistance (target 0.5–2 MΩ).
2. 电压电极刺入：在显微镜下将电压电极尖端对准卵母细胞动物极赤道附近，轻柔推进直至穿透卵膜（静息电位立即跳变至 −40 至 −70 mV 为穿刺成功标志）。Voltage electrode impalement: position tip near the equatorial zone of the animal pole; advance gently until membrane penetration — a sudden jump to −40 to −70 mV indicates success.
3. 电流电极刺入：在距电压电极 50–150 μm 处刺入第二根电极（电流电极），观察 V_m 是否维持稳定；若 V_m 剧烈波动则重新定位。Current electrode impalement: impale 50–150 µm from the voltage electrode; check V_m stability — large oscillations indicate poor impalement and require repositioning.
4. 建立钳制：启动钳位模式，将 V_cmd 设为保持电位（通常 −80 mV）；检查钳制质量——电流应在切换后 <1 ms 达到稳定，无持续振荡。Engage clamp: switch to voltage-clamp mode with holding potential −80 mV; verify clamp quality — current should settle in <1 ms with no oscillation.
5. 稳定期：在保持电位下等待 2–5 min，待漏电流（leak）稳定后开始施加刺激方案。Equilibration: wait 2–5 min at holding potential until leak current stabilises before applying stimulus protocols.
6. 实验与给药：通过灌流系统给予激动剂、拮抗剂或改变离子成分；每次给药前后均在保持电位下记录基线。Drug application: apply agonists, antagonists or ion substitutions via perfusion; record baseline at holding potential before and after each application.

7. 常用电压方案Voltage protocols

8. 常见问题排查Troubleshooting

参考文献References

1. Goldin AL (1992) Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol 207:266–279.
2. Sigel E (1990) Use of Xenopus oocytes for the functional expression of plasma membrane proteins. J Membr Biol 117(3):201–221.
3. Stühmer W (1992) Electrophysiological recording from Xenopus oocytes. Methods Enzymol 207:319–339.