Two-electrode voltage clamp (TEVC) and whole-cell patch clamp electrophysiology

TEVC recordings were performed in Xenopus oocytes using two microelectrode voltage clamp as described in our published protocols [57]. The microelectrodes had <1 MOhm tip resistances and were filled with 3 M KCl solutions. The voltage-clamp amplifier (Oocyte Clamp OC-725C, Warner Instruments) was under the control of WinWCP software (John Dempster, University of Strathclyde, Glasgow, UK). The data were digitized and low-pass filtered (model 902, Frequency Devices) at various frequencies depending on the sampling rate. The capacitive transient and linear leak subtraction were performed off-line. Recordings with offsets >3 mV were removed from data analysis, and the average leak current was <0.2 mA.

Mammalian cells and CG neurons were visualized using a fixed-state upright microscope (BX51WI, Olympus) equipped with 5x and 40x UMPlanFI objectives. Patch pipettes were fabricated by pulling standard borosilicate glass capillaries (1B150-4, World Precision Instruments) on a Flaming/Brown micropipette puller (model P-97, Sutter Instrument Co.). Pipette tip resistance was typically between 2–5 megaOhm. A MultiClamp 700B (Axon Instruments) was used to deliver voltage pulses under the control of MultiClamp Commander and pClamp10 (Axon Instruments) with the assistance of Digidata 1550 low-noise data acquisition system (Axon Instruments). The data were acquired on a desktop personal computer.

Tight-seal whole-cell recordings were obtained using standard patch clamp techniques. For cultured cells recordings, the patch electrodes were backfilled with a solution containing (in mM): 140 KCl, 1 CaCl₂, 1 MgCl₂, 10 EGTA, 10 HEPES (pH 7.4), and 5 Glucose; the bath solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES (pH 7.4), and 10 Glucose. For CG cell recordings, the patch electrodes were backfilled with a solution containing (in mM): 120 K-gluconate, 20 KCl, 4 NaCl, 10 HEPES (pH 7.25), 4 Mg-ATP, 0.3 Na-GTP, 7 Phosphocreatine, and the bath solution contained (in mM): 124 NaCl, 4.4 KCl, 0.1 CaCl₂, 3.1 MgSO4, 1 NaH2PO4, 0.1 4-AP, 0.0005 TTX, 26 NaHCO3, and 10 Glucose. Series resistance and whole-cell capacitance were determined by optimal cancellation of the capacitive transient. The series resistance was typically 6–10 megaOhm, about 2x the tip resistance. Series resistance compensation was typically around 75–80%. The data has not been corrected for liquid junction potential between the electrode and bath solution (about -14.2 mV calculated). All experiments were conducted at room temperature (22–23°C). Ohmic leak were subtracted off-line.