Dual Whole-Cell Patch Clamp Analysis of Transfected Cell Pairs

Transiently transfected N2a cells were re-plated onto glass coverslips and incubated for 2 h at 37°C and 5% CO₂. Coverslips were transferred to a recording chamber on a fluorescent microscope (Leica DM IRB) and subsequently washed and bathed in extracellular solution (140 mM NaCl, 2 mM CsCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, 4 mM KCl, 5 mM D-Glucose, 2 mM pyruvate, pH 7.4). Glass micropipettes were pulled with a PC-100 puller (Narishige, Amityville, NY, United States) configured to yield a patch pipette resistance in the range of 2–3 MΩ, and back-filled with intracellular solution (130 mM CsCl, 10 mM EGTA, 0.5 mM CaCl₂, 3 mM MgATP, 2 mM Na₂ATP, and 10 mM HEPES, pH 7.2). Isolated transfected cell pairs, identified via fluorescence from the covalently bound GFP tag, were selected for dual whole-cell patch clamp and a voltage clamp at 0 mV was applied to each cell. In an alternating fashion, one cell in the pair was held at a constant holding potential of 0 mV and junctional currents (I_j) were recorded in this cell during the application of 7 s long trans-junctional voltage (V_j) pulses (at ±20, ±60, and ±100 mV) to the other cell of the pair as previously described (Xin et al., 2010 (link); Tong et al., 2014 (link); Yue et al., 2021 (link); Jaradat et al., 2022 (link); Lucaciu et al., 2022 (link)). The I_j recorded during the ±20 mV V_j pulse was used to calculate the macroscopic junctional conductance (G_j = I_j ÷ V_j) (Musa et al., 2004 (link); Bai and Cameron, 2016 (link)).