Three way perfusion valve controller

Whole-cell patch-clamp recordings were performed in −60 mV clamped-cells by using the following solutions: extracellular (mM): HEPES 10, D-glucose 5, NaCl 140, KCl 3, MgCl₂ 2, and CaCl₂ 2 (pH 7.4). Pipette (mM): K-Aspartate 130, MgCl₂ 2, Na₂-ATP 5, Na₂-GTP 0.1, EGTA 11, HEPES 10 (pH 7.2). Data were acquired with an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA), low-pass filtered at 10 kHz, stored and analyzed with a pClamp 9.2 software (Axon Instruments). Detailed protocols used to evoke voltage-dependent K⁺ or Na⁺ currents are described in Supplementary Material. Drugs used were applied by superfusion with a three-way perfusion valve controller (Harvard Apparatus). Current-clamp recordings were performed by applying 12 steps of current injection (300 ms duration; 100 pA increment, from −100 pA to 1000 pA) from the resting membrane potential of the investigated cell, as already described (Coppi et al., 2012 (link)) and detailed in Supplementary Material.