2.6. Whole-cell patch-clamp electrophysiology

Recordings were made from GFP-fluorescing tsA201 cells, expressing the protein of interest using whole-cell patch-clamp in a voltage clamp configuration. Whole–cell currents were recorded from cells maintained at a holding potential of −60 mV at 20–24 °C (room temperature). A combined voltage-step and voltage-ramp protocol was applied using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA). Cells were first hyperpolarized to −80 mV for 100 ms and then stepped to −40 mV for 500 ms, then stepped to −120 mV for 100 ms (the “voltage-step” component). This was followed by a 500-ms ramp change in voltage to +20 mV and a step back to −80 mV for another 100 ms before being returned to the holding potential of −60 mV (the “voltage-ramp” component). This protocol was composed of sweeps lasting 1.5 s (s), including sampling at the holding voltage and was repeated once every 5 s. For all experiments, the external recording solution comprised of 145 mM NaCl, 2.5 mM KCl, 3 mM MgCl₂, 1 mM CaCl₂ and 10 mM HEPES (pH to 7.4, using NaOH). The internal recoding solution contained 150 mM KCl, 3 mM MgCl₂, 5 mM EGTA and 10 mM HEPES (pH adjusted to 7.4 with KOH).