Patch Clamp Analysis of P2X7 Receptor in HEK293 Cells

Patch clamp recording of HEK293 cells expressing pdP2X7 constructs was performed as previously described (Karasawa and Kawate, 2016 (link)). Briefly, HEK293 cells were transfected with 1 μg of the pdP2X7 constructs using FuGENE6 (Promega, Madison, WI). Cells were used 18–32 hr after transfection for measuring the pdP2X7 receptor activities using the whole cell patch clamp configuration. Membrane voltage was clamped at −60 mV with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), currents were filtered at 2 kHz (eight-pole Bessel; model 900BT; Devices, Ottawa, IL), and the recording data were sampled at 10 kHz using a Digidata 1440A and pCLAMP 10.5 software (Molecular Devices, Sunnyvale, CA). The extracellular solution contained 147 mM NaCl, 10 mM HEPES, 13 mM Glucose, 2 mM KCl, 0.1 mM CaCl₂, (pH 7.3). The pipette solution contained 147 mM NaCl, 10 mM HEPES, 10 mM EGTA, which was adjusted to pH7.0 using NaOH. Extracellular solutions were rapidly exchanged to the solutions containing 1 mM ATP using a computer-controlled perfusion system (RSC-200; Bio-Logic, France).