Whole-Cell Patch Clamp Recordings

L-type calcium currents (ICa, L) were recorded using the whole-cell patch-clamp configuration with the external recording solution of the following composition (in mmol/L): 140 NaCl, 6 KCl, 10 glucose, 10 HEPES, 1.5 MgCl₂, 1 CaCl₂, pH 7.4 with 1M NaOH. An internal pipette solution contained (in mmol/L): 100 Cs-methanesulfonate, 40 CsCl, 10 HEPES, 5 EGTA, 5 Mg-ATP free acid, 0.75 MgCl₂, pH 7.2 with CsOH. Patch pipettes had mean resistances of 3.5–5 MΩ. Currents were recorded using an Axopatch-1D amplifier connected to a Digidata 1322A acquisition system (Axon Instruments, Foster City, CA, USA). The bath was connected to the ground via a reference electrode containing Ag–AgCl pellet. Data were low-pass filtered at 2 kHz using the Bessel filter of the amplifier and sampled at 2 kHz. All recordings were performed at room temperature (22–24°C). ICa, L channel activity was recorded during 200 ms from a holding potential of -40 mV to test potentials ranging from -40 to +60 mV, with pulses applied every 2 s in 5 mV increments. Current amplitude at 0 mV was taken as a peak current and divided by a capacitance value for each cell (current density, pA/pF).