2.6. Whole cell patch clamp

Standard whole-cell patch clamp technique was used to measure Cav1.2 CACNA1C wild type and mutant calcium currents at room temperature (22–24 °C) with the use of an Axopatch 200B amplifier, Digidata 1440A and pclamp version 10.2 software (Axon Instruments, Sunnyvale, CA). The extracellular (bath) solution contained (mmol/L): 2 CaCl₂, 1 MgCl_2, 150 TEA-Cl and 10 HEPES, pH adjusted to 7.35 with CsOH. The intracellular (pipette) solution contained (mmol/L): 110 CsCl, 0.1 CaCl₂, 10 HEPES, 10 EGTA, 2 MgATP and 10 TEA-Cl, pH adjusted to 7.30 with CsOH as previously described [24]. Microelectrodes were pulled on a P-97 puller (Sutter Instruments, Novato, CA) and fire polished to a final resistance of 2–3 MΩ. Series resistance was compensated by 80–85%. Currents were filtered at 1 kHz and digitized at 5 kHz with an eight-pole Bessel filter. The voltage dependence of activation and inactivation was determined using voltage-clamp protocols described in the relevant figure legends. Data were analyzed using Clampfit (Axon Instruments, Sunnyvale, CA), Excel (Microsoft, Redmond, WA), and fitted with Origin 9.1 (OriginLab Corporation, Northampton, MA) software.

The voltage-dependence of activation curve was fitted with a Boltzmann function: G_Ca/G_Ca max = {1 + exp[(V − V_1/2) / k]}⁻¹, where V_1/2 and k are the half-maximal voltage of activation and the slope factor respectively. The steady-state inactivation curve was fitted with a Boltzmann function: I_Ca/I_Ca max = {1 + exp[(V − V_1/2) / k]}⁻¹, where V_1/2 and k are the half-maximal voltage of inactivation and the slope factor respectively. I_Ca decay was fitted with a two-exponential function: y = y₀ + {1 − [A_f exp(−t / τ_f)] + [As exp(−t / τ_s)]}, where A_f and As represent the amplitudes of the fast and the slow inactivating components respectively and τ_f and τ_s represent the fast and slow time constants of inactivation respectively. Sustained Cav1.2 current was measured at the end of 500 ms long depolarization of +20 mV.