Biophysical properties of the newly generated stable cell lines and time-dependent decrease of LTCC I_Ca during simulated regular SN DA-like pacemaking (2.5 Hz; 2 mm Ca²⁺). hCa_v1.2 (blue), hCa_v1.3_S (gray), and hCa_v1.3_L (black). A, Voltage dependence of activation and inactivation; normalized steady-state activation (circles) and inactivation (triangles) curves were obtained as described in Materials and Methods. Currents <200 and >3000 pA were prospectively excluded from analysis. For statistics see Table 1. B, Inactivation of stable cell lines during a 5-s-long depolarizing pulse from a holding potential of −89 mV to V_max. Insets show the first 150 ms. Vertical lines represent error bars (mean ± SEM). For statistics see Table 1. C, AP waveform of an SN DA neuron used as command voltage to mimic regular pacemaking (top) and representative traces of the first (100%) and 184th (equilibrium) sweep of I_Ca through hCa_v1.3_S (bottom). D, Representative current traces of hCa_v1.3_S, hCa_v1.3_L, and hCa_v1.2 are shown (with components insensitive to 3 μm ISR subtracted; for details, see Fig. 4A). All three LTCC isoforms conduct I_AP. Only Ca_v1.3 channel constructs give rise to I_ISI. E, Time course of I_Ca decay of representative recordings for each LTCC subtype. I_AP amplitude was normalized to the peak I_AP amplitude of the first SN DA AP after holding the cell at −89 mV. At the end of each recording, LTCC I_Ca was completely blocked by 3 μm ISR and remaining ISR-insensitive current components were subtracted off-line. The inset shows mean values ±SEM (and n-numbers) of each construct after 1, 3, and 5 min of pacemaking. F, Curves were best described by a double-exponential function, and parameters are given as median (10th to 90th percentile; outliers illustrated as points). Statistical significance was determined using the Kruskal–Wallis test (A_fast, A_slow, τ_fast, τ_slow: p < 0.0001) with Dunn's multiple-comparison post hoc test. ***p < 0.001; **p < 0.01; *p < 0.05.