Isolation of I_Na in Nav1.3 KO cells. (A) Noninactivating inward current (as shown in C) from seven Nav1.3 KO cells with no detectable fast inactivating inward current were used to provide an empirical description of macroscopic Ca²⁺ current. The rising phase of inward current was fit with a single exponential current to define maximal inward Cav current and the time constant of inactivation at membrane potentials from −40 through +20 mV. A plot of peak current density (mean ± SD) shows peak inward current from −10 to 0 mV. (B) Voltage dependence of average time constants (±SD) of Cav activation for seven cells. The averaged time constants were then used in conjunction with the empirically measured steady-state current to generate idealized Cav current for subtraction from individual cells as in C–E. (C) For a cell lacking observable fast inactivating inward current, panels show (left) raw current traces activated by steps from −40 through +20 mV, (center) fits of single exponential functions to the currents on the left, and (right) subtracted of best fits to the raw data reflecting removal of the Cav component from the traces. (D) For a cell with a modest fast inactivating inward current on the rising phase of Cav current, panels are as in C. Idealized Cav traces (center) generated based on measured activation time constants (B) were subtracted from raw current traces (left) to generate empirically isolated Nav currents lacking any contaminating Cav current. Red trace corresponds to voltage step to −10 mV, and blue trace to +10 mV. (E) For an Nav1.3 KO cell with one of the larger peak Nav currents, the same subtraction method was employed. Subtraction of the idealized Cav currents (center traces) from the measured currents (left traces) yielded isolated fast inactivated current without Cav contamination. (F) From traces with Cav currents removed (right panels in C–E), I-V curves were generated for voltages from −40 to +20 mV. Peak current was determined for each cell and current density (pA/pF) determined. Plot shows binned current densities. Cells with 0 Nav current were included in the bin corresponding to 0–25 pA/pF; bin width, 25 pA/pF. (G) Binned (100 pA/pF) current densities were normalized to generate separate probability density distributions for WT (gray) and Nav1.3 KO (red) cells, highlighting the lack of overlap between the two.