Extended biophysical characterization of DIV W[+2]A mutation. (A) Population r₈₀₀ data plotted as a function of test-pulse potential reveals reduced inactivation at higher voltages. Each dot, mean ± SEM. n = 7 cells. (B) A two-pulse protocol is used to dissect voltage dependence of inactivation. A brief 15-ms prepulse to 10 mV quantifies available current prior to inactivation. Subsequently, a family of 800-ms voltage steps (test pulse) is used to evoke steady-state inactivation and a 15-ms postpulse is then used to quantify the current remaining following VDI. The extent of inactivation is quantified as the ratio of peak currents during postpulse to prepulse. Exemplar currents at three different test pulse potentials (light gray, −50 mV; gray, 0 mV; and black +50 mV) are shown. Exemplar trace for wild-type Ca_V1.3. Further analysis in Fig. 2, C and E. (C) Exemplar trace for DIV W[+2]A mutation. Further analysis in Fig. 2, D and F. (D–F) VDI of DIV W[+2]A probed in the presence of Ca²⁺ as charge carrier. Panel D, exemplar trace. Panel E, comparison of Ca²⁺ currents during prepulse (left, I₁) versus postpulse (right, I₂). Note that the current magnitude during the postpulse is maximally reduced at intermediate voltages. Panel F, population data shows U-shaped dependence of inactivation with Ca²⁺ as the charge carrier (red). Black trace, relationship with Ba²⁺ as the charge carrier reproduced from Fig. 2 F. Each dot, mean ± SEM, n = 8 cells. (G–I) VDI of DIV W[+2]A mutation with Na⁺ (but no divalents) as charge carrier. Format as in panels D–F. n = 7 cells.