Figure 3. Steady-state Ca²⁺ sensitivity of Ca_V2.1 CaM regulation revealed by Ca²⁺ uncaging.

A, characterization of CDF using the prepulse protocol. With Ca²⁺ as the charge carrier, a step depolarization to 5 mV induces an inward current with rapid and slow components due to a superposition of voltage activation and CDF (left). With a preceding voltage step to 20 mV, sufficient Ca²⁺ enters to partially facilitate the channels, such that the subsequent step to 5 mV has a markedly blunted slow component (right). The area between the two current traces (ΔQ), divided by τ_slow, gives an approximation of the CDF triggered by the prepulse. B, population data (mean ± SEM) for CDF vs. prepulse voltage of the pore mutant E3A (red, n = 9) and WT (black, n = 8). The open circles denote the use of Ba²⁺ as the charge carrier (incapable of triggering CDF), while the filled circles denote the use of Ca²⁺. C, E3A mutant permeating Li⁺ current shows negligible Ca²⁺ block. Ca²⁺ uncaging during a voltage step of a CDF-incapable splice variant of Ca_v2.1 (EFb −47) induces neither block nor CDF (middle, right). D, Ca²⁺ uncaging with the EFa variant (−47) showcases large and rapid induction of CDF (middle, right). E, population data for Ca_V2.1 EFa −47 (black circles, mean ± STD, n = 12 cells), quantifying CDF as a function of different uncaged Ca²⁺ concentrations during voltage steps to −10 mV yields a Ca²⁺ sensitivity curve, with EC₅₀ = 0.6 μm and n_Hill = 1.8. Population data for Ca_V2.1 EFb −47 (grey circles, mean ± STD, n = 17 cells) show complete absence of CDF. F, similar experiments with Ca_V2.1 EFa −47 coexpressed with CaM₁₂₃₄, a mutant CaM incapable of binding Ca²⁺, resulted in the elimination of CDF (mean ± STD, n = 7 cells). G, similar experiments with Ca_V2.1 EFa –47 carrying an I(0)A mutation at the IQ domain show a knockout of CDF (mean ± STD, n = 8 cells).