Figure 7. The pre-IQ domain is required for Ca2+-CaM-mediated CaV1.3S coupling.
(A) Schematic of CaV1.3S mutations introduced to disrupt CaM binding to the IQ (I1608E) or the pre-IQ (AAE) domain; the position of the mutated amino acid is shown in the sequence below. (B) Normalized ICa currents evoked by a 30-ms depolarizing pulse from a holding potential of -80 mV to a test potential of 0 mV in tsA-201 cells expressing CaV1.3S (Control, black), CaV1.3S(I1608E) (blue), or CaV1.3S(AAE) (red). Currents analyzed for these experiments were in a range between 100 and 600 pA (C–E) TIRF images of Venus fluorescence reconstitution in the presence of 20 mM Ca2+ in tsA-201 cells expressing (C) CaV1.3S-VN and CaV1.3S-VC, (D) CaV1.3S(I1608E)-VN and CaV1.3S(I1608E)-VC, or (E) CaV1.3S(AAE)-VN and CaV1.3S(AAE)-VC. Fluorescence reconstitution was measured in response to depolarizing voltage steps from a holding potential of -80 mV to test potentials of -60 mV to +60 mV. (F) Voltage-dependence of Venus fluorescence reconstitution in the presence of 20 mM Ca2+ for control (black), I1608E mutant (blue), and AAE mutant (red) from the cells shown in (C–E). (G) Bar plot of averaged Venus fluorescence in the presence of 20 mM Ca2+ at -60 mV and +20 mV. Bars are averages of 5 cells ± SEM (*p<0.05).