Figure 8. Distal auto-regulatory domain (DCRD) is not responsible for the lack of coupling of CaV1.3L channels.
(A) Schematic of CaV1.3L channel splice variant (left), depicting the domains important for Ca2+-mediated regulation: pre-IQ (green), IQ (blue), proximal and distal C-terminal regulatory domains (PCRD, DCRD, gray. Schematic of the CaV1.3LΔC116 channel where the last 116 aa in the C-terminal were removed (right). (B) Representative currents of CaV1.3L (black) and CaV1.3L ΔC116 channels (red) expressed in tsA-201 cells. Currents were evoked by a 300-ms depolarization from holding potential of -80 mV to a test potential of 0 mV, with 2 mM Ca2+ as the charge carrier. Currents analyzed for these experiments were in a range between 0.3 and 1 nA (C) Bar plot of the% inactivation after 25 or 300 ms at 0 mV. Bars are averages of 5 cells ± SEM (*p < 0.001) (D–F) TIRF images of Venus fluorescence reconstitution in the presence of 2 mM Ca2+ in tsA-201 cells expressing CaV1.3S-VN and CaV1.3S-VC (D) CaV1.3L-VN and CaV1.3L-VC (E) or CaV1.3L ΔC116-VN and CaV1.3L ΔC116-VC (E). 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. (G) Bar plot of averaged Venus fluorescence at -60 mV and +20 mV for each of the aforementioned construct pairs. Bars are averages of 5 cells ± SEM (*p<0.05). Data for CaV1.3L ΔC116-VN and CaV1.3L ΔC116-VC Venus reconstitution with 20 mM Ca2+ is presented in Figure 8—figure supplement 1.
Figure 8—figure supplement 1. High Ca2+ concentration is not enough to induce coupling in CaV1.3LΔC116 channels.
