Abstract 1526: Synergistic Autoinhibition of the CaV1.2 Channel by its Proteolytically Cleaved Distal C-Terminus and Mg Bound to the C-Terminal EF-Hand
L-type Ca current through CaV1.2 channels initiates excitation-contraction coupling in the heart. Here we show that intracellular Mg (Mgi), acting via a C-terminal EF-hand motif, is required for normal autoinhibition of the CaV1.2 channel by its proteolytically cleaved Distal C-terminal (DCT) domain. We recently showed that Mgi and the DCT enhance voltage-dependent inactivation (VDI) of CaV1.2 current. We used Na as charge carrier in whole-cell voltage clamp experiments to eliminate divalent ion-dependent inactivation. VDI was quantified as the fraction of peak current remaining at the end of a 1000 ms depolarization to 0 mV (r1000). For truncated CaV1.2 channels co-expressed with DCT, reduction of Mgi from 7.2 mM to 0.26 mM did not alter the rapid VDI of CaV1.2(Na) current (r1000 = 0.28 ± 0.03 (21) for 0.26mM Mgi vs. 0.25 ± 0.03 (11) for 7.2 mM Mgi), but VDI was significantly slowed (r1000 = 0.60 ± 0.05 (5), p<0.01) when Mgi was reduced to 0.1 mM. These results show that the affinity for stimulation of VDI by Mgi is greater for the autoinhibitory complex of truncated CaV1.2 channels with DCT than for full-length CaV1.2 (EC50Δ0.15 mM vs. 0.9 mM), suggesting a synergistic interaction between DCT and the EF-hand. Since we showed previously that the C-terminal EF-hand is required for Mgi modulation of VDI, we tested the impact of the EF-hand mutation D1546R on modulation of VDI by the DCT. DCT[1801–2171] was unable to enhance VDI of EF-hand mutant CaV1.2(D1546R)Δ1800 (r1000= 0.70 ± 0.03 (15) vs. 0.72 ± 0.02 (17)) in contrast to its strong effect on wild-type CaV1.2. In addition, since the DCT modulates coupling of gating charge movement to opening the pore of CaV1.2 channels, we investigated the impact of the DCT on the coupling ratio of EF-hand mutant CaV1.2(D1546R) Δ1800 and found that the DCT was also unable to modulate the coupling ratio of CaV1.2(D1546R) Δ1800. Taken together our results suggest that the Mgi binding to the EF-hand of CaV1.2 channels is required for the DCT to enhance VDI and reduce the coupling ratio of CaV1.2 channels, demonstrating a strong synergistic autoinhibition of CaV1.2 channels by the EF-hand and the DCT.
This research has received full or partial funding support from the American Heart Association, AHA National Center.