Identification biochimique et fonctionnelle des domaines structuraux d’une sous-unité des canaux calciques

Voltage-gated calcium channels play a key role in the excitation-contraction coupling by promoting Ca2+ influx through CaV1.2 into the cardiac cells. A dysregulation in the function of this channel has been involved in cardiac arrhythmias. L-type voltage-gated calcium channels are formed by the main pore-forming CaVa1C subunit assembled with two auxiliary subunits. The cytoplasmic CaVb subunit strongly interacts with the intracellular loop between repeat I and II of CaVa1 and promote cell surface expression of the channel whereas the interaction with the extracellular CaVa2d1 subunit increases by 5- to 10-fold the peak current density and promote channel activation within a physiological range. The full set of subunits is required to reproduce the biophysical properties of the native channel. For L-type voltage-gated calcium channel, the mechanism whereby CaVa2d1 exerts its function is still not fully understood. A structural determination of CaVa2d1 conducted using a typical biochemical approach was initiated but many post-translational modifications (16 Nglycosylation sites, intra- and extra-disulfide bonds, glycosylphosphatidylinositol anchor) prevented the purification of the entire protein. We then devised a “divide-and-conquer” strategy that lead to the purification of the Cache2 domain of CaVa2d1. The low-resolution structure of the Cache2 domain by small angle X-rays scattering revealed a predominantly fold in b-sheet with multiple flexible regions. The publication in 2016 of the high-resolution structure of the homologous CaV1.1 channel complex at 3.6 Å of resolution solved by cryoelectronic microscopy provided crucial molecular details regarding the organization of the structural domains of CaVa2d1 but also its spatial orientation within the CaV1.1 complex. In particular, the VWA domain of CaVa2d1 appears to come within atomic distance of many extracellular loops of CaVa1 subunit. To establish whether physical interaction between the two proteins is essential to mediate the functional regulation of CaV1.2 current by CaVa2d1, we investigated the molecular determinants responsible for the interaction between CaVa1C (CaV1.2) and CaVa2d1. Using homology modeling, electrophysiology and coimmunoprecipitation experiments we demonstrated that residues in the first extracellular loop in repeat I of CaV1.2 play a dominant role in the interaction and modulation by CaVa2d1. In particular, CaV1.2 Asp-181 anchors the physical interaction with CaVa2d1 while CaV1.2 Asp- 180 mediates the channel activation in a physiological range of membrane potential. In addition, we have explored the contribution of metal ion-dependant adhesion site (MIDAS) residues within the Won Willebrand Factor-A (VWA) domain of CaVa2d1 in mediating channel modulation. Our results show that mutating side chain at position Asp-259 and Ser- 261 eliminate channel modulation although these residues are not predicted to be within atomic distance of CaVa1. Hence, we propose that CaVa2d1 Asp-259 captures the Ca2+ ion which in turn folds the MIDAS motif within the VWA domain of CaVa2d1. The folding of the MIDAS around the Ca2+ locks CaVa2d1 Ser-261 and Ser-263 within atomic distance of CaVa1C Asp-181. This network of molecular interactions stabilizes the protein-protein interface demonstrating that high affinity binding of CaVa2d1 induce a local structural rearrangement that facilitate CaV1.2 channel opening and consequently increase Ca2+ influx through the pore. Al together, the result emerging from these studies provide novel mechanism insight regarding the function of the cardiac CaV1.2 channel.

Keyphrases: CaV1.2-CaVa2d1, Calcium channels, Cache2 domain structure, macromolecular interface, protein-protein interaction, co-immunoprecipitation, electrophysiology, 3D homology modelling, molecular dynamics simulations