De novo CACAN1D Ca2+ channelopathies: clinical phenotypes and molecular mechanism

Figures

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  Fig. 1. Characteristic gating changes induced by high-risk pathogenic CACNA1D variants permitting enhanced channel function of Cav1.3 L-type Ca2+ channels (taken and modified from [66, 26]). Typical changes in the voltage dependence of activation (A+C) and inactivation (C) as well as inactivation kinetics during prolonged depolarizations (B+D) are shown for C-terminally short wild-type (black), G407R (red; type 1) and S652L Cav1.3 channels (turquoise; type 2). Note that for the almost non-inactivating G407R variant no steady-state inactivation curve was determined (A). The short Cav1.3 splice variant lacks an intramolecular interaction between the distal and proximal C-terminus which enhances its voltage-sensitivity and inactivation kinetics compared with full-length Cav1.3 channels [8, 77]; however, similar gating changes were also elicited in the long Cav1.3 variant [26, 64]

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  Fig. 2. Scheme of the position of de novo germline and de novo somatic APA/APCC variants within the Cav1.3 α1-subunit. The α1-subunit consists of four homologous repeats (I–IV), each comprising six transmembrane segments (S1–S6). From each repeat, S1–S4 form the voltage sensors (S4 contains the positive gating charges) and S5–S6 with their connecting loop build the ion conducting pore. The S4–S5 loop links the voltage-sensor movements to the pore opening. Color code: germline (orange), somatic (blue)

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  Fig. 3. CACNA1D structural models. (A) A homology model of the inactivated state based on rabbit Cav1.1 (PDB entry 5gjv, [93]). Transmembrane segments of homologous repeats I (cyan) and II (blue) are shown with germline de novo missense variants highlighted as orange sticks. They spatially cluster around the activation gate formed by S6-helices of repeats I and II. (B) The homology model of the CACNA1D resting state (based on PDB entry 6p6w, [92]) shows that A749 and G403 (orange spheres) tightly interact with surrounding residues on the S5 helix and S4-S5 linker (highlighted as spheres). Cartoon and carbon atoms depicted as spheres are colored according to the homologous repeats, whereas nitrogen and oxygen atoms of residues depicted as spheres are colored blue and red, respectively. (C) The pore-forming helix S6 requires conformational rearrangements of the backbone to facilitate pore opening in the activated state (green cartoon) compared with the resting state (cyan cartoon). G407 (S6 in repeat I) is highlighted in orange (resting state) and yellow (activated state)

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  Fig. 4. Modeling of the R619 variants. (A) R619 (orange sticks and highlighted by dotted lines) is the uppermost positively charged arginine in the VSD of repeat II. For reference, germline variants from Fig. 3A are shown as orange sticks as well. (B + C) Top view from the extracellular side onto the (B) wild-type R (orange sticks), and the W (green sticks) and (C) P (yellow sticks) variants in the Cav1.3 resting state model (gray). (D + E) In the resting state, both R619 (D, orange, highlighted by dotted lines) and the large hydrophobic side chain of the R619W variant (E, green, highlighted by dotted lines) provide a steric barrier that prevents ion leakage through the surface of the VSD (gray surface). (F) The R619P variant (yellow) lacks this large, hydrophobic sidechain, thus creating a tunnel via which ions can potentially pass through to the intracellular side. For comparison, this tunnel is depicted from both the top and bottom view