Peptide-Based Targeting of the L-Type Calcium Channel Corrects the Loss-of-Function Phenotype of Two Novel Mutations of the CACNA1 Gene Associated With Brugada Syndrome

Figures

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  Figure 1. Brugada patients with novel Cavα1.2 loss-of-function (LoF) mutations. (A) DNA sequence analysis showing the two heterozygous single nucleotide substitutions (arrows) in Cavα1.2 responsible for the T320M and Q428E missense mutations, and a schematic representation of Cavα1.2, indicating the location (stars) of mutants. (B) Baseline ECGs showing type 1 coved ST segment elevation pattern (arrows) in the two patients. AID, alpha-interaction domain.

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  Figure 2. Treatment with R7W-MP corrects Ca2+ alterations due to Cavα1.2 mutations in a heterologous cell model. (A) Current-voltage (I-V) relationships recorded in HEK293 cells transiently co-transfected with Cavα1.2 WT (WT, n = 14), Cavα1.2 T320M (T320M, n = 14), or Cavα1.2 Q428E (Q428E, n = 13) and Cavβ2 at baseline and 24 h after treatment with 1.3 μM R7W-MP (T320M + R7W-MP, n = 14; Q428E + R7W-MP, n = 12). (B) Single dots and histograms (average) showing ICa measured at a test pulse of +10 mV for all the above-mentioned experimental conditions. * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskal-Wallis test). (C) Graphical representation of ICa activation-curves (g/gmax) for WT (V1/2 = −4.09 ± 1.81, slope = 8.29 ± 0.37), T320M (V1/2 = −1.90 ± 0.93, slope = 8.79 ± 0.60), and Q428E (V1/2 = −5.10 ± 0.92, slope = 7.64 ± 0.95). Kinetic curves did not change after R7W-MP treatment (V1/2 = −2.19 ± 1.18, slope = 8.21 ± 0.37; V1/2 = −3.69 ± 0.64, slope = 7.71 ± 0.31 for T320M + R7W-MP and Q428E + R7W-MP, respectively).

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  Figure 3. R7W-MP corrects the reduced interaction between mutant Cavα1.2 and Cavβ2. Cavβ2-Cavα1.2 protein affinity as measured in a BRET assay in HEK293 cells transfected with Cavβ2-NanoLuc and Cavα1.2-Halo (WT, T320M, and Q428E mutant) and treated with increasing doses (0.12, 1.3, and 10.2 μM) of R7W-MP and R7W-Scr as indicated (n = 6). * p < 0.05; ** p < 0.01 (Mann-Whitney test). Black, red, and blue indicate untreated conditions.

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  Figure 4. R7W-MP corrects LTCC density at the plasma membrane in Cavα1.2 mutant-transfected cells. (A) Subcellular localization of Cavα1.2 (top) and line scan analyses (bottom) for each condition as indicated. AU, arbitrary units. Scale bars (in white), 10 μm (n = 50). Representative experiments are shown. Cells were treated with increasing doses (0.12, 1.3, and 10.2 μM) of R7W-MP or R7W-Scr. (B) Cell surface biotinylation assay followed by followed by Western blot analysis (a representative image is shown) on transfected cells treated as indicated (n = 3). (C) Cavα1.2 half-life as measured in a NanoLuc luciferase assay. HEK293 cells were transfected with Cavα1.2-NanoLuc (WT, T320M, and Q428E mutant) and treated with 1.3 μM R7W-MP or R7W-Scr as indicated (n = 6). * p < 0.05 (Mann-Whitney test).