Four TRPM4 Cation Channel Mutations Found in Cardiac Conduction Diseases Lead to Altered Protein Stability

Figures

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  Figure 1. ECG recordings and variants information. (A) Variants location in the TRPM4 protein. (B) ECG from patient 1 carrying p.A101T variant and showing a complete heart block phenotype. (C,D) ECG from patient 5 carrying p.A101T/P1204L variant at rest (C) and under physical exercise showing IVF phenotype (D). The arrowhead shows the administration of beta-blockers.

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  Figure 2. Western Blot analysis of HEK293 cells transiently expressing WT-TRPM4 and TRPM4 variants. Representative immunoblots of human TRPM4 expression at the total (A) and surface expression (B). Actin and alpha Na+/K+ ATPase were used for normalization of the total and the surface expression respectively. White and black arrowheads represent fully and core glycosylated band respectively. Quantification of the total expression (C) and surface expression (D) is shown as relative intensity of protein band for both full and core glycosylated forms of TRPM4 in each fraction. Data are represented as mean ± s.e.m. of n = 3; *P < 0.05; **P < 0.005; ***P < 0.001.

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  Figure 3. mRNA TRPM4 expression of WT and variants. qPCR analysis on TRPM4 gene expression in HEK 293 cells transiently transfected with WT-TRPM4 and TRPM4 variants. Data are represented as relative mRNA expression. The expression of GAPDH is used as reference (mean ± s.e.m. of n = 6). No significant differences are observed between WT and variants.

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  Figure 4. Inside-out patch clamp recordings of WT and TRPM4 variants. (A) Individual current traces of WT TRPM4 and TRPM4 variants after applying 300 μM Ca2+. (B) Quantification of currents of WT and cardiac conduction disorders variants. **P < 0.005; ***P < 0.001 (WT, n = 13; A101T, n = 7; Q854R, n = 7; S1044C, n = 6; P1204L, n = 7; A101T/P1204L, n = 11).

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  Figure 5. Half-life of WT and TRPM4 variants. Immunoblots showing immunoprecipitation with anti-TRPM4 antibody. Cells were harvested prior to treatment (0 h) and 4, 16, 24, and 36 h after treatment with 100 μg/ml cycloheximide. TRPM4 is detected using anti-HA antibody in presence of 100 μg/ml cycloheximide (left panel), or 0.3% DMSO (right panel). White and black arrowheads represent fully and core glycosylated band respectively. Data are representative of three independent experiments.

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  Figure 6. TRPM4 protein expression decay. Immunoblots from Figure 5 are quantified as relative intensity of protein band of the fully (A) and the core (B) glycosylated band. 0.3% DMSO values are used at each time point for normalization. Statistical analysis has been performed using one-way ANOVA and results are shown in Supplementary Table 1.