Mutations in NaV1.5 Reveal Calcium-Calmodulin Regulation of Sodium Channel

Figures

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  FIGURE 1. Clinical and genetic characteristics of the proband. (A) Left, Holter recording at rest. Right, 12-lead ECG during exercise. (B) Sequence chromatogram of SCN5A variants. (C) Schematic illustration of NaV1.5 protein and the location of the variants found. (D) Pedigree tree of the family. Circle indicates female, squares indicate male. The arrow points to the proband. In gray, suspected affected sibling. Red for the maternal allele variant, blue for the paternal allele variant.

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  FIGURE 2. Whole-cell INa of WT and mutant NaV1.5 in HEK and HL-1 cells. (A) Top, representative currents elicited from resting potential by depolarizing steps (–70 until –20 mV in 5 mV interval) in HEK cells. Bottom, voltage-dependent amplitudes and maximal current densities, in cells expressing GFP-NaV1.5WT, GFP-NaV1.5K1493del, and NaV1.5K1493del, with and without r/hNaVβ1 co-expressing RFP in the same vector. All groups were compared to GFP-WT for statistical significance. (B) WT and mutant NaV1.5 expressed in HEK cells (without NaVβ subunit). Current densities were normalized to the mean current density of NaV1.5WT in the same experiment and summarized from 9 experiments. Currents in A and the representative currents in B were recorded in [10 mM EGTA]in. (Ca), Gene expression levels of SCNB1-4 in HL-1 cells. Top, gene levels were quantified by real-time PCR and normalized to GAPDH. Bottom, semi-quantitative PCR. (b), INa measured in naïve and transfected HL-1 cells, in [10 mM EGTA]in. Mean current densities and representative cells are shown. Statistical significance was determined by one-way ANOVA, followed by Holm–Sidak test. ns, not significant.

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  FIGURE 3. Cell surface and total expression of NaV1.5WT and NaV1.5K1493del. (A,B) Representative biotinylation assays. (A) HEK cells were transfected with NaV1.5WT and NaV1.5K1493del. The control “no-biotin” group shows no signal from non-biotinylated channels. (B) HEK cells transfected with NaV1.5WT and NaV1.5K1493del (7.5 μg) +/– rNaVβ1 (2.5 and 7.5 μg). (C) Summary of % total protein (left) and % trafficking (right), normalized to NaV1.5WT without NaVβ1. N = 9 experiments without NaVβ. N = 4 experiments with rNaVβ1 expressed. Groups expressing rNaVβ1 in molar ratios β:α 0.6-2 were pulled together. (D) GFP-fused and non-tagged NaV1.5WT and NaV1.5K1493del were expressed (without NaVβ subunit). This is a summary of three experiments. Statistical significance was determined by one-way ANOVA, followed by Holm–Sidak test.

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  FIGURE 4. Ca2+-dependent gating properties of NaV1.5 variants. (A) Activation curves of conductance-voltage relationship. Current traces recorded in [10 μM Ca2+]in, and –40 mV trace is red. (B) SSI curves of voltage-dependent channel availability. Current traces during the test pulse at –20 mV are with (10 mM BAPTA)in. The test-pulse trace that follows –80 mV pre-pulse is red. (C) Recovery from inactivation. (D) Activation (top) and SSI (bottom) curves of NaV1.5WT +GFP and NaV1.5A1924T^*+GFP co-expressing rNaVβ1/RFP were plotted with the fitted curves of these groups without rNaVβ1/RFP (presented in A, B). Current traces recorded in [10 μM Ca2+]in. The –40 mV trace during activation and the test-pulse that follows the –80 mV pre-pulse during SSI are red. (E) Activation (left) and SSI (right) curves with 10 μM Ca2+ chelated with HEDTA in the internal solution. (F) Activation (left) and SSI (right) curves of NaV1.5 variants co-expressing rNaVβ1/RFP and GFP-NaV1.5K1493del were plotted with the fitted curves of these groups without GFP-NaV1.5K1493del (presented in D). Statistical significance of Va was determined by t-test: ns, not significant;*p < 0.05; ∗∗p < 0.01; ∗∗∗, p < 0.001 (see Table 2).

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  FIGURE 5. CaM-interaction with NaV1.5 variants (A). HEK cells were transfected with NaV1.5WT, NaV1.5K1493del, and NaV1.5A1924T^* together with rNaVβ1. Lysates from transfected groups were pulled-down with CaM-agarose beads in Ca2+ and EGTA. Left, a representative Western blot from a pull-down assay. Right, the CaM-bound fraction was normalized to input level, and presented as % of NaV1.5WT in 2 mM Ca2+, in the same experiment. Statistical significance was determined by one-way ANOVA followed by Holm–Sidak test. P-value compared to NaV1.5WT in 2 mM Ca2+ is indicated above each bar. (B) NaV1.5WT and NaV1.5K1493del with or without co-expressed CaM were pulled-down with CaM-agarose beads in 2 mM Ca2+. (a) CaM- bound NaV1.5, and total input levels of NaV1.5, CaM and γ-tubulin were detected in Western-blot. (b) Input NaV1.5+CaM levels were normalized to NaV1.5 without CaM, from the same experiment, N = 4. (c) CaM-bound fractions were normalized to input levels, and are presented as % of NaV1.5WT+CaM in the same experiment, N = 3. Statistical significance was estimated by t-test. (C) HEK cells transfected with equal total NaV1.5 α-subunit (7.5 μg; ratio 1:1, 3.75 μg of each construct; ratio 1:4,…

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  FIGURE 6. Functional and biochemical role of K1493 residue. (A) The amino acid sequence of residues 1484–1500 in NaV1.5. The double lysines 1492-3 are marked in a square. Amino acid’s charge (+ or–) or polarity (P) are indicated. (B) NaV1.5 variants co-expressed with rNaVβ1. (a) Representative INa traces recorded in (10 mM EGTA)in. (b) Maximal current densities. (c) Time constant (τ) obtained by fitting a single exponential to the decay phase of the currents in (10 mM EGTA)in at four voltages. Statistical significance was determined by one-way ANOVA followed by Holm–Sidak test (compared to NaV1.5WT). (C) Cells were transfected with NaV1.5K1493A/E/R together with rNaVβ1. (a) Example of a pull-down experiment with CaM-agarose beads, in Ca2+ and EGTA. (b) The total expression as quantified in EGTA, and normalized to NaV1.5WT (N = 3). (c) Pull-down experiments with CaM-agarose beads. The CaM-bound fraction was normalized to input level and quantified as % of NaV1.5WT in 2 mM Ca2+ (N = 3). Statistical significance was determined by one-way ANOVA, followed by Holm–Sidak test, compared to WT in 2 mM Ca2+. ∗∗p < 0.01; *p < 0.05. (D) GFP-NaV1.5WT (a) or GFP-NaV1.5K1493del (b) coexpressed with…