Biophysical Investigation of Sodium Channel Interaction with β-Subunit Variants Associated with Arrhythmias

Abstract

Background: Voltage-gated sodium (Na_V) channels help regulate electrical activity of the plasma membrane. Mutations in associated subunits can result in pathological outcomes. Here we examined the interaction of Na_V channels with cardiac arrhythmia-linked mutations in SCN2B and SCN4B, two genes that encode auxiliary β-subunits. Materials and Methods: To investigate changes in SCN2B ^R137H and SCN4B ^I80T function, we combined three-dimensional X-ray crystallography with electrophysiological measurements on Na_V1.5, the dominant subtype in the heart. Results: SCN4B ^I80T alters channel activity, whereas SCN2B ^R137H does not have an apparent effect. Structurally, the SCN4B ^I80T perturbation alters hydrophobic packing of the subunit with major structural changes and causes a thermal destabilization of the folding. In contrast, SCN2B ^R137H leads to structural changes but overall protein stability is unaffected. Conclusion: SCN4B ^I80T data suggest a functionally important region in the interaction between Na_V1.5 and β4 that, when disrupted, could lead to channel dysfunction. A lack of apparent functional effects of SCN2B ^R137H on Na_V1.5 suggests an alternative working mechanism, possibly through other Na_V channel subtypes present in heart tissue. Indeed, mapping the structural variations of SCN2B ^R137H onto neuronal Na_V channel structures suggests altered interaction patterns.

Keywords: NaV1.5; SCN2B; SCN4B; arrhythmia.

FIG. 1.

(A–C) Representative traces of hNa_V1.5 alone or coexpressed with hβ4 (red) or hβ4^I80T (magenta). Oocytes were held at −120 mV and pulsed from −90 to 15 mV for 50 ms. (D) Western blot probing for the C-terminal myc-tag of hβ4. Signal is seen for wild-type hβ4 as well as hβ4^I80T, in both whole cell (closed circles) and surface (open circle) fractions. (E) Normalized conductance voltage (G-V, filled circles) and channel availability (I-V, open circles) relationships for hNa_V1.5 either without hβ4 (black), with hβ4 (red), or with hβ4 I80T (magenta). Summary data of G-V and I-V V_1/2 values, slope values, and p-values determined by unpaired t-test are reported in Table 1. (F) Rate (τ) of channel fast inactivation using the same color scheme described above. Time constant was obtained using a single-exponential fit of the current decay phase over a 45 mV range. Error bars in all cases reflect SEM with n = 5–7; *p < 0.05 or **0.01 using one-way ANOVA. ANOVA, analysis of variance; hNa_V, human voltage-gated sodium; SEM, standard error of the mean.

FIG. 2.

(A, B) Representative traces of hNa_V1.5 coexpressed with hβ2 (red) or hβ2^R137H (cyan). Oocytes were held at −120 mV and pulsed from −90 to 15 mV for 50 ms. (C) Normalized conductance voltage (G-V, filled circles) and channel availability (I-V, open circles) relationships for hNa_V1.5 without a β-subunit (black) or with either hβ2 (red) or with hβ2 R137H (cyan). Summary data of G-V and I-V V_1/2 values, slope values, and p-values determined by unpaired t-test are reported in Table 1. (D) Rate (τ) of channel fast inactivation using the same color scheme described above. Time constant was obtained using a single-exponential fit of the current decay phase over a 45 mV range. (E) Western blot probing for the C-terminal myc-tag of hβ2. Signal is seen for wild-type hβ2 as well as hβ2^R137H, in both whole cell (closed circles) and surface (open circle) fractions. Error bars in all cases reflect SEM. with n = 5–7; *p < 0.05 or **0.01 using one-way ANOVA.

FIG. 3.

(A) Normalized persistent current of hNa_V1.5 (black), hNa_V1.5 +hβ4 (red), or hNa_V1.5 +hβ4^I80T (magenta) measured 20 ms after depolarization over a 30 mV range. (B) Normalized recovery from fast inactivation measured over a 50 ms timeframe using a double-pulse protocol to the maximum current of the I-V in Figure 1. Recovery voltage used for this protocol was −10 mV. (C, D) Normalized conductance voltage (G-V, filled circles) and channel availability (I-V, open circles) relationships of hβ4 (red) or hβ4^I80T (magenta) coexpressed with either hNa_V1.1 (C) or hNa_V1.2 (D). Both wild-type channels without β-subunit coexpression are shown in black. Summary data of G-V and I-V V_1/2 values, slope values, and p-values determined by unpaired t-test are reported in Table 1.

FIG. 4.

Crystallographic analysis of hβ2^R137H. Wild-type hβ2 (containing the C55A mutation to facilitate crystallization) is shown in blue, and the R137H mutant in green. R137 is shown in black sticks and H137 in pink. (A) Overall superposition of both structures. (B) Details around the site of the mutation. Selected residues are shown in sticks and labeled. Hydrogen bonds are shown with dotted lines. In wild-type hβ2, the Arg137 side chain is involved in a hydrogen bond network as well as a cation–π interaction with Y128. Substitution by H137 results in multiple changes in side chain orientations, as well as the main chain conformation of the N-terminal region around N28.

FIG. 5.

Crystallographic analysis of hβ4^I80T. Wild-type hβ4 (containing the C58A mutation to facilitate crystallization) is shown in blue, and the I80T mutant in orange. I80 is shown in black sticks, and T80 in pink. (A) Overall superposition of both structures. (B) Details around the site of the mutation. Select residues are shown in sticks and labeled. I80 is involved in packing of the hydrophobic core, making interactions with other hydrophobic residues. Substitution by Thr results in minor structural changes, including a rearrangement of the I101 side chain conformation.

FIG. 6.

Thermal melting curves monitored through CD recorded at 213 nm.(A) Wild-type hβ4 (containing the C58 mutation—black) and the I80T mutant (red). (B) Wild-type hβ2 (containing the C58A mutation—black) and the R137H mutant (green). The T_M values were obtained from the midpoints of the transitions. CD, circular dichroism.

FIG. 7.

Mapping the mutation sites on the Na_V1.2-β2 cryo-EM structure. (A) Overall cryo-EM structure of Na_V1.2 bound to hβ2 (PDB 6J8E). The pore-forming domain and VSDs are shown in gray and red, respectively, and hβ2 in blue. (B). Details of the interaction. Selected residues in hβ2 are labeled. Notice that several of the hβ2 residues, shown to be affected by the R137H mutation, are directly at the interface with Na_V1.2. EM, electron microscopy; VSDs, voltage-sensing domains.

FIG. 8.

(A) Normalized conductance voltage (G-V, filled circles) and channel availability (I-V, open circles) relationships for hNa_V1.2 without a β-subunit (black) or with either hβ2 (red) or with hβ2 R137H (cyan). Summary data of G-V and I-V V_1/2 values, slope values, and p-values determined by unpaired t-test are reported in Table 1. (B) Rate (τ) of channel fast inactivation using the same color scheme described above. Time constant was obtained using a single-exponential fit of the current decay phase over a 45 mV range. Error bars in all cases reflect SEM. with n = 5; *p < 0.05 or **0.01 using one-way ANOVA.