Biophysical and structural insights into the SCN4A E452K variant linked to myotonia and paramyotonia congenita

Figures

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  Fig. 1. Localization of the E452K variant in hNaV1.4. (A) Sanger sequencing of a portion of exon 9 revealed the heterozygous mutation c.1354G > A that causes the E452K substitution. (B) The E452K mutation is in a region that is highly conserved in all voltage-gated sodium channels in humans and other species. (C) Schematic representation of the hNaV1.4 α-subunit highlighting the localization of the E452K mutation on the intracellular loop between domain I and domain II. (D) Side view of the α-subunit illustrating its four domains (I to IV) along with its β1 regulatory subunit. (E) Intracellular view of the channel protein illustrating the open pore cavity in the center, the four domains (I to IV), and the inactivation gate (III-IV linker). (F) A higher resolution view of the I-II linker, pinpointing the position of the glutamate (E) residue.

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  Fig. 2. Analysis of whole-cell sodium currents recorded from HEK 293 cells expressing hNaV1.4/WT or hNaV1.4/E452K. (A) INa from hNaV1.4/WT and hNaV1.4/E452K were obtained using depolarizing pulses ranging from –140 mV to + 65 mV, with 5-mV increments at each step. (B) Current density at –15 mV. The current density of hNaV1.4/E452K decreased by 51% compared to hNaV1.4/WT (**p < 0.01). The current–voltage relationships of hNav1.4/WT (n = 15) and hNaV1.4/E452K (n = 16) were analyzed, with the current amplitude normalized to the membrane capacitance. (C) Voltage-dependence of fast inactivation was assessed by fitting current decays to an exponential function and was plotted against voltage.

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  Fig. 3. Gating properties of hNaV1.4/WT and hNaV1.4/E452K. (A) Voltage-dependence of steady-state activation and inactivation of hNaV1.4/WT (activation, n = 15; inactivation, n = 8) and hNaV1.4/E452K (activation, n = 16; inactivation, n = 9). The activation curves were obtained from I/V relationships, while the inactivation curves were derived using the protocol shown in the insets. The resulting data were fitted to a standard Boltzmann distribution. The slope factor k activation for hNav1.4/E452K was significantly greater than for hNav1.4/WT (**p < 0.01). (B) The predicted window current was obtained using the following equation: (1/(1 + exp ((V0.5activation—V)/k activation)) x ((1—C)/(1 + exp ((V -V0.5inactivation)/kinactivation)) + C) × 100. The open probability was 2.2 times higher in hNav1.4/E452K than in hNav1.4/WT. (C) Time courses of recovery from inactivation in hNaV1.4/WT (n = 7) and hNaV1.4/E452K (n = 8).

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  Fig. 4. Gating properties of the slow inactivation. (A) Closed-state inactivation in hNaV1.4/WT (–100 mV, n = 12; –80 mV, n = 7; –60 mV, n = 8) and hNaV1.4/E452K (–100 mV, n = 5; –80 mV, n = 6; –60 mV, n = 6). (B) Recovery from slow inactivation of hNaV1.4/WT (n = 7) and hNaV1.4/E452K (n = 13). (C) Onset of slow inactivation in hNaV1.4/WT (n = 4) and hNaV1.4/E452K (n = 4). All the protocols used to generate the currents are shown in the insets. The slow time constant (τSlow) was faster in hNaV1.4/E452K than in hNaV1.4/WT (*p < 0.05).

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  Fig. 5. Temperature influences the biophysical properties of the hNaV1.4. (A) Examples of currents recorded from hNaV1.4/WT and hNaV1.4/E452K at –10 mV at different temperatures (22 °C, 15 °C, and 10 °C). (B, C) Current density at –10 mV. The current density of hNaV1.4E452K decreased by 46% (**p < 0.01) at 15 °C (B) and by 52% (**p < 0.01) at 10 °C (C) compared to hNaV1.4/WT. For comparison purposes, the I/V curves of hNaV1.4/WT and hNaV1.4/E452K at 22 °C are shown in gray. The current amplitude was normalized to membrane capacitance. (D, E) Relationships between the inactivation time constant and voltage. These relationships were obtained by fitting the time constant of current decay to an exponential function and then plotted against voltage.

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  Fig. 6. Structural investigation by MD simulation. (A) Alpha subunit of hNaV1.4/WT in a lipid bilayer channel (dark gray), OL (transparent gray), PA (transparent yellow), PC (transparent purple) E452 (blue), and K249 (cyan). (B) Zoomed in region highlighting the E452 and K249 interaction for hNaV1.4/WT. (C) Distances between the charged atoms of the side chains at positions 249 and 452 plotted for each frame of the MD calculation (1-μs simulation with coordinates sampled every 100 ps). Data color-coded hNaV1.4/WT (dark blue) and hNaV1.4/E452K (orange). (D) Overlay of hNaV1.4/WT (grey) and hNaV1.4/E452K (purple) with K249 (cyan for both), WT E452 (blue), and K452 mutation (red).