Clinical, electromyographic, and biophysical characterization of the rare Nav1.4 channel mutation SCN4A L1436P

Figures

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  FIGURE 1. Location of the L1436P and R1448H variants (A) 2D representation of α-Subunit of Nav1.4 (Jurkat-Rott et al., 2010). (B) 3D representation of human Nav1.4-β1 complex based on cryo-EM structure (Research Collaboratory for Structural Bioinformatics Protein Data Bank).

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  FIGURE 2. Compound muscle action potential (CMAP) amplitude related to time after short and long exercises (Case 3). The CMAP of the abductor digiti minimi muscle is recorded following stimulation of the ulnar nerve at the wrist. (A) The short exercises consist of a maximal voluntary muscle contraction against resistance for 10 s (represented by the red rectangles), repeated three times at 1-min intervals. Prior to this, the supramaximal intensity of nerve stimulation is determined by setting it at 150% of the intensity at which the CMAP amplitude no longer increases. The first 10-s exercise is performed, and the CMAP amplitude is measured immediately after this effort and then four more times every 10 s. The CMAP amplitude is normalized (%) relative to the maximal CMAP amplitude established before the exercises. The same sequence is repeated twice. (B) The same test is repeated on the same muscle after 7 min of muscle cooling (cold pack). (C) The long exercise is performed on the other abductor digiti minimi muscle to avoid interference from the short exercises and muscle cooling. This exercise consists of a maximal voluntary muscle contraction against resistance for 25 s, followed by 5 s…

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  FIGURE 3. Activation and fast inactivation of L1436P, R1448H and WT channels (A) Raw traces of Nav currents that were recorded at 22°C in HEK293T cells expressing WT or mutant hNav1.4 channels. One representative cell is shown for each subtype. Ten mV steps from −120 to 30 mV evoked a rapidly inactivating current with slower inactivation kinetics in the mutants (especially R1448H). Traces have been leak-subtracted (see Methods) (B) Normalized conductance (G/Gmax) versus voltage curves obtained for L1436P (n = 10); R1448H (n = 6), and WT channels (n = 6) using the equation G = I/(V-Vrev), with Vrev being +69 mV under our conditions). R1448H channels activated at more negative potentials than the other species at 22°C, but not at 32 °C. Bars represent interquartile range. The voltage commands are shown below.

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  FIGURE 4. Effect of temperature on fast inactivation of the various channels and steady-state inactivation (A) Representative raw traces recorded at −10 mV of WT, L1436P, and R1448H currents at 15°C, 22°C and 32°C. The amplitudes of the currents have been scaled to the same maximal amplitude to better compare the channel kinetics. Note the marked slowing down of fast inactivation (and of activation) in the three channel species when temperature was lower. (B) Summary of voltage- and temperature-dependence of inactivation time constants. The decay of the current at depolarized voltages was best described by a single exponential fit, with the resulting time constants (τ) plotted as a function of voltage. Note the differences in the vertical scales at the three temperatures. The voltage command is identical to that in Figure 3. (C) and (D) The steady-state inactivation curve of the L1436P mutant was slightly more depolarized than the one of the other species at 22°C, but not at 32°C. The voltage command is inserted in panel (D). In (B), (C) and (D), bars represent interquartile range.