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. 2018 Jul 9:11:232.
doi: 10.3389/fnmol.2018.00232. eCollection 2018.

Gain of Function for the SCN1A/hNav1.1-L1670W Mutation Responsible for Familial Hemiplegic Migraine

Sandra Dhifallah  1 , Eric Lancaster  2 , Shana Merrill  2 , Nathalie Leroudier  1 , Massimo Mantegazza  1 , Sandrine Cestèle  1
Affiliations

Gain of Function for the SCN1A/hNav1.1-L1670W Mutation Responsible for Familial Hemiplegic Migraine

Sandra Dhifallah et al. Front Mol Neurosci. .

Abstract

The SCN1A gene encodes for the voltage-dependent Nav1.1 Na+ channel, an isoform mainly expressed in GABAergic neurons that is the target of hundreds of epileptogenic mutations. More recently, it has been shown that the SCN1A gene is also the target of mutations responsible for familial hemiplegic migraine (FHM-3), a rare autosomal dominant subtype of migraine with aura. Studies of these mutations indicate that they induce gain of function of the channel. Surprisingly, the mutation L1649Q responsible for pure FHM-3 showed a complete loss of function, but, when partially rescued it induced an overall gain of function because of modification of the gating properties of the mutant channel. Here, we report the characterization of the L1670W SCN1A mutation that has been previously identified in a Chinese family with pure FHM-3, and that we have identified also in a Caucasian American family with pure FHM-3. Notably, one patient in our family had severe neurological deterioration after brain radiation for cancer treatment. Functional analysis of L1670W reveals that the mutation is responsible for folding/trafficking defects and, when they are rescued by incubation at lower temperature or by expression in neurons, modifications of the gating properties lead to an overall gain of function. Therefore, L1670W is the second mutation responsible for FHM-3 with this pathophysiological mechanism, showing that it may be a recurrent mechanism for Nav1.1 hemiplegic migraine mutations.

Keywords: GABAergic neurons; cortical spreading depression; epilepsy; migraine with aura; sodium channels.

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Figures

Figure 1
Figure 1
L1670W familial hemiplegic migraine (FHM-3) family. (A) Family pedigree. The proband is patient II-2. No genetic test was performed on patient I-1. Squares are representative of men and circles of women. Plain symbols represent affected individuals and empty symbols unaffected individuals. (B) Schematic representation of NaV1.1 alpha subunit and localization of FHM-3 mutations identified thus far; the mutation L1670W that we have studied here is highlighted with an arrow. Voltage-gated sodium channels are formed by four main domains (DI-IV), each one formed by six transmembrane segments (S1–6) connected by intracellular and extracellular loops; within each domain, S1–4 form the voltage sensing module (in which the positively charged S4 is the voltage sensor) and S5–6 form the pore module, with the intracellular loop between S4 an S5 that is the linker between the two modules. The intracellular loop between DIII-IV is the inactivation gate, which blocks the pore in the fast-inactivated states of the channels acting as a hinged lid. Slow inactivation depends on rearrangements of the pore domain. DIV is important for the coupling between activation and inactivation. Thus, most of the FHM mutations are in regions that are important for inactivation.
Figure 2
Figure 2
hNav1.1-L1670W is a rescuable folding/trafficking defective mutant. Representative families of whole-cell sodium current traces recorded applying depolarizing steps from −70 to +60 mV in 5 mV increments (from a holding potential of −120 mV) in tsA-cells transfected with hNav1.1-WT and incubated at 37°C (A) transfected with hNav1.1-L1670W and incubated at 37°C (B) transfected with hNav1.1-WT and incubated at 30°C (C) transfected with hNav1.1-L1670W and incubated at 30°C (D); Scale bars: 1 nA, 1 ms. (E) Data and box chart (the bar represents the median, the square the mean, the box the SEM and the whiskers the 10–90% range) of the mean maximum current density of hNav1.1-WT incubated at 37°C (106.9 ± 21.0 pA/pF, n = 11), hNav1.1-L1670W incubated at 37°C (24.7 ± 1.7 pA/pF, n = 29), hNav1.1-WT incubated at 30°C (167 ± 27 pA/pF, n = 21), and hNav1.1-L1670W incubated at 30°C (89.7 ± 12.5 pA/pF, n = 22); statistical comparison: hNav1.1-WT-37°C vs. hNav1.1-L1670W-37°C p = 1.2 * 10−5, hNav1.1-WT-37°C vs. hNav1.1-WT-30°C n.s., hNav1.1- L1670W-37°C vs. hNav1.1-L1670W-30°C p = 8 * 10−7, hNav1.1-WT-30°C vs. hNav1.1-L1670W-30°C n.s. (Kruskall-Wallis test followed by Mann-Whitney test with Bonferroni correction for four comparisons). ns, non significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(F) Fold increase in current density with incubation at 30°C.
Figure 3
Figure 3
Functional effects of hNav1.1-L1670W on fast gating properties upon rescue in tsA-201 cells. (A) Mean voltage dependence of activation and fast inactivation, lines are mean Boltzmann fits; mean parameters: voltage of half activation Va and slope Ka for hNav1.1-WT (n = 18), Va = −21.6 ± 1.2 mV, Ka = 6.6 ± 0.4, hNav1.1-L1670W (n = 22), Va = −16.8 ± 1.0 mV (p = 0.004), Ka = 6.1 ± 0.5 mV; voltage of half inactivation (Vh), slope (Kh) and baseline for hNav1.1-WT (n = 12), Vh = −55.6 ± 0.3 mV, Kh = 4.9 ± 0.3 mV, baseline = 0.04 ± 0.01, L1670W (n = 16), Vh = −47.3 ± 0.9 mV (p = 5 * 10−5), Kh = 6.1 ± 0.5 mV, baseline = 0.19 ± 0.05 (p = 2 * 10−5); Welch t-test. Inset: normalized current-voltage (I-V) plot of the peak transient current. (B) Comparison of mean normalized current traces elicited with a command step to −10 mV from a holding potential of −120 mV; the dotted line is the 0. n = 13 for WT and L1679W. (C) Time (ms) of half-activation of the current at the indicated potentials: −30 mV WT 0.46 ± 0.03, L1670W 0.51 ± 0.03; −20 mV WT 0.36 ± 0.03, L1670W 0.45 ± 0.03 (p = 0.02); −10 mV WT 0.29 ± 0.02, L1670W 0.36 ± 0.03 (p = 0.04); 0 mV WT 0.25 ± 0.02, L1670W 0.30 ± 0.02 (p = 0.05); 10 mV WT 0.22 ± 0.01 L1670W 0.25 ± 0.02; 20 mV WT 0.20 ± 0.01 L1670W 0.23 ± 0.02; Welch t-test, n = 13 for all groups. (D) Voltage dependence of the time constant (τ in ms) of the current decay (single exponential fits at the indicated potentials): −30 mV WT 1.5 ± 0.3, L1670W 2.8 ± 0.4 (p = 0.02); −20 mV WT 0.63 ± 0.06, L1670W 1.2 ± 0.1 (p = 5 * 10−4); −10 mV WT 0.45 ± 0.04, L1670W 0.59 ± 0.03 (p = 0.008); 0 mV WT 0.37 ± 0.03, L1670W 0.41 ± 0.02; 10 mV WT 0.31 ± 0.03 L1670W 0.34 ± 0.02; 20 mV WT 0.30 ± 0.03 L1670W 0.32 ± 0.02; Welch t-test, n = 13 for all groups. (E) τ (ms) of the development of fast inactivation at the indicated potentials: −60 mV, τDEV-WT = 25.6 ± 2.2 (n = 12), τDEV-L1670W = 8.6 ± 1.4 (n = 4; p = 0.009); −50 mV, τDEV-WT = 11.3 ± 2.0 (n = 10), τDEV-L1670W = 2.4 ± 0.8 (n = 7; p = 0.002); −40 mV, τDEV-WT = 2.8 ± 1.1 (n = 6), τDEV-L1670W = 3.6 ± 0.4 (n = 7); Mann-Whitney test. (F) τ (ms) of the recovery from fast inactivation at the indicated potentials: −120 mV, τREC-WT = 1.4 ± 0.2 (n = 7), τREC-L1670W = 0.8 ± 0.2 ms (n = 4); −110 mV, τREC-WT = 1.7 ± 0.2 ms (n = 7), τREC-L1670W = 0.79 ± 0.09ms (n = 3; p = 0.02); −100 mV, τREC-WT = 2.7 ± 0.3 ms (n = 8), τREC-L1670W = 1.2 ± 0.2 ms (n = 4; p = 0.014); −90 mV, τREC-WT = 5.6 ± 0.9 ms (n = 7), τREC-L1670W = 1.7 ± 0.6 ms (n = 5; p = 0.02); −80 mV, τREC-WT = 9.8 ± 1.9 ms (n = 7), τREC-L1670W = 2.9 ± 0.8 ms (n = 6; p = 0.02); Mann-Whitney test. (G) Left panel, comparison of the same mean normalized current traces displayed in (B), but with a longer time scale in order to compare INaP; right panel, mean current-voltage plots for INaP measured after 5 min from the establishment of the whole-cell configuration and expressed as percentage of the transient current (maximal INaP: 4.0 ± 1.5% n = 21 WT, 14.9 ± 2.1% n = 22 L1670W; p = 0.002 Mann-Whitney test). Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 4
Figure 4
Functional effects of hNav1.1-L1670W on slow inactivation upon rescue in tsA-201 cells. (A) Voltage dependence of development of slow inactivation; the lines are mean Boltzmann fits: mean parameters, hNav1.1-WT, Vh = −53.5 ± 2.0 mV, Kh = 7.6 ± 0.9 mV, baseline 0.14 ± 0.03 (n = 11); hNav1.1-L1670W, Vh = −34.8 ± 2.7 mV (p = 2 * 10−4), Kh = 10.0 ± 1.4, baseline 0.13 ± 0.03 (n = 6). ***p < 0.001. (B) Mean kinetics of development of slow inactivation at 0 mV for the indicated durations, the lines are mean fits of single exponentials: mean parameters, τDEV-WT = 1310 ± 184 ms, baseline 0.08 ± 0.02 (n = 8), τDEV-L1670W = 2070 ± 283 ms, baseline 0.15 ± 0.04 (n = 6). (C) Voltage dependence of recovery from slow inactivation; the lines are mean Boltzmann fits: mean parameters, WT, Vh = −66.9 ± 2.4 mV, Kh = 7.4 ± 1.2 mV, baseline 0.15 ± 0.03 (n = 8); L1670W, Vh = −68.3 ± 2.9 mV, Kh = 7.5 ± 0.9 baseline 0.15 ± 0.04 (n = 6). (D) Mean recovery at −90 mV from slow inactivation; the lines are mean fits of single exponentials: mean parameters, τDEV-WT = 3252 ± 892 ms (n = 6), τDEV-L1670W = 3449 ± 383 ms (n = 5). Welch t-test for all the comparisons. The insets show the voltage stimuli; the relative durations of the steps are not in scale (see the durations indicated). Data are shown as mean ± SEM.
Figure 5
Figure 5
Overall effect of hNav1.1-L1670W expressed in tsA-201 cells. (A–C) Use dependence (current normalized to the first stimulus in the train) induced by trains of 200 depolarizing steps 2 ms long to 0 mV from the holding potential of −70 mV at the frequencies of 10 Hz (A) 50 Hz (B) and 100 Hz (C) for hNav1.1-WT (black symbols) and hNav1.1-L1670W (open symbols). Comparison of the last stimulation in the train: 10 Hz WT 0.83 ± 0.06 (n = 5), L1670W 0.91 ± 0.05 (n = 5); 50 Hz WT 0.65 ± 0.05 (n = 5), L1670W 0.87 ± 0.04 (n = 5; p = 0.03); 100 Hz 0.45 ± 0.05 (n = 5), L1670W 0.77 ± 0.04 (n = 5; p = 0.02); Mann-Whitney test. *p < 0.05. (D–F) Action Na+ currents (expressed as mean current density; error bars are not shown for clarity) recorded using as voltage stimulus an action potential discharge recorded from a GABAergic fast spiking neuron in neocortical brain slices (D) for hNav1.1-WT (E) n = 7, and hNav1.1-L1670W. **p < 0.01. (F) n = 7. Insets show the first five (left) and the last five (right) action currents; the time scale is the same for all the insets. The amplitude of the first three action currents was not different: (pA/pF) WT −49 ± 13, −16 ± 8, −14 ± 7, L1670W −30 ± 6, −27 ± 6, −26 ± 7; the first difference was observed for the 4th peak action current: WT −12 ± 4, L1670W −27 ± 6 (p = 0.045); comparison of the amplitude of the last five peak action currents pooled: WT 9.6 ± 1.4 (n = 7), L1670W 28 ± 7 (n = 7; p = 0.01); Mann-Whitney test for all the comparisons. Data are shown as mean ± SEM.
Figure 6
Figure 6
Functional effects of hNav1.1-L1670W expressed in neocortical GABAergic neurons. (A) Representative whole-cell sodium current traces recorded in the presence of 1 μM tetrodotoxin (TTX) from fusiform bipolar GABAergic neurons selected in primary cultures of neocortical neurons transfected with hNav1.1-F383S-WT (left) or hNav1.1-F383S-L1670W (right); the inset displays the mean current density-voltage plots for hNav1.1-F383S-WT and hNav1.1-F383S-L1670W, and the comparison of the mean maximum current density yielded: WT 41.9 ± 5.7 pA/pF n = 22; L1670W 19.4 ± 1.5 pA/pF n = 20 (p = 7 * 10−4), Mann-Whitney test. (B) Mean voltage dependence of activation and fast inactivation, lines are mean Boltzmann fits; mean parameters: voltage of half activation (Va) and slope (Ka) for WT (n = 16), Va = −17.5 ± 1.3 mV, Ka = 5.1 ± 0.3, L1670W (n = 15), Va = −8.4 ± 1.4 mV (p = 10−4), Ka = 7.7 ± 0.8 mV (p = 0.01); voltage of half inactivation (Vh), slope (Kh) and baseline for WT (n = 15), Vh = −48.1 ± 1.9 mV, Kh = 4.3 ± 0.2 mV, baseline = 0.09 ± 0.03, L1670W (n = 9), Vh = −27.9 ± 1.5 mV (p = 3 * 10−4), Kh = 5.1 ± 0.7 mV, baseline = 0.26 ± 0.06 (p = 5*10−4); Welch t-test. (C) Mean current-voltage plots for INaP measured after 5 min from the establishment of the whole-cell configuration and expressed as percentage of the transient current; comparison of maximal INaP: WT 5.6 ± 0.9% n = 21, L1670W 13.6 ± 1.9% n = 22, p = 0.005, Mann-Whitney test). (D) Time (ms) of half-activation of the current at the indicated potentials: −30 mV WT 0.81 ± 0.14, L1670W 1.05 ± 0.09; −20 mV WT 0.70 ± 0.08, L1670W 0.9 ± 0.1; −10 mV WT 0.53 ± 0.05, L1670W 0.69 ± 0.07; 0 mV WT 0.41 ± 0.04, L1670W 0.52 ± 0.04; 10 mV WT 0.34 ± 0.04 L1670W 0.43 ± 0.03; 20 mV WT 0.29 ± 0.03 L1670W 0.35 ± 0.03; non-significant differences, Welch t-test, n = 15 for all groups. (E) Voltage dependence of the time constant (τ in ms) of the current decay (single exponential fits at the indicated potentials): −20 mV WT 1.6 ± 0.3, L1670W 2.3 ± 0.3; −10 mV WT 0.86 ± 0.18, L1670W 1.77 ± 0.26 (p = 0.02); 0 mV WT 0.59 ± 0.07, L1670W 0.88 ± 0.07; 10 mV WT 0.51 ± 0.06 L1670W 0.49 ± 0.05; 20 mV WT 0.45 ± 0.05 L1670W 0.48 ± 0.16; Welch t-test, n = 15 for all groups. Whole-cell action sodium currents (expressed as mean current density; error bars are not shown for clarity) recorded as in Figures 5D–F using as voltage stimulus an action potential discharge recorded in a neocortical mouse brain slice from a GABAergic fast spiking neuron for WT (n = 11; F) and L1670W (n = 9; G): comparison of the amplitude of the first peak action current in the discharge, WT 19.6 ± 2.9 pA/pF; L1670W 12.7 ± 1.8 pA/pF; the first action current with amplitude statistically different was the 3rd in the discharge: WT 5.9 ± 1.5 pA/pF; L1670W 10.5 ± 1.4 pA/pF (p = 0.048); comparison of the average of the last five peak action currents (pooled) in the discharge, WT 5.2 ± 0.8 pA/pF; L1670W 9.0 ± 1.0% pA/pF (p = 0.01); Welch t-test for all the comparisons. Data are shown as mean ± SEM.
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References

    1. Bechi G., Rusconi R., Cestele S., Striano P., Franceschetti S., Mantegazza M. (2015). Rescuable folding defective NaV1.1 (SCN1A) mutants in epilepsy: properties, occurrence, and novel rescuing strategy with peptides targeted to the endoplasmic reticulum. Neurobiol. Dis. 75, 100–114. 10.1016/j.nbd.2014.12.028 - DOI - PubMed
    1. Bechi G., Scalmani P., Schiavon E., Rusconi R., Franceschetti S., Mantegazza M. (2012). Pure haploinsufficiency for Dravet syndrome NaV1.1 (SCN1A) sodium channel truncating mutations. Epilepsia 53, 87–100. 10.1111/j.1528-1167.2011.03346.x - DOI - PubMed
    1. Bernier V., Lagacé M., Bichet D. G., Bouvier M. (2004). Pharmacological chaperones: potential treatment for conformational diseases. Trends Endocrinol. Metab. 15, 222–228. 10.1016/j.tem.2004.05.003 - DOI - PubMed
    1. Catterall W. A. (2012). Voltage-gated sodium channels at 60: structure, function and pathophysiology. J. Physiol. 590, 2577–2589. 10.1113/jphysiol.2011.224204 - DOI - PMC - PubMed
    1. Catterall W. A. (2014). Structure and function of voltage-gated sodium channels at atomic resolution. Exp. Physiol. 99, 35–51. 10.1113/expphysiol.2013.071969 - DOI - PMC - PubMed