Structural basis for the rescue of hyperexcitable cells by the amyotrophic lateral sclerosis drug Riluzole

Abstract

Neuronal hyperexcitability is a key element of many neurodegenerative disorders including the motor neuron disease Amyotrophic Lateral Sclerosis (ALS), where it occurs associated with elevated late sodium current (I_NaL). I_NaL results from incomplete inactivation of voltage-gated sodium channels (VGSCs) after their opening and shapes physiological membrane excitability. However, dysfunctional increases can cause hyperexcitability-associated diseases. Here we reveal the atypical binding mechanism which explains how the neuroprotective ALS-treatment drug riluzole stabilises VGSCs in their inactivated state to cause the suppression of I_NaL that leads to reversed cellular overexcitability. Riluzole accumulates in the membrane and enters VGSCs through openings to their membrane-accessible fenestrations. Riluzole binds within these fenestrations to stabilise the inactivated channel state, allowing for the selective allosteric inhibition of I_NaL without the physical block of Na⁺ conduction associated with traditional channel pore binding VGSC drugs. We further demonstrate that riluzole can reproduce these effects on a disease variant of the non-neuronal VGSC isoform Nav1.4, where pathologically increased I_NaL is caused directly by mutation. Overall, we identify a model for VGSC inhibition that produces effects consistent with the inhibitory action of riluzole observed in models of ALS. Our findings will aid future drug design and supports research directed towards riluzole repurposing.

Fig. 1. Prokaryotic VGSCs are structurally simpler compared to eukaryotic VGSCs but share basic functional architecture.

a Basic topology in the membrane of a single chain of the prokaryotic VGSC NavMs, showing the transmembrane helices (labelled 1-6 for helices S1-S6) which forms one domain of the homotetrameric channel, (top left), next to the single chain of the eukaryotic VGSC Nav1.4 which forms all 4 domains (top right) (b) Four individual subunits of NavMs produce the functional homotetrameric channel in a domain-swapped arrangement (a single domain coloured in deeper blue), c Nav1.4 folds from the single chain to form the functional pseudo-tetrameric channel.

Fig. 2. Riluzole interaction with NavMs reveals an atypical VGSC binding site.

a ¹⁹F-¹⁹F STD build-up over lengthening saturation times produces a curve for the interaction of riluzole with NavMs_F-Phe with an STDmax at plateau ∼3.3% (inset, riluzole coloured by heteroatom, with numbering as used throughout this study). b Riluzole binding site in NavMs produced from x-ray crystal analysis. Two views of the rilzuole binding site in NavMs (pink and yellow denoting sidechains from interacting residues from neighbouring domains) with 2Fo-Fc map (contoured at 1σ, grey), and sulphur anomalous signals (contoured at 3.5σ, red). c ¹⁹F-¹⁹F STD NMR using a site specific probe orientates riluzole in its binding site in NavMs. Subtraction of riluzole ¹⁹F signal produced from irradiation at the NavMs-BTFA fluorine resonance, (ON RESONANCE left, middle) from that produced from irradiation at the control resonance, (OFF RESONANCE left, bottom) results in a large STD peak ( ∼11% top, left) indicating strong saturation transfer between NavMs-BTFA and positioning the CF3-group of riluzole at the membrane side of the fenestration binding site (right panel). The grey sphere represents the radius of 7 Å which is the maximum distance for the STD effect from the fluorines on the C204-BTFA probe (shown as the modified sidechain at the centre of the sphere) (d) The binding site of riluzole within NavMs. (left), The complete structure of NavMs is shown cut through at fenestration depth with riluzole bound. (left,) with zoomed in view (right). e Ligplot analysis of the riluzole binding site with hydrophobic contacts from protein residues shown as red eyelashes. Interacting residues are labelled for their corresponding domains if transposed onto the D_III-D_IV fenestration of eNavs (f) Surface view of the fenestration binding site for riluzole coloured by hydrophobicity scale (low, white to high, red) looking outward from the pore.

Fig. 3. Effects of riluzole binding eNavs are reproduced in NavMs.

a Riluzole produces a dose-dependent hyperpolarised shift in the SSI curve for NavMs. Voltage dependence of inactivation of HEK293T cells transfected with NavMS after perfusion of the vehicle (black circles) or increasing concentrations of riluzole. Data points were fit to a Boltzmann equation to calculate V_1/2 (Supplementary Information Table 2). The normalised current data is displayed as mean ± S.E.M. SSI V_1/2 after perfusion with the vehicle (black circles) was −84.5 ± 1.4 mV (n = 11), but was −86.9 ± 2.6 mV (n = 5), −94.1 ± 2.5 mV (n = 18), −95.5 ± 1.8 mV (n = 8), and −97.3 ± 3.2 mV (n = 6) after perfusion with 1 µM (blue triangles), 2.5 µM (red squares), 5 µM (green triangles), and 25 µM (purple squares) riluzole respectively. One-way ANOVA and post-hoc Tukey test found that 2.5, 5 and 25 µM riluzole significantly shifted SSI compared to vehicle (p = 0.025, 0.038, and 0.024 respectively). b Riluzole slows NavMs recovery from inactivation in HEK293T cells. The t_fast component of a biphasic recovery process was significantly increased by 3-fold. Data displayed as mean ± S.E.M, *p < 0.05, **p < 0.01. τ_fast after perfusion with the vehicle was 0.009 ± 0.002 s (n = 8), but was 0.034 ± 0.007 s (n = 8), 0.050 ± 0.004 s (n = 5), and 0.033 ± 0.008 s (n = 6) after perfusion with 2.5, 5 and 25 µM riluzole respectively. One-way ANOVA and post-hoc Tukey test found that that 2.5, 5 and 25 µM riluzole significantly shifted τ_fast compared to vehicle (p = 0.008, 0.0002, and 0.027 respectively). c Riluzole only blocks NavMs in HEK293T cells at the high concentration (100 µM) with 1–25 µM producing no significant occlusion of the channel pore. Data points shown with mean ± S.E.M indicated, ***p < 0.0001. Fractional block after perfusion with the vehicle was 0.002 ± 0.016 (n = 11), and was −0.027 ± 0.021 (n = 5), −0.007 ± 0.024 (n = 18), 0.055 ± 0.068 (n = 8), −0.011 ± 0.050 (n = 6), and 0.239 ± 0.064 (n = 6) for 1 µM (blue triangles), 2.5 µM (red squares), 5 µM (green triangles), 25 µM (purple squares), and 100 µM (pink diamond) riluzole respectively. One-way ANOVA and post-hoc Tukey test found that only 100 µM riluzole significantly blocked NavMS compared to vehicle (p = 0.0001). d Top-down sliced view of NavMs at fenestration depth showing riluzole bound in all four fenestrations. e Side view from Hole2 analysis showing that even with all 4 fenestrations occupied, riluzole does not block Na⁺ conduction. A pore radius of > 2.3 Å is required for Na+ conduction and is represented by blue in the channel tunnel cartoon (left panel) and delineated by the broken vertical line in the plot of pore radius along the pore axis (right panel). The green shaded area in the plot (right panel) represents Na⁺ conduction pathway at fenestration depth.

Fig. 4. MD simulation of riluzole with NavMs.

a Clustering analysis of riluzole and NavMs, all members of the primary cluster (depicted in grey) and NavMs WT (depicted as pink helices in a top-down view at the depth of the fenestrations. The primary cluster represents the riluzole location for 26% of the total simulation time, b The primary cluster overlays with the binding location of riluzole in the X-ray structure. c Contact map of riluzole atoms with T176 and M204 from NavMs calculated from the simulation, coloured by the average number of contacts per timestep, showing that riluzole orientation is the same in both structurally-determined and MD simulation-determined binding sites.

Fig. 5. Importance of the LA binding site for riluzole binding and action in NavMs and the overall mechanism of interaction.

a LA binding site mutation T207A abrogates the riluzole effect on SSI of NavMs expressed in HEK293T cells. Data points for WT NavMs (circles) and NavMS T207A (squares) were fit to a Boltzmann equation to calculate V_1/2 (Supplementary Information Table 2). The normalised current data is displayed as mean ± S.E.M. Data points were fit to a Boltzmann function to calculate V_1/2. Unpaired Student’s t-test of the riluzole effect on SSI of NavMs T207A found no significant difference compared to NavMs T207A without riluzole (n = 5, p > 0.5), b Contact map for riluzole interaction with NavMs T207A over the course of the simulation showing that the average number of riluzole atoms which are in simultaneous contact with residues T176 and M204 are greatly reduced compared to WT c Top-down view at fenestration depth of all members of the primary cluster (grey) from the NavMs T207F MD simulation. d Riluzole binding site within this primary cluster represents riluzole binding in the eNav D_III-IV-mimicking fenestration of NavMs T207F with the additional π–π stacking interaction indicated by a broken yellow line e Bar chart of the maximum riluzole residence times observed in the binding sites of each NavMs variant (f) Pathway taken by a typical NavMs binding riluzole molecule during the MD simulations. Black lines represent the membrane boundary.

Fig. 6. MD simulation shows two riluzole binding sites in the hNav1.4 D_III-D_IV fenestration.

a Cluster 1 (grey) for riluzole interaction in the hNav1.4 D_III-D_IV fenestration. b Interacting residues at binding site 1 showing a hydrogen bond between riluzole and S1283. c Surface view  of riluzole bound in the fenestration at binding site 1 looking outward from the pore. d Cluster 2 (grey) for riluzole interaction. e Interacting residues at binding site 2 features a π–π stacking interaction between the benzothiazole group of riluzole and F1586. f Surface view of riluzole in the fenestration at binding site 2 looking outward from the pore. Figure 6c, f surfaces are coloured for hydrophobicity on the scale shown in Fig. 2f.

Fig. 7. Riluzole stabilises inactivation in a hNav1.4 myotonia mutant.

a Effect of riluzole (1 µM) or its vehicle solvent (DMSO) on the SSFI of hNav1.4 P1158S expressed in HEK293T cells. Data is plotted as average ( ± S.E.M) with the insert showing the protocol and representative currents (n = 5, each). Data points were fit to a Boltzmann function. b Representative I_NaL showing the effect of riluzole (1 or 5 µM) or its vehicle solvent on hNav1.4 P1158S expressed in HEK293T cells. The inset bar graph shows the effect of riluzole on the percentage ( ± S.E.M) of I_NaL (n = 5, each). One-way ANOVA and post-hoc Tukey test found that hNav1.4 P1158S significantly increased I_NaL (p < 0.0001) compared to WT, and that application of 1 and 5 µM riluzole significantly decreased I_NaL (p < 0.0001 for both). I_NaL levels produced on addition of 1 and 5 µM riluzole to hNav1.4 P1158S were not significantly different from WT vehicle (p > 0.9 for both).