XB-ART-57298
Br J Pharmacol
2020 Oct 01;17719:4481-4496. doi: 10.1111/bph.15196.
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Uncoupling sodium channel dimers restores the phenotype of a pain-linked Nav 1.7 channel mutation.
Rühlmann AH
,
Körner J
,
Hausmann R
,
Bebrivenski N
,
Neuhof C
,
Detro-Dassen S
,
Hautvast P
,
Benasolo CA
,
Meents J
,
Machtens JP
,
Schmalzing G
,
Lampert A
.
???displayArticle.abstract???
BACKGROUND AND PURPOSE: The voltage-gated sodium channel Nav 1.7 is essential for adequate perception of painful stimuli. Mutations in the encoding gene, SCN9A, cause various pain syndromes in humans. The hNav 1.7/A1632E channel mutant causes symptoms of erythromelalgia and paroxysmal extreme pain disorder (PEPD), and its main gating change is a strongly enhanced persistent current. On the basis of recently published 3D structures of voltage-gated sodium channels, we investigated how the inactivation particle binds to the channel, how this mechanism is altered by the hNav 1.7/A1632E mutation, and how dimerization modifies function of the pain-linked mutation. EXPERIMENTAL APPROACH: We applied atomistic molecular simulations to demonstrate the effect of the mutation on channel fast inactivation. Native PAGE was used to demonstrate channel dimerization, and electrophysiological measurements in HEK cells and Xenopus laevis oocytes were used to analyze the links between functional channel dimerization and impairment of fast inactivation by the hNav 1.7/A1632E mutation. KEY RESULTS: Enhanced persistent current through hNav 1.7/A1632E channels was caused by impaired binding of the inactivation particle, which inhibits proper functioning of the recently proposed allosteric fast inactivation mechanism. hNav 1.7 channels form dimers and the disease-associated persistent current through hNav 1.7/A1632E channels depends on their functional dimerization status: Expression of the synthetic peptide difopein, a 14-3-3 inhibitor known to functionally uncouple dimers, decreased hNav 1.7/A1632E channel-induced persistent currents. CONCLUSION AND IMPLICATIONS: Functional uncoupling of mutant hNav 1.7/A1632E channel dimers restored their defective allosteric fast inactivation mechanism. Our findings support the concept of sodium channel dimerization and reveal its potential relevance for human pain syndromes.
???displayArticle.pubmedLink??? 32663327
???displayArticle.pmcLink??? PMC7484505
???displayArticle.link??? Br J Pharmacol
???displayArticle.grants??? [+]
MA 7525/1-1 Deutsche Forschungsgemeinschaft, SCHM 536/12-1 Deutsche Forschungsgemeinschaft, LA 2740/3-1 Deutsche Forschungsgemeinschaft, MA 7525/1-2 Deutsche Forschungsgemeinschaft, HA 6095/1-2 Deutsche Forschungsgemeinschaft, Jülich-Aachen Research Alliance Center, School for Simulation and Data Sciences, IZKF TN1-8/IA 532008 Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the RWTH Aachen University
Species referenced: Xenopus laevis
Genes referenced: nav1 serpine2
GO keywords: voltage-gated sodium channel activity
???displayArticle.disOnts??? paroxysmal extreme pain disorder [+]
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Figure 1. Expression of hNav1.7/A1632E channels causes a prominent persistent current compared to that in a Nav1.7 channel stable cell line. (a) Representative traces of hNav1.7/WT (middle panel) and hNav1.7/A1632E (bottom panel) sodium currents. The persistent current was measured in the indicated range (between the red bars). The voltage protocol is shown in the top panel. Concentration of transfected cDNA: WT: Nav1.7 stable cell line; A1632E: 1.25 μg hNav1.7/A1632E + 0.25 μg GFP (HEK293T cells). (b) Relative persistent current of HEK cells expressing the indicated hNav1.7 channels. The measured persistent current at each voltage step was normalized to the peak inward current of the cell. Data were not statistically analysed. For means, SD, and N for each data point, please refer to Tables S3‐S4. (c) Maximal relative persistent current of each cell as used for statistical comparison. Data shown are individual values with means ± SD; hNav1.7/WT, N = 28; hNav1.7/A1632E, N = 17. Difference of means: 19.8%, 95% CI of difference of means: {17.5; 22.2}. *P < 0.05, significantly different as indicated | |
Figure 2. The A1632E mutation impedes binding of the IFM motif via steric repulsion and hydrophobic mismatch. (a) Overview of the cryo‐EM structure of the hNav1.7 α‐subunit from the intracellular side (PDB ID: 6J8I). Spheres, A1632 of DIV S5; orange sticks, the IFM motif. (b) Close‐up view of the IFM‐binding pocket, a hydrophobic cavity formed by residues from the DIII S4–S5 linker, DIII S5, DIII S6, DIV S5, and D‐IV S6 in both WT (upper) and A1632E Nav1.7 (lower). Thin sticks, pocket‐forming residues; thick sticks, IFM‐motif residues; spheres, residue 1632. Intensity of green colour indicates the hydrophilicity/hydrophobicity. (c) Solvent‐accessible surface of the IFM‐binding pocket from the same perspective as shown in (b). Water densities from unguided 200‐ns all‐atom MD simulations of WT and A1632E Nav1.7 (with the DIII ‐ DIV linker removed) were mapped onto the protein surface, with mapping strength indicated by the colour‐scale bar. The IFM motif was not present in these simulations, but its position in the IFM‐bound structure is indicated by orange lines | |
Figure 4. Nav1.5 and Nav1.7 migrate as non‐covalent homodimers under native PAGE. (a) SDS‐urea‐PAGE of hNav1.5 and hNav1.7 in the presence or absence of the reducing agent DTT. (b) and (c) hrCN‐PAGE of hNav1.5and hNav1.7 with various detergents used to turn dimers into monomers. The indicated channel proteins were extracted from Xenopus laevis oocytes with digitonin (a, b) or one of the indicated detergents (c), resolved by hrCN‐PAGE, and the GFP tags were visualized by Typhoon fluorescence scanning. Protein migration is shown under native conditions and after partial denaturation (1‐h incubation with 0.1% LiDS at 37°C). Numbers in the right margins in (b) and (c) indicate the sequence‐calculated masses (protomers to tetramers) of the partially denatured hTrpV1‐GFP channel. Numbers in the left margin in (c) correspond to the sequence‐calculated masses of the hNav1.7‐GFP protomer and homodimer. NG310, lauryl maltose neopentyl glycol; GDN, glyco‐diosgenin; DIG, digitonin | |
Figure 5. Loss‐of‐function mutations support dimerization of hNav1.7 channels in a Nav1.7stable cell line. (a) Representative traces of (top to bottom): hNav1.7/WT, hNav1.7/R896Q, hNav1.7/G375Afs, hNav1.7/WT + hNav1.7/R896Q, and hNav1.7/WT + hNav1.7/G375Afs. Concentration of transfected cDNA: WT: Nav1.7 stable cell line + 1.5 μg GFP or difopein; WT + R896Q ± difopein: 1.25 μg hNav1.7/R896Q + 0.25 μg GFP or difopein (Nav1.7 stable cell line); WT + G37Afs ± difopein: 1.25 μg hNav1.7/G375Afs + 0.25 μg GFP or difopein (Nav1.7 stable cell line); R896Q ± difopein: 1.25 μg hNav1.7/R896Q + 0.25 μg GFP or difopein (HEK293T cells); G375Afs ± difopein: 1.25 μg hNav1.7/G375Afs + 0.25 μg GFP or difopein (HEK293T cells). (b) Schematic representation of hNav1.7 showing the sites mutated in hNav1.7/G375Afs, hNav1.7/R896Q (red dots) and the possible dimerization site in the linker region between DI and DII (grey rectangle). (c) Peak current density in pA/pF was used for statistical analysis. Data shown are individual values with means ± SD; hNav1.7/R896Q: 10.8 ± 4.3, N = 16; hNav1.7/G375Afs: 12.4 ± 5.8, N = 13; hNav1.7/R896Q + difopein: 11.9 ± 3.4, N = 11; hNav1.7/G375Afs + difopein: 11.3 ± 5.6, N = 11.n.s. = not significant as indicated. (d) Peak current density in pA/pF was used for statistical analysis. Data shown are individual values with means ± SD; hNav1.7/WT + hNav1.7/R896Q, N = 16; hNav1.7/WT, N = 17; hNav1.7/WT + hNav1.7/R896Q + difopein, N = 16; hNav1.7/WT + difopein, N = 13. *P < 0.05. Difference of means: hNav1.7/WT − hNav1.7/WT + hNav1.7/R896Q: −231.4, 95% CI of difference of means: {−398.7; −64.1}; hNav1.7/WT + difopein − hNav1.7/WT + hNav1.7/R896Q + difopein: −60.1 {−239.4; 119.3}. *P < 0.05, significantly different, n.s. = not significant, as indicated. (e) Peak current density in pA/pF was used for statistical analysis: Data shown are individual values with means ± SD. hNav1.7/WT + hNav1.7/G375Afs, N = 13; hNav1.7/WT, N = 17; hNav1.7/WT + hNav1.7/G375Afs + difopein, N = 15; hNav1.7/WT + difopein, N = 14. Difference of means: hNav1.7/WT − hNav1.7/WT + hNav1.7/G375Afs: 2.4, 95% CI of difference of means: {−224.8; 229.6}; hNav1.7/WT + difopein − hNav1.7/WT + G375Afs + difopein: −62.6 {−291.7;166.6}. n.s. = not significant as indicated | |
Figure 6. Difopein reduces the size of the persistent current of hNav1.7/A1632E channels in HEK293T cells. (a) Relative persistent current of HEK cells expressing the hNav1.7/A1632E channel in absence or presence of difopein. The measured persistent current at each voltage step was normalized to the peak inward current of the cell. Concentration of transfected cDNA: A1632E: 1.25 μg hNav1.7/A1632E + 0.25 μg GFP or difopein (HEK293T cells). Data were not statistically analysed. For mean, SD and N of each data point, please refer to Tables S8‐S9. (b) Maximal relative persistent current of each cell is shown, as it was used for statistical comparison. Data shown are individual values with means ± SD; hNav1.7/A1632E, N = 17; hNav1.7/A1632E + difopein, N = 13. Difference of means: 13.2%, 95% CI of difference of means: {−17.2; −9.3}. *P < 0.05, significantly different as indicated | |
Figure 7. Expression of hNav1.7/WT and hNav1.7/A1632E channels in Xenopus laevis oocytes. (a) Representative current traces of X. laevis oocytes expressing hNav1.7/WT (left panel), hNav1.7/A1632E (right panel) or the combination of WT and A1632E (middle panel) elicited by stepwise depolarizations from −90 mV to +40 mV in 10 mV increments (from a holding potential of −100 mV). (b) Relative persistent current of oocytes expressing the indicated hNav1.7 channels. The measured persistent current at each voltage step was normalized to the maximum peak inward current of each oocyte. Oocytes were injected with the following amounts of cRNA(s) encoding the indicated Nav‐channel: hNav1.7/WT: 30 ng; hNav1.7/A1632E: 30 ng; hNav1.7/WT + hNav1.7/A1632E: 60 ng (30 ng each). (c) Maximal relative persistent current of each cell is shown as it was used for statistical comparison. Data shown are individual values with means ± SD; hNav1.7/WT, N = 14; hNav1.7/WT + hNav1.7/A1632E, N = 13; hNav1.7/A1632E, N = 19. Difference of means: hNav1.7/WT − hNav1.7/WT + hNav1.7/A1632E: −8.8%, 95% CI of difference of means {−13.9; −3.8}; hNav1.7/WT − hNav1.7/A1632E: −29.3 {−33.9; −24.7}; hNav1.7/WT + hNav1.7/A1632E − hNav1.7/A1632E: −20.5 {−25.2; −15.7}. *P < 0.05, significantly different as indicated. (d) Relative persistent current of oocytes expressing the hNav1.7/A1632E channel in absence or presence of difopein. The measured persistent current at each voltage step was normalized to the maximum peak inward current of each oocyte. Oocytes were injected with the following amounts of cRNA(s) encoding either the hNav1.7/A1632E channel or the hNav1.7/A1632E channel + difopein: hNav1.7/A1632E: 30 ng; hNav1.7/A1632E + difopein: hNav1.7/A1632E 30 ng + difopein 12 ng. (e) Maximal relative persistent current of each cell is shown as it was used for statistical comparison. Data shown are individual values with means ± SD; hNav1.7/A1632E, N = 19; hNav1.7/A1632E + difopein, N = 16. Analyzed using a Mann‐Whitney test. Difference between medians: −13.6%, 95% CI of difference of means: {−18.5; −11.4}. For exact numbers of mean, SD, and N of each data point (b, d), please refer to Tables S10‐S14 *P < 0.05, significantly different as indicated | |
FIGURE 1. Expression of hNav1.7/A1632E channels causes a prominent persistent current compared to that in a Nav1.7 channel stable cell line. (a) Representative traces of hNav1.7/WT (middle panel) and hNav1.7/A1632E (bottom panel) sodium currents. The persistent current was measured in the indicated range (between the red bars). The voltage protocol is shown in the top panel. Concentration of transfected cDNA: WT: Nav1.7 stable cell line; A1632E: 1.25 μg hNav1.7/A1632E + 0.25 μg GFP (HEK293T cells). (b) Relative persistent current of HEK cells expressing the indicated hNav1.7 channels. The measured persistent current at each voltage step was normalized to the peak inward current of the cell. Data were not statistically analysed. For means, SD, and N for each data point, please refer to Tables S3‐S4. (c) Maximal relative persistent current of each cell as used for statistical comparison. Data shown are individual values with means ± SD; hNav1.7/WT, N = 28; hNav1.7/A1632E, N = 17. Difference of means: 19.8%, 95% CI of difference of means: {17.5; 22.2}. * P < 0.05, significantly different as indicated | |
FIGURE 2. The A1632E mutation impedes binding of the IFM motif via steric repulsion and hydrophobic mismatch. (a) Overview of the cryo‐EM structure of the hNav1.7 α‐subunit from the intracellular side (PDB ID: 6J8I). Spheres, A1632 of DIV S5; orange sticks, the IFM motif. (b) Close‐up view of the IFM‐binding pocket, a hydrophobic cavity formed by residues from the DIII S4–S5 linker, DIII S5, DIII S6, DIV S5, and D‐IV S6 in both WT (upper) and A1632E Nav1.7 (lower). Thin sticks, pocket‐forming residues; thick sticks, IFM‐motif residues; spheres, residue 1632. Intensity of green colour indicates the hydrophilicity/hydrophobicity. (c) Solvent‐accessible surface of the IFM‐binding pocket from the same perspective as shown in (b). Water densities from unguided 200‐ns all‐atom MD simulations of WT and A1632E Nav1.7 (with the DIII ‐ DIV linker removed) were mapped onto the protein surface, with mapping strength indicated by the colour‐scale bar. The IFM motif was not present in these simulations, but its position in the IFM‐bound structure is indicated by orange lines | |
FIGURE 3. Co‐expression of hNav1.7/WT and hNav1.7/A1632E in a Nav1.7 stable cell line disproportionally affects persistent current. (a) Relative persistent current of HEK cells expressing the indicated hNav1.7 channels. The measured persistent current at each voltage step was normalized to the peak inward current of the cell. Concentration of transfected cDNA: WT: Nav1.7 stable cell line; WT + A1632E: 1.25 μg hNav1.7/A1632E + 0.25 μg GFP (Nav1.7 stable cell line); A1632E: 1.25 μg hNav1.7/A1632E + 0.25 μg GFP (HEK293T cells). Data were not statistically analysed. For mean, SD, and N of each data point, please refer to Tables S5‐ S7. (b) Maximal relative persistent of each cell as used for statistical analysis. Data shown are individual values with means ± SD; hNav1.7/WT, N = 28; hNav1.7/WT + hNav1.7/A1632E, N = 35; hNav1.7/A1632E, N = 17. Difference of means: hNav1.7/WT − hNav1.7/WT + hNav1.7/A1632E: −2.9%, 95% CI of difference of means: {−5.4; −0.4}; hNav1.7/WT − hNav1.7/A1632E: −19.8% {−22.9; −16.8}; hNav1.7/WT + hNav1.7/A1632E − hNav1.7/A1632E: −16.9% {−19.8; −14.0}. * P < 0.05, significantly different as indicated | |
FIGURE 4. Nav1.5 and Nav1.7 migrate as non‐covalent homodimers under native PAGE. (a) SDS‐urea‐PAGE of hNav1.5 and hNav1.7 in the presence or absence of the reducing agent DTT. (b) and (c) hrCN‐PAGE of hNav1.5and hNav1.7 with various detergents used to turn dimers into monomers. The indicated channel proteins were extracted from Xenopus laevis oocytes with digitonin (a, b) or one of the indicated detergents (c), resolved by hrCN‐PAGE, and the GFP tags were visualized by Typhoon fluorescence scanning. Protein migration is shown under native conditions and after partial denaturation (1‐h incubation with 0.1% LiDS at 37°C). Numbers in the right margins in (b) and (c) indicate the sequence‐calculated masses (protomers to tetramers) of the partially denatured hTrpV1‐GFP channel. Numbers in the left margin in (c) correspond to the sequence‐calculated masses of the hNav1.7‐GFP protomer and homodimer. NG310, lauryl maltose neopentyl glycol; GDN, glyco‐diosgenin; DIG, digitonin | |
FIGURE 5. Loss‐of‐function mutations support dimerization of hNav1.7 channels in a Nav1.7stable cell line. (a) Representative traces of (top to bottom): hNav1.7/WT, hNav1.7/R896Q, hNav1.7/G375Afs, hNav1.7/WT + hNav1.7/R896Q, and hNav1.7/WT + hNav1.7/G375Afs. Concentration of transfected cDNA: WT: Nav1.7 stable cell line + 1.5 μg GFP or difopein; WT + R896Q ± difopein: 1.25 μg hNav1.7/R896Q + 0.25 μg GFP or difopein (Nav1.7 stable cell line); WT + G37Afs ± difopein: 1.25 μg hNav1.7/G375Afs + 0.25 μg GFP or difopein (Nav1.7 stable cell line); R896Q ± difopein: 1.25 μg hNav1.7/R896Q + 0.25 μg GFP or difopein (HEK293T cells); G375Afs ± difopein: 1.25 μg hNav1.7/G375Afs + 0.25 μg GFP or difopein (HEK293T cells). (b) Schematic representation of hNav1.7 showing the sites mutated in hNav1.7/G375Afs, hNav1.7/R896Q (red dots) and the possible dimerization site in the linker region between DI and DII (grey rectangle). (c) Peak current density in pA/pF was used for statistical analysis. Data shown are individual values with means ± SD; hNav1.7/R896Q: 10.8 ± 4.3, N = 16; hNav1.7/G375Afs: 12.4 ± 5.8, N = 13; hNav1.7/R896Q + difopein: 11.9 ± 3.4, N = 11; hNav1.7/G375Afs + difopein: 11.3 ± 5.6, N = 11.n.s. = not significant as indicated. (d) Peak current density in pA/pF was used for statistical analysis. Data shown are individual values with means ± SD; hNav1.7/WT + hNav1.7/R896Q, N = 16; hNav1.7/WT, N = 17; hNav1.7/WT + hNav1.7/R896Q + difopein, N = 16; hNav1.7/WT + difopein, N = 13. * P < 0.05. Difference of means: hNav1.7/WT − hNav1.7/WT + hNav1.7/R896Q: −231.4, 95% CI of difference of means: {−398.7; −64.1}; hNav1.7/WT + difopein − hNav1.7/WT + hNav1.7/R896Q + difopein: −60.1 {−239.4; 119.3}. * P < 0.05, significantly different, n.s. = not significant, as indicated. (e) Peak current density in pA/pF was used for statistical analysis: Data shown are individual values with means ± SD. hNav1.7/WT + hNav1.7/G375Afs, N = 13; hNav1.7/WT, N = 17; hNav1.7/WT + hNav1.7/G375Afs + difopein, N = 15; hNav1.7/WT + difopein, N = 14. Difference of means: hNav1.7/WT − hNav1.7/WT + hNav1.7/G375Afs: 2.4, 95% CI of difference of means: {−224.8; 229.6}; hNav1.7/WT + difopein − hNav1.7/WT + G375Afs + difopein: −62.6 {−291.7;166.6}. n.s. = not significant as indicated | |
FIGURE 6. Difopein reduces the size of the persistent current of hNav1.7/A1632E channels in HEK293T cells. (a) Relative persistent current of HEK cells expressing the hNav1.7/A1632E channel in absence or presence of difopein. The measured persistent current at each voltage step was normalized to the peak inward current of the cell. Concentration of transfected cDNA: A1632E: 1.25 μg hNav1.7/A1632E + 0.25 μg GFP or difopein (HEK293T cells). Data were not statistically analysed. For mean, SD and N of each data point, please refer to Tables S8‐S9. (b) Maximal relative persistent current of each cell is shown, as it was used for statistical comparison. Data shown are individual values with means ± SD; hNav1.7/A1632E, N = 17; hNav1.7/A1632E + difopein, N = 13. Difference of means: 13.2%, 95% CI of difference of means: {−17.2; −9.3}. * P < 0.05, significantly different as indicated | |
FIGURE 7. Expression of hNav1.7/WT and hNav1.7/A1632E channels in Xenopus laevis oocytes. (a) Representative current traces of X. laevis oocytes expressing hNav1.7/WT (left panel), hNav1.7/A1632E (right panel) or the combination of WT and A1632E (middle panel) elicited by stepwise depolarizations from −90 mV to +40 mV in 10 mV increments (from a holding potential of −100 mV). (b) Relative persistent current of oocytes expressing the indicated hNav1.7 channels. The measured persistent current at each voltage step was normalized to the maximum peak inward current of each oocyte. Oocytes were injected with the following amounts of cRNA(s) encoding the indicated Nav‐channel: hNav1.7/WT: 30 ng; hNav1.7/A1632E: 30 ng; hNav1.7/WT + hNav1.7/A1632E: 60 ng (30 ng each). (c) Maximal relative persistent current of each cell is shown as it was used for statistical comparison. Data shown are individual values with means ± SD; hNav1.7/WT, N = 14; hNav1.7/WT + hNav1.7/A1632E, N = 13; hNav1.7/A1632E, N = 19. Difference of means: hNav1.7/WT − hNav1.7/WT + hNav1.7/A1632E: −8.8%, 95% CI of difference of means {−13.9; −3.8}; hNav1.7/WT − hNav1.7/A1632E: −29.3 {−33.9; −24.7}; hNav1.7/WT + hNav1.7/A1632E − hNav1.7/A1632E: −20.5 {−25.2; −15.7}. * P < 0.05, significantly different as indicated. (d) Relative persistent current of oocytes expressing the hNav1.7/A1632E channel in absence or presence of difopein. The measured persistent current at each voltage step was normalized to the maximum peak inward current of each oocyte. Oocytes were injected with the following amounts of cRNA(s) encoding either the hNav1.7/A1632E channel or the hNav1.7/A1632E channel + difopein: hNav1.7/A1632E: 30 ng; hNav1.7/A1632E + difopein: hNav1.7/A1632E 30 ng + difopein 12 ng. (e) Maximal relative persistent current of each cell is shown as it was used for statistical comparison. Data shown are individual values with means ± SD; hNav1.7/A1632E, N = 19; hNav1.7/A1632E + difopein, N = 16. Analyzed using a Mann‐Whitney test. Difference between medians: −13.6%, 95% CI of difference of means: {−18.5; −11.4}. For exact numbers of mean, SD, and N of each data point (b, d), please refer to Tables S10‐S14 * P < 0.05, significantly different as indicated |
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