Uncoupling sodium channel dimers restores the phenotype of a pain-linked Nav 1.7 channel mutation.

Annika H Rühlmann,Petra Hautvast,Jan‐Philipp Machtens,Nikolay Bebrivenski,Jannis Körner,Angelika Lampert,Ralf Hausmann,Jannis Meents,Christian Neuhof,Silvia Detro‐Dassen,Carène A Benasolo,Günther Schmalzing

doi:10.1111/bph.15196

Abstract

Background and PurposeThe voltage‐gated sodium channel Nav1.7 is essential for adequate perception of painful stimuli. Mutations in the encoding gene, SCN9A, cause various pain syndromes in humans. The hNav1.7/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 hNav1.7/A1632E mutation, and how dimerization modifies function of the pain‐linked mutation.Experimental ApproachWe 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 hNav1.7/A1632E mutation.Key ResultsEnhanced persistent current through hNav1.7/A1632E channels was caused by impaired binding of the inactivation particle, which inhibits proper functioning of the recently proposed allosteric fast inactivation mechanism. hNav1.7 channels form dimers and the disease‐associated persistent current through hNav1.7/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 hNav1.7/A1632E channel‐induced persistent currents.Conclusion and ImplicationsFunctional uncoupling of mutant hNav1.7/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.

Highlights

Conference proceedings: Partial results of this work were presented at the 63th Annual Meeting of the Biophysical Society in Baltimore: Biochemical and Functional Evidence for Homodimerization of Voltage-Gated Sodium Channels (Navs)
We study the previously reported gain-of-function mutation hNav1.7/A1632E mutation, which induces a combination of inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD) in the heterozygous carrier and is characterized by an incomplete fast inactivation leading to a prominent persistent current (Estacion et al, 2008; Eberhardt et al, 2014)
We demonstrate that the increase of size and addition of negative charge at position 1632 impedes binding of the IFM motif to a hydrophobic binding pocket, preventing fast inactivation, supporting the new allosteric inactivation mechanism suggested by the recent cryo-EM structures (Pan et al, 2018; Yan et al, 2017; Shen et al, 2017, 2019; Clairfeuille et al, 2019)

Summary

Results

Using patch-clamp of HEK cells overexpressing the pain-linked mutation hNav1.7/A1632E, we confirmed its reported prominent persistent current (Fig. 1A-C), suggesting an impaired fast inactivation as major disease-linked gating change (Dib-Hajj et al, 2013). Taking into account the recently proposed allosteric inactivation mechanism (Yan et al, 2017), this constellation predicts impaired fast inactivation, resulting in persistent current for the hNav1.7/A1632E mutation: the larger glutamic acid and the increased hydration of the binding pocket impairs the binding of the IFM motif. Discussion inactivation mechanism of the channel, we demonstrate that Nav1.7 forms functional dimers and that uncoupling dimers reduces disease-relevant persistent current of the hNav1.7/A1632E mutation. Substitution of A1632 with glutamate adds additional size, which may sterically impair IFM binding, but it increases hydration of the pocket This seems to severely interfere with IFM binding and likely causes the prominent persistent current of hNav1.7/A1632E. Co-expression of the mutation and dimerization with the WT could retain the functioning channels within the cell This would explain why sodium current increases in the presence of difopein the transfected cDNA-concentration remains stable. Even diseases caused by different Nav-subtypes might need to be re-evaluated, considering the possibility of channel dimerization

Conclusion

Materials and Methods

A Boltzmann equation was used for fitting