Molecular determinants of resurgent sodium currents mediated by Navβ4 peptide and A-type FHFs

Resurgent current (I_NaR ) generated by voltage-gated sodium channels (VGSCs) plays an essential role in maintaining high-frequency firing of many neurons and contributes to disease pathophysiology such as epilepsy and painful disorders. Targeting I_NaR may present a highly promising strategy in the treatment of these diseases. Navβ4 and A-type fibroblast growth factor homologous factors (FHFs) have been identified as two classes of important I_NaR mediators; however, their receptor sites in VGSCs remain unknown, which hinders the development of novel agents to effectively target I_NaR .

Navβ4 and FHF4A can mediate I_NaR generation through the amino acid segment located in their C-terminus and N-terminus, respectively. We mainly employed site-directed mutagenesis, chimera construction and whole-cell patch-clamp recording to explore the receptor sites of Navβ4 peptide and FHF4A in Nav1.7 and Nav1.8.

We show that the receptor of Navβ4-peptide involves four residues, N395, N945, F1737 and Y1744, in Nav1.7 DI-S6, DII-S6, and DIV-S6. We show that A-type FHFs generating I_NaR depends on the segment located at the very beginning, not at the distal end, of the FHF4 N-terminus domain. We show that the receptor site of A-type FHFs also resides in VGSC inner pore region. We further show that an asparagine at DIIS6, N891 in Nav1.8, is a major determinant of I_NaR generated by A-type FHFs in VGSCs.

Cryo-EM structures reveal that the side chains of the critical residues project into the VGSC channel pore. Our findings provide additional evidence that Navβ4 peptide and A-type FHFs function as open-channel pore blockers and highlight channel inner pore region as a hotspot for development of novel agents targeting I_NaR .