Xiao Yucheng, Pan Yanling, Xiao Jingyu, Cummins Theodore R
Biology Department, School of Science, Indiana University Indianapolis, Indianapolis, IN, United States.
School of Engineering, Purdue University, West Lafayette, IN, United States.
Front Mol Neurosci. 2024 Oct 2;17:1433981. doi: 10.3389/fnmol.2024.1433981. eCollection 2024.
Resurgent current ( ) 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 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 mediators; however, their receptor sites in VGSCs remain unknown, which hinders the development of novel agents to effectively target .
Navβ4 and FHF4A can mediate 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 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 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 .
电压门控钠通道（VGSCs）产生的复苏电流（ ）在维持许多神经元的高频放电中起着至关重要的作用，并导致诸如癫痫和疼痛性疾病等疾病的病理生理过程。靶向 可能是治疗这些疾病的一种极具前景的策略。Navβ4和A型成纤维细胞生长因子同源因子（FHFs）已被确定为两类重要的 介质；然而，它们在VGSCs中的受体位点仍然未知，这阻碍了有效靶向 的新型药物的开发。
Navβ4和FHF4A可分别通过位于其C末端和N末端的氨基酸片段介导 的产生。我们主要采用定点诱变、嵌合体构建和全细胞膜片钳记录来探索Navβ4肽和FHF4A在Nav1.7和Nav1.8中的受体位点。
我们发现Navβ4肽的受体涉及Nav1.7的DI-S6、DII-S6和DIV-S6中的四个残基，即N395、N945、F1737和Y1744。我们发现产生 的A型FHFs取决于FHF4 N末端结构域起始处而非远端的片段。我们发现A型FHFs的受体位点也位于VGSC内孔区域。我们进一步表明，Nav1.8中DIIS6处的天冬酰胺N891是VGSCs中A型FHFs产生 的主要决定因素。
冷冻电镜结构显示关键残基的侧链伸入VGSC通道孔中。我们的研究结果提供了额外的证据，证明Navβ4肽和A型FHFs作为开放通道孔阻滞剂发挥作用，并突出了通道内孔区域作为开发靶向 的新型药物的热点。
