A Novel Spider Toxin Inhibits Fast Inactivation of the Nav1.9 Channel by Binding to Domain III and Domain IV Voltage Sensors.

Shuijiao Peng,Zhen Xiao,Songping Liang,Minzhi Chen,Xin Xiao,Sen Luo,Xi Zhou,Zhonghua Liu

doi:10.3389/fphar.2021.778534

Abstract

Venomous animals have evolved to produce peptide toxins that modulate the activity of voltage-gated sodium (Nav) channels. These specific modulators are powerful probes for investigating the structural and functional features of Nav channels. Here, we report the isolation and characterization of δ-theraphotoxin-Gr4b (Gr4b), a novel peptide toxin from the venom of the spider Grammostola rosea. Gr4b contains 37-amino acid residues with six cysteines forming three disulfide bonds. Patch-clamp analysis confirmed that Gr4b markedly slows the fast inactivation of Nav1.9 and inhibits the currents of Nav1.4 and Nav1.7, but does not affect Nav1.8. It was also found that Gr4b significantly shifts the steady-state activation and inactivation curves of Nav1.9 to the depolarization direction and increases the window current, which is consistent with the change in the ramp current. Furthermore, analysis of Nav1.9/Nav1.8 chimeric channels revealed that Gr4b preferentially binds to the voltage-sensor of domain III (DIII VSD) and has additional interactions with the DIV VSD. The site-directed mutagenesis analysis indicated that N1139 and L1143 in DIII S3-S4 linker participate in toxin binding. In sum, this study reports a novel spider peptide toxin that may slow the fast inactivation of Nav1.9 by binding to the new neurotoxin receptor site-DIII VSD. Taken together, these findings provide insight into the functional role of the Nav channel DIII VSD in fast inactivation and activation.

Highlights

Voltage-gated sodium (Nav) channels are important transmembrane proteins that play a vital role in the generation and propagation of action potentials in excitable cells, such as central and peripheral neurons, cardiac and skeletal muscle myocytes, and neuroendocrine cells (Goldin 2001; Catterall 2012; Mantegazza and Catterall 2012)
This fraction was further purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and approximately 8 μg of purified peptide toxin was obtained from 1 mg crude spider venom (Figure 1C)
A mutation of Nav1.9 (S1145A) enhanced sensitivity to Gr4b (Figures 6I,J and Table 4). These results suggest that Gr4b preferentially binds to the DIII voltage-sensing domain (VSD) and has additional interactions with the DIV VSD, while two residues (N1139 and L1143) in the DIII S3-S4 linker may affect the interaction of Nav1.9 with Gr4b

Summary

Introduction

Voltage-gated sodium (Nav) channels are important transmembrane proteins that play a vital role in the generation and propagation of action potentials in excitable cells, such as central and peripheral neurons, cardiac and skeletal muscle myocytes, and neuroendocrine cells (Goldin 2001; Catterall 2012; Mantegazza and Catterall 2012). The subtypes can be divided into two categories according to their sensitivity to TTX: TTX-sensitive (Nav1.1–1.4, Nav1.6, and Nav1.7) or TTXresistant (Nav1.5, Nav1.8, and Nav1.9) These subtypes have different tissue-specific localization and functions. Nav channel consist of an approximately 260 kDa pore-forming α-subunit and one or more associated β-subunits of 30–40 kDa (Catterall 2012; Bennett et al, 2019). The gating charges move outward upon membrane depolarization and initiate the voltage-dependent activation and inactivation of Nav channels (Jiang et al, 2020). These characteristics endow the various conformation transformations of Nav channel via an electromechanical coupling mechanism to open and close the pore. The three major states are defined as resting, activation, and inactivation

Methods

Results

Conclusion