Molecular Mechanism For Jingzhaotoxin-34 Inhibiting Soduim Channel Subtype Nav1.7

Spider peptide toxins are regarded as important molecular probes for investigating the structure-function relationship of voltage-gated sodium channels (VGSCs) due to molecular diversity, high affinity and isoform-spicific selectivity. However, the molecular mechanism for the interaction between spider toxins and VGSCs is often poorly understood. Jingzhaotoxin-34 (JZTX-34) is an inhibitors cysteine knot motif peptide isolated from Chinese tarantula Chilobrachys jingzhao venom that could selectively block tetraodotoxin-sensitive (TTX-S) voltage-sensitive sodium channels from dorsal root ganglia (DRG) neurons, but had no obvious effect on tetraodotoxin-resistant (TTX-R) sodium channel.In this study, JZTX-34 was chemically synthesized by solid-phase chemistry method with Fmoc-protected amino acids. We found that the refold JZTX-34 and native peptides had the same relative molecular mass and bioactivity on DRG cells determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined and clamp patch. The refold JZTX-34 selectively inhibited Nav1.3 and Nav1.7 with IC50 value of 7.95μM and 610 nM respectively, while having no effect on Nav1.5 and Nav1.8. JZTX-34 caused hyperpolarization shift about 9mV in steady-state inactivation of Nav1.7 without affecting the voltage-current (Ⅰ-Ⅴ) curve or activation, the currents of chimeric Nav 1.5 (Nav1.7-DⅡ-S3-S4) and chimeric Nav1.7 (Navl.5-DⅣ-S3-S4) were obviously blocked by JZTX-34 with an IC50 value of 706.3 nM and 1.05μM respectively, indicating that the binding site of JZTX-34 on Nav1.7 is neurotoxin receptor site 4. Mutant D816R in Nav 1.7 decreased the sensitivity of JZTX-34 to Nav 1.7 by 32 fold. Meanwhile, the R800D reverse mutation in Nav 1.5 markedly increased the toxin affinity for Navl.5. Compared with tarantula toxin ProTx-Ⅱand Domain Ⅳ to influence sodium channel fast inactivation and scorpion β-toxin targeting Domain Ⅱ to affect sodium channel activation, Our data showes that JZTX-34 maybe adopt a different molecular mechanism for sodium channel inhibition by animal toxins involving binding to neurotoxin receptor site 4.