| Chinese tarantula Chilobrachys jingzhao, one of the largest and most venomous spiders of China, is an attractive source of venom material because of its large size, the ability to live in captivity and the availability of a large amount of venom. In order to understand the envenoming and composition of the venom from C.jingzaho, we have performed an overview analysis of this venom with high performance liquid chromatogrophy and whole cell patch clamp methods. It has been found that peptides are the predominant components (69% in crude venom,w/w).We isolated the venom of the Chinese tarantula Chilobrachys jingzhao by positive ion-exchange chromatography and reverse-phase high performance liquid chromatography. In total, individual masses of a host of peptides range 2-8 kDa. Of dozens of peptides, most peptides have six or eight Cycs,and have three or four disulfides and about one third are found to have C-terminal amidation. JZTX-Ⅺwith high abundance was selected for further structure and function analysis.JZTX-Ⅺ, a novel neurotoxic peptide was isolated further fro m the venom from C.jingzaho through positive ion-exchange chrom atography and reverse-phase high performance liquid chromatography. It is composed of 34 Amino acids residues with three disulfide bo nds, Its aimno acid sequence of JZTX-Ⅺis:ECRKMFGGCSVDS DCCAHLGCKPTLKYCAWDGTF, The molecular mass of naturally occurring toxin was determined to be 3728.6 Da by MALDI-TOF mass spectrometry. JZTX-XI has no C-terminal amidation. It has six Cycs and three disulfide bonds. JZTX-Ⅺis one of main compo-ents, about 2.3%(w/w). The earlier studies indicated that JZTX-Ⅺcould significantly inhibit the currents from the Nav channel in the rat cardiac myocytes and the rKv2.1 channel expressed in Xenpus Laevis oocytes, but it had no effect on the Nav and Cav channels currents in adult rat dorsal root ganglion neurons. JZTX-Ⅺselectiv ely inhibits rKv2.1 and hNav1.5, and is concentration-dependent and voltage-dependent. It couldn’t inhibit the currents from Kv1、Kv3、Kv4 expressed. JZTX-Ⅺcould inhibit weakly rNavl.4 channel curr ents, but compeletely inhibit those of hNavl.5 channels expressed in HEK293T cells with an IC50 value of 0.44μM. The application of JZTX-Ⅺaffected the activation and inactivation characteristics of hNav1.5 channel, and caused a depolarizing shift of the current-voltage relationship curve and a hyperpolarizing shift of the steady-state inactivation curve by approximately 8 mV and 10 mV respecti vely. Moreover, JZTX-Ⅺcould bias the activities of hNav1.5 chan-nels towards closed state because the time constant for decay (chan nel deactivation) of tail currents became faster in the presence of toxin. We speculate that JZTX-Ⅺis a gating modifier of sodium channels and traps the voltage sensor at closed state. Data showed that certain amino acid residues in the S3-S4 extracellular linker of a subunit impact intensivity between Nav1.5 and JZTX-Ⅺ. The key residuses that influence intensivity are Ser802, Asn8O3, and Ser805.Despite significant difference between JZTX-Ⅲand JZTX-Ⅺ, both spatial structure are typical of the ICK motif structure. Both toxins has a hydrophobic patch and charged residues surrounding t he patch. Electrophysiological experiments indicate that this structure is that JZTX-Ⅺand JZTX-Ⅲplay a structural basis for biological activity. The above study showed the peptide activity and some structural features systematicly by means of high performance liquid chromatography and electrophysiological methods. This study will not only enable us to further understand its mechanism of toxicity on the molecular level, but also lay a solid foundation for screeni ng and developing of pharmacological significant peptide toxin in future. Our studies provides the most direct data for JZTX-Ⅺdeve-loping into ion channel reagent on the rKv2.1 and hNav1.5. |
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