A Study Of The Interaction Sites Between The Voltage-gated Sodium Channels And The Toxins From The Chinese Bird Spider

The spiders(Haplopelma schmidti, Chilobrachys guangxiensis, and Haplopelma hainanum) distributing in the south of China are the relatively big, venomous, and rare bird spiders. In our previous studies, huwentoxin-I (HWTX-I), huwentoxin-IV (HWTX-IV), hainantoxin-IV (HNTX-IV), and jingzhaotoxin-III (JZTX-III) have been demonstrated that they have different selectivity and inhibiting ability on voltage-gated sodium channels (VGSCs) using whole-cell patch clamp assays. However, the interaction mechanism between spider toxins and VGSCs is still unclear. Here, we tried to solve it partially using yeast two hybird assay. The coding sequences of mature peptides of toxins were inserted to the yeast two hybird vector pGBKT7, and the coding sequences of outer-membrane segments of VGSCs were ligated to another vector pGADT7. Some potential interaction sites between these toxins and VGSCs (hNav1.5 and hNav1.7) were detected using yeast two hybird assays. The potential interaction site between HWTX-IV and hNav1.7 is only at the extracellular S3-S4 linker of domain III (DIII-S3-S4). The potential interaction sites between HWTX-I and hNav1.7 are at DI-S3-S4, DIII-S1-S2, DIII-S5-S6, and DIV-S5-S6. The potential interaction sites between HNTX-IV and hNav1.7 are at DI-S3-S4, DII-S1-S2, DIII-S1-S2, and DIII-S5-S6. The potential interaction sites between JZTX-III and hNav1.7 are at DI-S3-S4, DI-S5-S6, DII-S1-S2, DIII-S5-S6, DIV-S1-S2, and DIV-S5-S6. The potential interaction sites between JZTX-III and hNav1.5 are at DⅠ-S1-S2, DⅡ-S3-S4, and DⅢ-S3-S4.Previous studies in our laboratory have demonstrated that IC50 value of HWTX-IV is 26 nM for wild type (WT) hNav1.7. The IC50 value of JZTX-III is approximately 300 nM for WT hNav1.5 (unpublished datas). The inhibiting abilities of HWTX-I, HNTX-IV, and JZTX-III on the WT hNav1.7 were detected using electrophsiolgy assay. It was demonstrated that the IC50 value of HWTX-I and HNTX-IV were 640 nM and 22 nM for WT hNav1.7 channel, respectively. The hNav1.7 currents almost could not be inhibited after exposure to 1μM JZTX-III. The order of inhibition ability of toxins to hNav1.7 channel is HNTX-Ⅳ≈HWTX-Ⅳ>HWTX-Ⅰ>>JZTX-Ⅲ.According to the results of the electrophysiology and yeast two hybrid assays, key points can be concluded as follows:1) There is almost no interaction site between the outer-membrane S5-S6 segments of hNav1.7 and HNTX-IV or HWTX-Ⅳ, respectively.2) It is indicated that the inhibition ability of HNTX-Ⅳare identical to that of HWTX-IV for the hNav1.7 channel using electrophsiolgy assay, but the yeast two hybird results demonstrate that there are significant differences between them. The potential interaction site between HWTX-IV and hNav1.7 is only one, but HWTX-IV has four potential interaction sites to hNav1.7.3) The differences of segments of DII-S3-S4 and DIII-S3-S4 between hNav1.5 and hNav1.7 might result in the different sensitivity to JZTX-III. Outer-membrane S1-S2 and S3-S4 segments of VGSCs are almost no more than ten amino acids. We predicted that the core motif of these short out-membrane segments was about six amino acids by the analysis of related papers. The random-6-peptide library were constructed by inserting the coding sequences of the random-6-peptide into the expression vector-pGADT7, so the library could be used for the yeast two hybrid experiments. There were two different ways to build the random-6-peptide library. The first one was the regular method using the restriction endonuclease and T4 DNA ligase as some paper mentioned. But the second way was a novel method to build the library using the site-directed insertion mutagenesis. Two random-6-peptide libraries were constructed by two different ways mentioned above. Independent clones of each random-6-peptide library was over 100,000. Twenty clones were sequenced randomly from the two libraries mentioned above, respectively. The results of sequencing demonstrated that seventy percent sequences could be used for library screening, and there was no repetitive sequence, respectively. It was indicated that the libraries were of high quality.An efficiency method to express the small molecular bioactive peptides was developed. The coding sequences of HWTX-I, HNTX-Ⅳ, and JZTX-V were amplified by PCR. The cloned fragment was inserted into the expression vector pET-40b, and then the constructor was transformed into E.coli strain BL21(DE3), respectively. The expression of the soluble fusion protein was auto-induced into the periplasm of E. coli by lactose in the absence of IPTG. It was demonstrated that the three kinds of fusion proteins were expressed by the analysis of SDS-PAGE assay. After a partial purification using a Ni-NTA column and dialysis, the expressed fusion protein was digested using the enterokinase to release heteroexpressed HWTX-Ⅰ(rHWTX-Ⅰ) and further purified using RP-HPLC. The molecular weight of the rHWTX-I was 3750.69 using TOF/TOF MS spectrometer assays, which is identical to that of the natural toxin isolated from the spider venom. It was indicated there was three disulfide bonds formation of the rHWTX-I. The physiological properties of the rHWTX-I was further analyzed using the whole-cell patch clamp assay. The rHWTX-I was able to block the currents generated by human Nav1.7 at an IC50 of 640 nM, similar to that of the natural huwentoxin-I, which is 630 nM.