Activity And Applied Fundamental Research Of Two Spider Toxins

Hainan Tarantulas(Haplopelma hainanum [Selenocosmia hainana]) are mainly distributed in southern China’s forest area and prey on birds and small mammals, Hainantoxin-I(HNTX-I) is isolated from the venom of Hainan Tarantulas’ neurotoxin. The research first get lyophilized powder by electrical stimulation of the spider venom chelicerae enrichment, then getting purified single component HNTX-I by using cation exchange and reversed-phase high-performance liquid chromatography. We next identified the impact of the toxin molecule conductance calcium-activated potassium channel gating properties by using the patch-clamp technique(intermediate-conductance calcium-activated potassium channel, IK) on. The research shows that the HNTX-I had amino acid residues, 6 cysteines(Cys) form three disulfide bonds, a sequence comprising two adjacent cysteine, disulfide paring mode for I-IV, II- V, III-VI, relative molecular mass of 3607.1 Da, Hainan tarantula venom components highest abundance. HNTX-I could activate IK channels open, and HNTX-I in rat dorsal root for voltage-gated sodium channels on the ganglion. In addition, saturation dose HNTX-I could only produce a weak activating effect when voltage-gated calcium channels on HEK293 T cells heterologous expression of slowly activating delayed rectifier potassium channel no The same family for another high homology small conductance(small-conductance calcium-activated potassium channel, SK) calcium-activated potassium channels. And HNTX-I was almost no effect obvious effect when a member-large conductance(big-conductance calcium-activated potassium channel, BK) calcium-activated potassium channels. At the same time, there was no obvious fatal by mouse diaphragm- phrenic nerve specimens and toxicology studies, because the HNTX-I could not block the conduction of nerve signals. IK channels played an important role in the relaxation process of vascular endothelial cells, its impaired function could cause hypertension and other cardiovascular diseases. Experiments proved HNTX-I can activate specific IK channel, not produce significant side effects.Jingzhao Tarantulas(Chilobrachys guangxiensis [Chilobrachys jingzhao]) were found in Hainan province.Jingzhao Tarantulas toxin-V(JZTX-V) is a broad spectrum neurotoxin that can suppress most of the voltage-gated sodium channels and potassium channels. It has been proved that the toxin can interact with phospholipids. We obtained synthesis JZTX-V toxin by synthetizing peptide solid-phase and oxidative refolding the synthesis of linear products. Next we used rhodamine B(Rhodamine B) a fluorescent to mark it. The results revealed that the JZTX-V with fluorescently labeled(R-JZTX-V) of electrophysiological activity and secondary structure elements of basic and natural toxins had the same toxin molecule through the same patch clamp experiments and circular dichroism(circular dichroism, CD) detection. It proved that the fluorescent labeling did not cause a significant impact on the function and structure of the toxin molecule. The experiment revealed that the JZTX-V could interact with vivo cell membrane. We used total internal reflection fluorescence microscopy(total internal reflection fluorescence microscopy, TIRFM) to track single JZTX-V fluorescence-labeled with toxin molecules in a living cell toxin by the Loss The track movement. By analyzing the trajectories, we found a single toxin molecules in the cell surface had three main movement patterns: random rapid activity, defined region of low-speed movement and stationary with respect to the film surface. Based on these experiments, we established an intuitive interaction method of researching toxin molecules and cell membranes, which was the foundation of the research of preliminary elaborated toxin molecules in the cell surface specific movement for the interaction of the toxin molecules and membrane ion channels.In summary, this study found the interaction of HNTX-I and IK channel, and we used peptide IK channel peptide activator for the first time. We found the application value of single JZTX-V toxin molecule in the study of cell surface interactions in vivo model. It was the first time in living cell systems using single-molecule fluorescence technique study toxin molecules.