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Functional Studies on Three Tarantula Toxins Inhibiting Kv4 and Sodium Channels Expressing on Xenopus Oocytes

Author: KuangFang
Tutor: XiaoYuCheng；LiangSongPing
School: Hunan Normal University
Course: Biochemistry and Molecular Biology
Keywords: spider venoms potassium channels voltage-clamp technique sodium channels Xenopuslaevis oocytes
CLC: R96
Type: Master's thesis
Year: 2008
Downloads: 11
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Abstract

The Chinese tarantula Ornithoctonus hainana belonging to the family of Theraphosidae mainly distributes in the hilly area of Hainan Province and habitually lives in holes underground. Our previous work has demonstrated that its crude venom contains a number of biologically active components inhibiting neuronal sodium channels. However, the effects of the spider venom on potassium channels remain unknown so far. In this study, the crude venom of Ornithoctonus hainana was fractionated to five fractions （Fraction I-V） on a C18 column using reverse-phase high performance liquid chromatography, and then we further characterized the biological functions of these five freeze-dries fractions on nine outward rectifier-delayed （Kv1.1, 1.2,1.3, 2.1 and 3.1） or A type （Kv1.4, 4.1,4.2 and 4.3） potassium channels expressed on Xenopus laevis oocytes. Using two-electrode voltage clamp recording technique, five fractions at the concentration of 10μg/mL almost showed no significant effect on all outward delay-rectified potassium channels, but exhibited distinct affinities to four A-type potassium channels. The result implied that the crude venom of the tarantula might have no component affecting delay-rectified potassium channels or at least delay-rectified potassium channel inhibitors should be very poor in the spider venom whereas the active components should be richer. On the basis of this finding, we further examine the effects two purified toxins Hainantoxin-III （HNTX-III） and Hainantoxin-IV （HNTX-IV）, which appeared in Fraction I, on four different A-type potassium channels. The two toxins are found to contain 33 and 35 amino acid residues, respectively. Both of them have three intracellular disulfide bonds （C1-C4, C2-C5, C3-C6） and fold into a typical inhibitor cystine knot that frequently emerges in spider toxins and conotoxins. Electrophysiological recordings canied out on Xenopus laevis oocytes showed that HNTX-III and HNTX-IV exhibited similar selectivity for A-type potassium channel subtypes. The two toxins showed no significant effect on Kv4.1 or Kv4.2, but could block Kv4.3 channel in a time- and concentration-dependent manner. Their IC₅₀ values on Kv4.3 were 2.19 and 0.47μM, respectively. The treatment of HNTX-III and HNTX-IV also caused a depolarizing shift of the current-voltage relationship of Kv4.3. The selectivity and the mechanism of the two toxins for different potassium channel subtypes are very similar to that of Phrixotoxin 1 and Phrixotoxin 2, a well-known potassium channel gating modifiers that bind preferentially to the closed state of the channel.Our previous work also demonstrated that HNTX-III and HNTX-IV could inhibit TTX-sensitive sodium channels expressing in adult rat dorsal root ganglion neurons. In order to clarify the selectivity of the two toxins for different sodium channel subtypes, here we investigated their actions on rat Nav1.2 （brain） and Nav1.4 （skeletal）, Nav1.2 and Nav1.4 are believed not to express in mammalian peripheral neurons. Our preliminary result indicated that each toxin inhibited both sodium channel isoforms, but the affinity of HNTX-III and HNTX-IV for Nav1.2 was 10-fold higher than that for Nav1.4. The IC₅₀ values for HNTX-III and HNTX-IV on Nav1.2 were 0.6 and 2.8μM, respectively, while the IC50 values on Nav1.4 were 10.6 and 19.7μM, respectively. Two toxins at 5μM depressed the amplitude of inward currents through Nav1.3 by around 50% and 30%, respectively.Chilobrachys jingzhao is a newly identified tarantula species in the hilly area of Hainan Province of China. From its venom we have isolated and characterized a novel neurotoxic peptide named Jingzhaotoxin-IX （JZTX-IX）. Whole-cell configuration indicated that JZTX-IX inhibited both TTX-resistant and TTX-sensitive sodium channels in adult rat dorsal root ganglion neurons with IC₅₀ values of 0.53 and 0.36μM, respectively. The toxin could act as a gating modifier of sodium channels by shifting the voltage dependence of activation to more positive voltages. Moreover, JZTX-IX was found to selectively inhibit Kv2.1 expressed in Xenpus Laevis oocytes (IC₅₀=1.6μM), too, but had no effect on other outward delay-rectified potassium channels （Kv1.1, 1.2, 1.3）. Many research groups have proved that two TTX-sensitive （Nav1.3, 1.7） and TTX-resistant sodium channel subtypes are involved in pain sensation and their inhibitors can effectively stop the transduction of pain in inflammatory animal models. Thus, JZTX-IX might be a potential candidate for the development of analgesic pharmaceutics and be an interesting tool for exploring the mechanisms across ion channels.

Functional Studies on Three Tarantula Toxins Inhibiting Kv4 and Sodium Channels Expressing on Xenopus Oocytes

Abstract

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