Study Of The Mechanisms Of The Interaction Between JZTX-V And Kv4.3 Patassium Channel

Jingzhaotoxin-V(JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. It's molecular weight was determined to be 3605.73 Da. Whole-cell patch clamp recording showed that JZTX-V could partly inhibited the transient outward potassium channels currents but had no effects on voltage-gated calcium channels or outward delay-rectified potassium channels currents in adult rat dorsal root ganglion neurons. This was further confirmed by testing the effects of JZTX-V on Kv1.4, Kv4.1 and Kv4.2 transient outward potassium channels currents expressed in Xenopus oocytes. As a result, JZTX-V could completely inhibited Kv4.2 channel current but had no effect on Kv1.4 or Kv4.1 channels currents. Kv4.3 potassium channel is one of the transient outward potassium channels, it is responsible for the regulation of heart rate and is a promising target for anti-arrhythmic drug development.In this study, we performed alanine scanning of JZTX-V using the solid-phase chemical synthesis methods and obtained 18 alanine mutants. These peptides were separated by reverse phase high performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectromrtry). Then two-electrode voltage-clamp technique was used to evaluate the inhibitory effect of JZTX-V and its mutations on Kv4.3 potassium channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation, and it could also inhibit Kv4.3 channel currents in a time and concentration dependent manner with an IC50 value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 potassium channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV respectively. Substitution of Trp5 of JZTX-V with Ala had led to almost totally loss of inhibitory activity, it increased the IC50 value by 38-fold. But the two mutants E17A-JZTX-V and K27A-JZTX-V decreased their IC50 values a little. Other alanine mutants and 3 splice variants caused no distinct change to their IC50 values. So when the molecular design of JZTX-V is performed in the future, the amino acids Yl and 129 can be cut to save production cost. Present findings indicated that Trp5 is a key amino acid residue relative to the selective binding of JZTX-V to Kv4.3 potassium channel.To further characterize the molecular determinants of interaction between JZTX-V and Kv4.3 potassium channel, alanine-scaning mutagenesis on amino acids in S1-S2, S3b-S4 regions of Kv4.3 potassium channel was performed and 12 alanine mutants had been constructed. The work will lay a good foundation for the study of the bingding sites of the interaction between JZTX-V and Kv4.3 potassium channel by using patch clamp technique.