Study Of The Mechanisms Of The Interaction Between JZTX-V And Kv4.2 Potassium Channel

Jingzhaotoxin-V (JZTX-V) is a peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Its molecular weight was determined to be 3605.73 Da. It was confirmed that JZTX-V could completely inhibited transient outward potassium channels currents, Kv4.2 channel current but had no effect on Kv1.4 or Kv4.1 channels currents. Kv4.2 channel is transient outward potassium channel, An increasing number of observations suggest indicate that Kv4.2 is expressed in both heart and brain cells and played an important role for voltage-gated potassium (Kv) channels in epilepsy and arrhythmia. Kv4 channel activity is modulated by several types of ancillary subunits, the most prominent of which are the Kv4 channel-interacting proteins (KChIPs), The products of three KChIP genes are predominant in the central nervous system (KChIPs1,3, and 4), whereas KChIP2 is more abundant in the heart, in where it contributes to the cardiac I to. To understand Kv4.2-based transient out-ward currents in native tissues, we tested the affinity of JZTX-V for Kv4.2 coexpressed with KChIP2.In this study, we showed that JZTX-V could inhibit Kv4.2+ KChIP2 with the IC50 value of 13nM, making it a useful probe for understanding Kv4.2 structure and drug binding. But 10nM JZTX-V did not affected the activation and inactivation characteristics of Kv4.2 potassium channel. we performed alanine-scanning of Kv4.2 S3b region and S3-S4 linker Mutations I275A and L277A on S3b could increase toxin affinity by～10 and ～20 fold respectively. While the mutation L277V have lower affinity for JZTX-V than did mutation L277A CO.2 fold) four mutations (V285A, F289A and V290A) on S3-S4 linker, formed a hydrophobic patch, could greatly decrease toxin affinity by ～7,～11 and～12 fold respectively. And the mutations V290L increased affinity by 2-fold. The result indicated that hydrophobic residues especially the four hydrophobic residues mutations (V285, F289 and V290), formed a hydrophobic patch, play a key role in the binding of JZTX-V to Kv4.2Further evidence can be provided by mutating JZTX-V, we performed alanine scanning of JZTX-V using the solid-phase chemical synthesis methods and obtained 15 alanine mutants. CD spectra of JZTX-V mutants indicate that all synthesis toxins did not significantly change the secondary structure of JZTX-V. Alanine-scanning analysis indicated that W5A, M6A, W7A, R20A, K23A increased affinity by 13,23,13,9,14-fold respectively, two alkaline residues (R20 and K22) and an exposed hydrophobic patch, formed by three residues (W5, M6 and W7), play very important roles in the binding of JZTX-V to Kv4.2These data show that JZTX-V could effectively inhibited transient outward potassium channels currents Kv4.2, binding to it by two aspects at least. The one is that the toxin interactive with channel by hydrophobic patchs, an exposed hydrophobic patch on Kv4.2, formed by three residues (W5, M6 and W7) interacted with a hydrophobic patch, formed from three mutations (V285A, F289A and V290A) on S3-S4 linker; the other is the long β-carbon chain of the amino acid limiting the introduction of secondary conformational and physiochemical changes...