Analysis of the Interaction of Tarantula Toxin Jingzhaotoxin-III (β-TRTX-Cj1α) with the Voltage Sensor of Kv2.1 Uncovers the Molecular Basis for Cross-Activities on Kv2.1 and Nav1.5 Channels

• Published in: Biochemistry (Easton) 2013-10, Vol.52 (42), p.7439-7448
• Main Authors: Tao, Huai, Chen, Jin J, Xiao, Yu C, Wu, Yuan Y, Su, Hai B, Li, Dan, Wang, Heng Y, Deng, Mei C, Wang, Mei C, Liu, Zhong H, Liang, Song P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Electrophysiology; Female; Ion Channel Gating - drug effects; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation - genetics; NAV1.5 Voltage-Gated Sodium Channel - chemistry; NAV1.5 Voltage-Gated Sodium Channel - genetics; NAV1.5 Voltage-Gated Sodium Channel - metabolism; Oocytes - cytology; Oocytes - drug effects; Oocytes - metabolism; Patch-Clamp Techniques; Peptides - pharmacology; Protein Conformation; Saccharomyces cerevisiae - metabolism; Sequence Homology, Amino Acid; Shab Potassium Channels - chemistry; Shab Potassium Channels - genetics; Shab Potassium Channels - metabolism; Spider Venoms - pharmacology; Spiders - metabolism; Xenopus laevis
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi4006418

Animal venoms contain a fascinating array of divergent peptide toxins that have cross-activities on different types of voltage-gated ion channels. However, the underlying mechanism remains poorly understood. Jingzhaotoxin-III (JZTX-III), a 36-residue peptide from the tarantula Chilobrachys jingzhao, is specific for Nav1.5 and Kv2.1 channels over the majority of other ion channel subtypes. JZTX-III traps the Nav1.5 DII voltage sensor at closed state by binding to the DIIS3-S4 linker. In this study, electrophysiological experiments showed that JZTX-III had no effect on five voltage-gated potassium channel subtypes (Kv1.4, Kv3.1, and Kv4.1–4.3), whereas it significantly inhibited Kv2.1 with an IC50 of 0.71 ± 0.01 μM. Mutagenesis and modeling data suggested that JZTX-III docks at the Kv2.1 voltage-sensor paddle. Alanine replacement of Phe274, Lys280, Ser281, Leu283, Gln284, and Val288 could decrease JZTX-III affinity by 7-, 9-, 34-, 12-, 9-, and 7-fold, respectively. Among them, S281 is the most crucial determinant, and the substitution with Thr only slightly reduced toxin sensitivity. In contrast, a single conversion of Ser281 to Ala, Phe, Ile, Val, or Glu increased the IC50 value by >34-fold. Alanine-scanning mutagenesis experiments indicated that the functional surface of JZTX-III bound to the Kv2.1 channel is composed of four hydrophobic residues (Trp8, Trp28, Trp30, and Val33) and three charged residues (Arg13, Lys15, and Glu34). The bioactive surfaces of JZTX-III interacting with Kv2.1 and Nav1.5 are only partially overlapping. These results strongly supported the hypothesis that animal toxins might use partially overlapping bioactive surfaces to target the voltage-sensor paddles of two different types of ion channels. Increasing our understanding of the molecular mechanisms of toxins interacting with voltage-gated sodium and potassium channels may provide new molecular insights into the design of more potent ion channel inhibitors.