title: 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.
abstract: 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.
authors.affiliation: Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University , Changsha, Hunan 410081, China.
authors.forename: Huai
authors.initials: H
authors.lastname: Tao
authors.forename: Jin J
authors.initials: JJ
authors.lastname: Chen
authors.forename: Yu C
authors.initials: YC
authors.lastname: Xiao
authors.forename: Yuan Y
authors.initials: YY
authors.lastname: Wu
authors.forename: Hai B
authors.initials: HB
authors.lastname: Su
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Heng Y
authors.initials: HY
authors.lastname: Wang
authors.forename: Mei C
authors.initials: MC
authors.lastname: Deng
authors.forename: Mei C
authors.initials: MC
authors.lastname: Wang
authors.forename: Zhong H
authors.initials: ZH
authors.lastname: Liu
authors.forename: Song P
authors.initials: SP
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004594:Electrophysiology; D005260:Female; D015640:Ion Channel Gating / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051669:Shab Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013111:Spider Venoms / Q000494:pharmacology*; D013112:Spiders / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D062554:NAV1.5 Voltage-Gated Sodium Channel; D010455:Peptides; D051669:Shab Potassium Channels; D013111:Spider Venoms; C487820:jingzhaotoxin-III, Chilobrachys jingzhao