title: Mapping the interaction site for the tarantula toxin hainantoxin-IV (β-TRTX-Hn2a) in the voltage sensor module of domain II of voltage-gated sodium channels.
abstract: Peptide toxins often have pharmacological applications and are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a group of potential VGSC inhibitors have been reported from tarantula venoms, little is known about the mechanism of their interaction with VGSCs. In this study, we showed that hainantoxin-IV (β-TRTX-Hn2a, HNTX-IV in brief), a 35-residue peptide from Ornithoctonus hainana venom, preferentially inhibited rNav1.2, rNav1.3 and hNav1.7 compared with rNav1.4 and hNav1.5. hNav1.7 was the most sensitive to HNTX-IV (IC50∼21nM). In contrast to many other tarantula toxins that affect VGSCs, HNTX-IV at subsaturating concentrations did not alter activation and inactivation kinetics in the physiological range of voltages, while very large depolarization above +70mV could partially activate toxin-bound hNav1.7 channel, indicating that HNTX-IV acts as a gating modifier rather than a pore blocker. Site-directed mutagenesis indicated that the toxin bound to site 4, which was located on the extracellular S3-S4 linker of hNav1.7 domain II. Mutants E753Q, D816N and E818Q of hNav1.7 decreased toxin affinity for hNav1.7 by 2.0-, 3.3- and 130-fold, respectively. In silico docking indicated that a three-toed claw substructure formed by residues with close contacts in the interface between HNTX-IV and hNav1.7 domain II stabilized the toxin-channel complex, impeding movement of the domain II voltage sensor and inhibiting hNav1.7 activation. Our data provide structural details for structure-based drug design and a useful template for the design of highly selective inhibitors of a specific subtype of VGSCs.
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, 410081 Hunan, China.
authors.forename: Tianfu
authors.initials: T
authors.lastname: Cai
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, 410081 Hunan, China.
authors.forename: Ji
authors.initials: J
authors.lastname: Luo
authors.affiliation: Research Center of Biological Information, College of Science, National University of Defense Technology, Changsha, 410073 Hunan, China.
authors.forename: Er
authors.initials: E
authors.lastname: Meng
authors.affiliation: Key Laboratory of Molecular Biophysics, Huazhong University of Science and Technology, Ministry of Education, College of Life Science and Technology, Wuhan, Hubei 430074, China.
authors.forename: Jiuping
authors.initials: J
authors.lastname: Ding
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, 410081 Hunan, China.
authors.forename: Songping
authors.initials: S
authors.lastname: Liang
authors.affiliation: Key Laboratory of Molecular Biophysics, Huazhong University of Science and Technology, Ministry of Education, College of Life Science and Technology, Wuhan, Hubei 430074, China. Electronic address: shengwang@hust.edu.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, 410081 Hunan, China. Electronic address: liuzh@hunnu.edu.cn.
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D013111:Spider Venoms / Q000737:chemistry* / Q000494:pharmacology; D061567:Voltage-Gated Sodium Channel Blockers / Q000737:chemistry* / Q000494:pharmacology; D061566:Voltage-Gated Sodium Channels / Q000737:chemistry* / Q000378:metabolism
chemical_list: D013111:Spider Venoms; D061567:Voltage-Gated Sodium Channel Blockers; D061566:Voltage-Gated Sodium Channels; C474037:hainantoxin-IV, Seleconosmia hainana
keywords: Nav1.7; Tarantula toxin; VGSCs; Voltage sensor