Natural mutations change the affinity of μ-theraphotoxin-Hhn2a to voltage-gated sodium channels

Fan Zhang; Yu Liu; Changxin Zhang; Jing Li; Zuqin Yang; Xue Gong; Yunxiang Gan; Ping Chen; Zhonghua Liu; Songping Liang

doi:10.1016/j.toxicon.2014.11.220

Natural mutations change the affinity of μ-theraphotoxin-Hhn2a to voltage-gated sodium channels

Toxicon. 2015 Jan:93:24-30. doi: 10.1016/j.toxicon.2014.11.220. Epub 2014 Nov 6.

Authors

Fan Zhang¹, Yu Liu², Changxin Zhang¹, Jing Li¹, Zuqin Yang¹, Xue Gong¹, Yunxiang Gan¹, Ping Chen¹, Zhonghua Liu³, Songping Liang⁴

Affiliations

¹ College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
² College of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414006, China.
³ College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China. Electronic address: liuzh@hunnu.edu.cn.
⁴ College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China. Electronic address: liangsp@hunnu.edu.cn.

PMID: 25447770
DOI: 10.1016/j.toxicon.2014.11.220

Abstract

μ-Theraphotoxin-Hhn2a (HNTX-III) isolated from the venom of the spider Ornithoctonus hainana is a selective antagonist of neuronal tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channels (VGSCs). Intriguingly, previous transcriptomic study revealed HNTX-III family consists of more than 15 precursors, in which the 20(th) and 24(th) residues of the mature sequences are variable. Try20 and Ser24 of HNTX-III are mutated to His20 and Asn24 of other members, respectively. In addition, the alkaline residue His26 of the potent VGSC inhibitor HNTX-III is substituted by acidic residue Asp of the weak VGSC inhibitor HNTX-I. Therefore, four mutants of HNTX-III, HNTX-III-Y20H, -S24N, -H26D and -Y20H/24N, were synthesized to examine the effects of these natural mutations on the inhibitory activity of HNTX-III. They were subjected to an electrophysiological screening on five VGSC subtypes (Nav1.3-1.5, Nav1.7 and Nav1.8) expressed on HEK293 cells by whole-cell patch clamp. Like HNTX-III, all mutants only displayed inhibitory activity on Nav1.3 and Nav1.7 among the five subtypes, but the inhibitory potency was much lower than that of HNTX-III. Regarding Nav1.7, the IC50 values of HNTX-III-Y20H, -S24N, -H26D and -Y20H/S24N were increased by approximately 62-, 8.4-, 49- and 19.5-folds compared with that of HNTX-III, respectively. Similar data were obtained for Nav1.3. Our results provide new insights into the activity-related residues of HNTX-III at genic level. Furthermore, the reduced potency of the four mutants probably reflects natural selection might favor and reserve the most potent bioactivity of HNTX-III which is one of the most abundant fractions of the venom.

Keywords: Natural mutations; Spider toxin; Voltage-gated sodium channels.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Base Sequence
Circular Dichroism
Evolution, Molecular
HEK293 Cells
Humans
Molecular Sequence Data
Mutation / genetics*
NAV1.3 Voltage-Gated Sodium Channel / metabolism
NAV1.7 Voltage-Gated Sodium Channel / metabolism
Patch-Clamp Techniques
Sequence Analysis, DNA
Sodium Channels / metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Spider Venoms / biosynthesis
Spider Venoms / genetics*
Spider Venoms / pharmacology*
Voltage-Gated Sodium Channels / metabolism*

Substances

NAV1.3 Voltage-Gated Sodium Channel
NAV1.7 Voltage-Gated Sodium Channel
SCN3A protein, human
SCN9A protein, human
Sodium Channels
Spider Venoms
Voltage-Gated Sodium Channels
hainantoxin III, Seleconosmia hainana