title: Natural mutations change the affinity of μ-theraphotoxin-Hhn2a to voltage-gated sodium channels.
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.
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.affiliation: College of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414006, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors.forename: Changxin
authors.initials: C
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors.forename: Zuqin
authors.initials: Z
authors.lastname: Yang
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Gong
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Gan
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China. Electronic address: liuzh@hunnu.edu.cn.
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Liu
authors.affiliation: College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China. Electronic address: liangsp@hunnu.edu.cn.
authors.forename: Songping
authors.initials: S
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D019143:Evolution, Molecular; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D062552:NAV1.3 Voltage-Gated Sodium Channel / Q000378:metabolism; D062556:NAV1.7 Voltage-Gated Sodium Channel / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017422:Sequence Analysis, DNA; D015222:Sodium Channels / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013111:Spider Venoms / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology*; D061566:Voltage-Gated Sodium Channels / Q000378:metabolism*
chemical_list: D062552:NAV1.3 Voltage-Gated Sodium Channel; D062556:NAV1.7 Voltage-Gated Sodium Channel; C424715:SCN3A protein, human; C486866:SCN9A protein, human; D015222:Sodium Channels; D013111:Spider Venoms; D061566:Voltage-Gated Sodium Channels; C488683:hainantoxin III, Seleconosmia hainana
keywords: Natural mutations; Spider toxin; Voltage-gated sodium channels