Molecular Basis Of Voltage-gated Sodium Channel Interaction With Peptide Toxins From The Spider Ornithoctonus Hainana

Peptide toxins from spider venom have high-affinity for their targets and isoform-specific selectivity.They 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 has been reported from tarantula venom,little is known about the mechanism of their interaction with VGSCs.In the present study,we investigated the solution structure and function of Hainantoxin-III(HNTX-III),a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana.It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels.The mechanism underlying the inhibitory effect of HNTX-III on Navl.7 expressed in HEK 293 cells was examined.HNTX-III suppressed Navl.7 current amplitude without significantly altering the activation,inactivation,and repriming kinetics.The inhibition was not rapid,with a time constant of 20.5±0.3s,and it was reversible upon washing in the presence of 1 ? M HNTX-?.The IC50 value was calculated to be approximately 1.27?M for Navl.1,275 nM for Nav1.2,491 nM for Navl.3 and 232 nM for Navl.7.Short extreme depolarizations partially activated the toxin-bound channel,indicating voltage-dependent inhibition of HNTX-?.HNTX-? increased the deactivation of the Navl.7 current after extreme depolarizations.The HNTX-III-Navl.7 complex was gradually dissociated upon prolonged strong depolarizations in a voltage-dependent manner,and the unbound toxin rebound to Navl.7 after a long repolarization.Moreover,analysis of chimeric channels showed that the DIIS3-S4 linker was critical for HNTX-? binding to Navl.7.These data are consistent with HNTX-? interacting with Navl.7 site 4 and trapping the domain? voltage sensor in the closed state.In our previous study,The solution structure of HNTX-III was determined by two-dimensional NMR and shown to possess an inhibitor cystine knot motif.Structural analysis indicated that certain basic,hydrophobic,and aromatic residues mainly localized in the C terminus may constitute an amphiphilic surface potentially involved in HNTX-? binding to Nav1.7.In site-directed mutagenesis analysis,we found E818 was especially crucial for HNTX-IV inhibition of Navl.7IC50 values was more than 50-fold higher than the effect of the toxin on the WT channel.These data suggest that HNTX-? might inhibit Navl.7 activation through a novel mechanism similar to that of huwentoxin-IV,which binds to Navl.7 site 4 in the closed state,trapping the voltage sensor of domain II in the inward position.Taken together,our results show that HNTX-III and HNTX-IV are distinct from?-scorpion toxins and other ?-spider toxins in its mechanism of action and binding specificity and affinity.The present findings contribute to our understanding of the mechanism of toxin-sodium channel interaction and provide a useful tool for the investigation of the structure and function of sodium channel isoforms and for the development of analgesics.In this study,we showed that hainantoxin-IV(?-TRTX-Hn2a,HNTX-IV in brief),a 35-residue peptide from Ornithocotonus hainana venom,preferentially inhibited Navl.2,Navl.3 and Navl.7 compared with Navl.4 and Navl.5.Among the five VGSCs tested,hNavl.7 was the most sensitive to HNTX-IV(IC50?21 nM).In contrast to many other tarantula toxins that affect VGSCs,HNTX-IV at subsaturating concentrations did not alter current-voltage curves,or activation and inactivation kinetics.Site-directed mutagenesis indicated that the toxin bound to site 4 of the neurotoxin receptor,which was located on the extracellular S3-S4 linker of domain II of hNavl.7.Mutants E753Q,D816N and E818Q of hNavl.7 decreased toxin affinity for hNavl.7 by 2.0-,3.3-and 130-fold,respectively.Other residues in this region had small effects on the inhibitory activity of the toxin.In silico docking indicated that a three-toed claw substructure formed by residues with close contacts in the interface between HNTX-? and Nav1.7 domain ? stabilized the toxin-channel complex,impeding movement of the domain ? voltage sensor and inhibiting Nav1.7 activation.In contrast to scorpion ?-toxins that trap the IIS4 voltage sensor in an outward configuration,we propose that HNTX-? traps the voltage sensor of domain ? in the inward,closed configuration.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.In our previous research,a group of toxins have been discovered in the venom of Ornithoctonus hainana to have effect on TTX-S voltage gated sodium channel,but until now,little is found to be able to inhibit TTX-R VGSC.In my study,I tested the venom of Ornithoctonus hainana on TTX-R VGSC expressed on rat DRG cells and Nav1.8 current heterologously expressed on ND7-23 cells at crude level.Four kinds of peptides have been discovered and analyzed to have effect on Nav1.8 current,whose molecular weight are 3977,3605.6,3537.5 and 3828.6Da,respectively.One kind of peptide,with molecular weight of 3828.6Da,has been reported in our previous study and named as HNTX-VII in brief(ECRYWLGTCSKTGDCCSHLSCSPKHGWCVWDWT).It has been proven that one peptide,with molecular weight of 3977,has the strongest effect on Navl.8 and the amino acid sequence was determined by Edam degradation(QCRYWLGTCSKTGDCCSQL-SCSPKQHWCVWDWW).