Identification Of The Interaction Interface Between Hainantoxin-Ⅲ And Sodium Channel Isoform Nav1.7

Hainantoxin-Ⅲ(HNTX-Ⅲ)is a 33-residue polypeptide derived from the venom of the spider Ornithoctonus hainana.The molecular weight of HNTX-Ⅲ is 3607 Da.It possesses six cysteine residues which form three disulfide bonds.Applied 2D-NMR techniques to determine the solution structure of HNTX-Ⅲ,revealed that HNTX-Ⅲ is a typical inhibitor cystine knot motif(ICK motif)polypeptide.It selectively suppresses neuronal tetrodotoxin-sensitive voltage-gated sodium channel(TTX-S)current amplitude,with IC50 values of 181 nM(to Nav1.7),235 nM(to Nav1.2),500 nM(to Nav1.3).According to previous research,HNTX-Ⅲ inhibits Nav1.7 activation through binding to site-4 in the closed state.Nav1.7 primarily exists in the peripheral nervous system and involved in the initiation and propagation of action potentials in excitable cells.Previous study indicates that Nav1.7 involved in signal transduction of pain,which made Nav1.7 a new target for analgesic drug develop.To achieve more specific inhibitors of Nav1.7,we designed some HNTX-Ⅲ mutants and Nav1.7 mutants and then tested their mutual affinity.Mutants of HNTX-Ⅲ were synthesized by solid-phase synthesis of peptide and their binding affinities with Nav1.7 were detected by patch clamp technique.Mutants of Navl.7 were obtained by site-directed PCR mutagenesis mutant cycle analysis was used to study the strength of interaction between residues in HNTX-Ⅲ and Nav1.7,we successfully identified the binding mode of HNTX-Ⅲ and Navl.7.The results show that the amino acid residues E818,D890,D891 on Nav1.7 act with basic amino acid residues K3,K25,H26,K30 on HNTX-Ⅲ by electrostatic;Hydrophobic amino acid residues L823,V824,F826 on the Navl.7 act with hydrophobic amino acid residues on the F5,W28 on HNTX-Ⅲ by strong hydrophobic interaction;V31 bind to V1408 via direct interaction,while Y32 and L33 can not interact with V1408;In addition,both V1410 and D1411 interact with V31,Y32 and L33 by direct interaction.The neutral amino acid residues N19,Y20,S24 mutates to A caused reduction of the affinity of HNTX-Ⅲ with Nav1.7 by 20-fold at least,perhaps because of the decreased hydrogen bonds caused by these mutation.Taken together,HNTX-Ⅲ inhibit Nav1.7 by electrostatic and hydrophobic interaction and the neutral amino acid residues N19,Y20,S24 may be essential by interacting with Navl.7 via hydrogen bonds.Overall,the binding mechanism of HNTX-Ⅲ and Navl.7 was successfully illustrated and HNTX-Ⅲ can be a useful tool for studying toxin-VGSC interaction and a potential prototype analgesic.To get more specific inhibitors of Navl.7 based on the framework of HNTX-Ⅲ,it is necessary to determine the difference of the molecular mechanisms between HNTX-Ⅲ binding to Nav1.7 and Nav1.3 and therefore,we examined a series of HNTX-Ⅲ mutations inhibition to Nav1.3.The results showed that the affinity of F5A,W28A to Nav1.3 reduced to 100-fold similar to Nav1.7,indicating that F5,W28 are critical residues for HNTX-Ⅲ binding to both channels;The binding affinity of L33A to Navl.7 and Nav1.3 reduced by 4-fold and 40-fold,respectively.A 10-fold difference suggesting that L33 is more essential for HNTX-III binding to Nav1.3 rather than Nav1.7;Basic amino acid residues K25,H26 mutated to A caused significant affinity decreased with affinity of K25A to Nav1.3 and Nav1.7 reduced by 100-fold and 23-fold,respectivly.On the contrary,the affinity of H26A to Nav1.7 decreased by 40-fold,5 times more than that of Nav1.3.Which suggest that K25 is more important to the binding of HNTX-Ⅲ to Nav1.3,however,H26 is more important to the binding of HNTX-Ⅲ to Nav1.7;Both N19A and Y20A caused affinity to Nav1.7 and Nav1.3 sharply decreased,at least by 20-fold,moreover,the decrease in affinity to Nav1.3 is 2.7 times of the decreasing degree to Nav1.7,indicating that N19,Y20 are more important to binding to Nav1.3;And K3A,S24A,K30A caused changes of binding affinity to both channel reduced by 3-fold;Intreastingly,Y32A results in a 2-fold increase of inhibitory activity to Nav1.7 and K13A results in a 2-fold increase of inhibitory activity to Nav1.3.Taken together,the molecular mechanisms of HNTX-Ⅲ binding to Nav1.7 and Nav1.3 are quite different,but the clear mechanisms need more efforts.