The Study Of The Mechanism Of HNTX-? Binding The Intermediate Conductance Calcium-activated Potassium Channel

The intermediate conductance calcium-activated potassium channels(IK,KCa3.1)are one of the most significant family of calcium-activated potassium channels and existed widely in humans.The activation of IK channels plays an crucial role in the relaxation process of the smooth muscle,meanwhile it also can stable the asthma and reduce the blood pressure.In recent years,many studies have discovered that the changes and abnormities of IK channels' quantity,structure and function can lead to the generation of some diseases and even malignant tumor.Many researches have reported that IK channels highly expressed in several cancers and involved in the process of cancer development,such as initial,progress,metastasis,multi-drug resistance of cancer.In the study of tumor,the abnormal expression of IK channels can be used as one of most important diagnostic indicators and parameters.Furthermore,it also has theoretical and clinical significances for searching effective targeted and anti-angiogenic therapy to tumors.HNTX-? is a neurotoxin isolated from the spider's venom of Selenocosmia hainanum,which is the highest abundance in the crude toxin of Selenocosmia hainanum.HNTX-? consists of 33 amino acid residues and has a relative molecular weight(MW)of 3608.1 Da.The amino acid sequence of HNTX-? is ECKGFGKSCVPGK NECCSGYACNSRDKWCKVLL,of which 6 cysteines(Cys)make up three pairs of disulphide bonds in a typical ?-???-???-? coulped mode,with three pairs of anti-parallel ?-fold to form a typical Inhibitor Cystine Knot motif(ICK).HNTX-? is a peptide activator of IK channels which was discovered for the first time.In the present study,IK-transfected HEK293T cells were studied in the whole-cell configuration of the patch-clamp technique,and the EC50 value of the wild type HNTX-? activated IK channels is about 26?M.It is useful and helpful to find some IK channels' activators with stronger activity,better selectivity and higher safety by studying the mechanism of HNTX-? on IK channels.In this study,molecular dynamics simulation and molecular docking techniques were used to find the critical binding sites on the IK channels and the key amino acid residues of HNTX-? relatively.The interactions between HNTX-? and IK channels have two modes,namely,electrostatic interaction and hydrophobic interaction.Previous experiments indicated that the tryptophane at position 54 is a key interactional site for IK channels.The toxin's mutants which based on prokaryotic expression and chemical synthesis were designed to study the hydrophobic interaction between W54 of IK channels and the corresponding residues of HNTX-? by molecular dynamics stimulation and molecular docking technique.The result showed that IK channels can be activated by these mutants which including HNTX-?-S8F?HNTX-?-S8A?HNTX-?-K13F and HNTX-?-W28A.Their EC50 values are 24.10?M,26.51?M,23.64?M,27.02?M respectively,and make no significant difference with the EC50 value of HNTX-?.However,the point is that the mutant named HNTX-1-F5A can not activate IK channel,the 5th tryptophan residue of HNTX-? plays a key role in the activation of IK channel obviously.The tryptophan residue at the 54th site of IK channel is a hydrophobic amino acid,and the phenylalanine residue at 5th position of HNTX-? is also hydrophobic.There is a strong hydrophobic interaction in particular which exists between the W54 site of IK channel and the F5 amino acid residue,because of their side chains' hydrophobicity.