Structure And Function Of FS50,A Salivary Protein From The Flea Xenopsylla Cheopis That Blocks The Sodium Channel Nav1.5

BACKGROUNDBiological toxins such as scorpion toxin,spider toxins,snake toxins and some reported marine biological toxins are almost from nature resources.So many peptides and small molecules had been Separated and purificated from the venom and the activity was identified,most of them were interacting with the ion channel receptor to change the function,trigger downstream signal pathway,cause a great discomfort of Bitten person.So these materials were patrly used as a tool to study ion channels'structure and function.Ion channels are pore-forming membrane proteins whose functions include establishing a resting membrane potential,shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane,controlling the flow of ions across secretory and epithelial cells,and regulating cell volume.Ion channels are present in the membranes of all cells.Ion channels are one of the two classes of ionophoricproteins,along with ion transporters.The special protein produced by cells consist several subunits around formed the pore,embedded into the cell membrane.Ions were passived transport across the membrane ion-channel-pore.Ion channels can be classified into voltage gated ion channel,ligand gated ion channels,mechanically gated ion channel,and so on.Ion channel's activity is through the opening and closing of channel to regulate the abilityof changing the materials in and out of the cell.Blood-sucking-arthropod animal in the nature,which contains mosquitoes,flies,fleas,gadfly and so on.This kind of insect mouthparts,penetrate into the human body with some material throughing,which will be released to induce the pain feel,itching skin disease and other symptoms were reduced or delayed,so that it will be more convenient tosuck blood.Some team has purificated variety of peptide from these salivary gland secretion,containing anticoagulant,antiplatelet agents,vasodilator substances,can not only affect the coagulation system,host immune response,and inflammation.,but also few peptide influence the function of ion channels.This already-published papers point out that if we can find some other ion channel regulators from the separated peptides from x.cheopis salivary gland secretion.The bioactive peptide can improve which existing human disease?OBJECTIVES1)To find out whether there is an active component on voltage-gated ion channel activity from the polypeptide FS50.2)To find out the mechanism of the interaction between FS50 and ion channel3)FS50 could improve which existing ion channel diseasesMETHODS1)Rat DRG neurons or HEK293T cells were cultured to verify whether FS50 has voltage-gated ion channel activity by patch clamp technique.2)The key residuces of FS50 on Nav1.5 channel were verified by patch clamp technique.3)The arrhythmia-model of rats or rhesus monkeys which were induced by BaCl2 were used to define whether FS50 can improve this disease.4)The proliferation effect of FS50 on MDA-MB-231 cells were verified by two different methods:MTT method and Cell-lumi ATP method.5)The effect of FS50 on cell motility were verified by wound-healing assay and migration assay.6)Immunocytochemistry,confocal microscopy,image analysis,and western blotting were used to define the channel recptor protein expression level.7)Q-PCR assay was used to define the channel recptor mRNA expression level.CONCLUSIONS:Our results found the expression and purification of peptide FS50 from x.cheopis salivary gland can specifically inhibit Nav1.5 receptor channel function;and the mechanism of ion channel receptor blocking effect is to play as a steric blocker,does not affect the characteristics of channel selectivity of ion channels,does not affect the steady state the activation and inactivation of key residues could significantly inhibit sodium currents and.FS50 channel receptors R6,H11,K32,the residues distribution in FS50 polypeptide structure surface and are located in the positive charge distribution intensive areas.FS50 for Nav1.5 channel function caused by abnormal arrhythmia has good curative effect,can significantly delay the time.Navl.5 channel arrhythmia occurrence time and arrhythmia duration as a cell movement of important regulatory targets,FS50 can weaken the Nav1.5 channel function weakened high Expression of metastasis and invasion of tumor cells,the MDA-MB-231 receptor.