Effects Of Buthus Martensi Karsch Polypeptides On Ion Channels In Neurons

Part one:The purification,isolation and identification of Buthus martensi Karsch(BmK)neurotoxinsTwo long-chain polypeptides BmK2255 and BmK2256 were purified from the venom of Asian scorpion Buthus martensi Karsch(BmK)by a combination of gel-filtration,ion-exchange chromatography and reversed-phased high-performance liquid chromatography.We studied the primary characterization and identification of the two peptides.It showed:The molecular weight of BmK2255 and BmK2256 were found to be 7223.0Da and 7036.85 Da by electrospray ionization mass spectrometry,respectively;The sequence of BmK2255 was determined to be VRDGY IADDK NCAYF CGRNA YCDEE CIING AESGY CQQAG VYGNA CWCYK LPDKV PIRVS GECQQ by Edman degradation,which was same as bukatoxin(BKTx)from BmK.The first 15 N-terminal amino acid sequence of BmKNJX11 was determined to be GRDAY IADSE NCTYT,which was same as Makatoxin I(MkTx I)and BmK9(3)-2 from BmK.BmK2256 was named BmKNJX11.Part two:Effects of BmK neurotoxins on action potential in rat dorsal root ganglion neuronsAction potential(AP)was successfully was recorded in freshly isolated rat dorsal root ganglion(DRG)neurons using whole-cell patch clamp technique.The firing frequency and the amplitude of AP could be reversibly inhibited by Tetrodotoxin(TTX)which is antagonist of Na⁺ channels.We investigated the effects of bukatoxin and BmKNJX11 on APs in rat DRG neurons respectively.The results showed both the two peptides could inhibit APs firing.The firing frequency and the amplitude of APs were decreased with 20,40μg/ml bukatoxin and BmKNJX11, respectively.There was almost no APs firing in DRG neurons with 80μg/ml bukatoxin and BmKNJX11. Part three:Effects of BmK neurotoxins on Na⁺ currents in neuronsThe effects of BmKNJX11 on voltage-depedent sodium currents(I_Na) including TTX-S I_Naand TTX-R I_Nawere studied in rat DRG neurons using the patch-clamp technique in the whole-cell configuration.The results showed that BmKNJX11 could inhibit the amplitudes of both TTX-S I_Naand TTX-R I_Nain a concentration- and voltage-dependent manner.At the concentration of 40μg/ml BmKNJX11 lowered the activation threshold and produced negative shifting of TTX-S I_Na activation curve,induced shifting of the steady-state inactivation curve to left,delayed the recovery of TTX-S I_Nafrom inactivation,and also reduced the fraction of available sodium channels.At the concentration of 40μg/ml BmKNJX11 produced positive shifting of TTX-R I_Naactivation curve,induced shifting of the steady-state inactivation curve to left, delayed the recovery of TTX-R I_Nafrom inactivation,and also reduced the fraction of available sodium channels.To observe the selective alterations of Na_v 1.3 and Na_v 1.8 expression in PC12 cells induced by bukatoxin,Real-Time RT-PCR was performed to detect alterations of Na_v 1.3 and Na_v 1.8 expression in PC12 cells at mRNA level.The results showed that at the concentration of 80μg/ml, bukatoxin increased expression of Na_v 1.3 at level of mRNA in 30min, then decreased expression of Na_v 1.3 at level of mRNA in 60min and 7hr. At the concentration of 80μg/ml,bukatoxin decreased expression of Na_v 1.8 at level of mRNA,but at 7hr expression of Na_v 1.8 at level of mRNA increased by contrast to at 60min.Part four:Effects of BmK neurotoxins on K⁺ currents in rat dorsal root ganglionIn this part,we observed the effects of bukatoxin and BmKNJX11 on the voltage dependent potassium channels in rat DRG neurons using whole-cell patch clamp technique.The results indicated as follows:even at 80μg/ml bukatoxin did not exert significant effects on the potassium channels and the peak K⁺ currents(I_K);but 80μg/ml BmKNJX11 showed slightly inhibiting effects on the I_K including the delayed rectifier potassium currents(I_KDR)and the fast inactivating potassium currents(I_A). I_KDRand I_A were decreased to 85.3±1%and 78.12±2%,respectively.Part five:Effects of BmK neurotoxins on GABA-activated currents in rat dorsal root ganglionWhole cell patch clamp technique was performed on isolated rat DRG neurons to investigate GABA-activated membrane currents (I_GABA).1,10,100,1000μmol/L GABA activated a dose-dependent inward current which had an obvious desensitization.I_GABAis a inward current at-100～0 mV,while it is a outward current at 0～+40 mV,which suggests I_GABAhas voltage-dependent property.I_GABAcould be almost completely blocked by bicuculine and increased by isoguvacine.These results confirmed that the current we recorded was I_GABA.We also investigated the effects of bukatoxin and BmKNJX11 on I_GABAin rat DRG neurons respectively.The results indicated as follows: I_GABAwas inhibited by bukatoxin,with IC₅₀and h were 42.26μg/ml and 2.01,respectively.I_GABAwas increased by BmKNJX11,with IC₅₀and h were 60.4μg/ml and 3.4,respectively.