The Research Of Electrophysiological Activities Of Several Toxins

Almost all centipedes are predators and have venom glands.The primary purpose of centipedes' venoms is to kill or paralyze prey.Centipedes,venoms are complex chemical cocktails in which proteins are the principal constituents.Using the whole-cell patch-clamp,,the effcts of centipede crude venom on adult rat dorsal root ganglion(DRG)neurons were analyzed,showing that the crude venom can inhibit both voltage-gated sodium channels and potassium channels.Therefore,we presumed that in centipede crude venom there are some neurotoxins that can inhibit both channels.By using reverse phase high performance liquid chromatography,we collected 43 fractions from the venom of the centipede Scolopendra subspinipes mutilans L.Koch.Under the whole-cell patch-clamp,we identified five peptide neurotoxins acting on voltage-gated sodium currents and four inhibiting potassium currents in adult rat dorsal root ganglion(DRG)neurons.Both of their inhibitions showed a dose-dependent manner.One neurotoxin,named SsmTx-I,has effect on delayed rectifier potassium currents and is the main component in crude venom.Therefor,further research of the physiological and biochemical properties was performed.The amino acid sequence is EESMLLSCPDLSCPTGYTCDVLTKKCKRLS DELWDH,as determined by Edman,and the molecular weight was determined as 4114.06Da by MALDI-TOF mass spectrometry.Electrophysiological assays indicated that its IC50 value on voltage-gated potassium channels on rat DRG cells was about 200nM.SsmTx-I had little effct on the current-voltage relationship of potassium channels,but it could shift depolarizatedly the voltage dependence of activation about 8.04mV.SsmTx-I could selectively inhibit wild Kv2.1 currents expressed in HEK293 with an IC50 value of about 41.7nM,but it had no obvious effects on Kv4.1?Kv4.2 and Kv4.3.Taken together,SsmTx-I is a a novel peptide toxin isolated from the venom of the centipede Scolopendra subspinipes mutilans L.Koch,it was able to selectively and potently inhibit Kv2.1,and therefore its would be helpful probe for investigating the structure and function of Kv2.1.Consequently,centipede venoms are a rich source of novel pharmacologically and agrochemically interesting compounds that have received increased attention from pharmacologists and biochemists in recent years.Besides,we determined electrophysiological activities of several Hainanatoxin-III isoforms.HNTX-III was purified from the venom of the spider Selenocosmia hainana Liang,its molecular weight is 3607 Da.It could inhibit voltage-gated tetrodotoxin-sensitive(TTX-S)sodium currents(IC50=43.6nmol/L),but not the voltage-gated TTX-resistant(TTX-R)sodium currents.From its cDNA library,we found many cDNA sequences with high sequence identities with HNTX-?.As their amounts in the venom are very low,four isoforms,HNTX-? S24N?HNTX-? H26D?HNTX-? Y20H and HNTX-? Y20H/S24N,were synthesized and refolded.Like HNTX-?,all the four isoforms could inhibit TTX-S sodium currents in rat DRG cells.Their IC50 values were calulcated to be 138nmol/L,2.69 ?mol/L,4.37 ?mol/L,1.95 ?mol/L,respectively,lower than that of HNTX-?.The result shows that His26 and Tyr20 might be key residues of HNTX-? binding to TTX-S sodium channels on rat DRG cells.