An Investigation Of Inhibitory Actitvity Of Peptide Toxins On Potassium

Animal venoms contain abundant neural polypeptide toxins.With high binding affinity and a variety of pharmacological properties,peptide toxins are powerful probes for investigating the structure-function relationships of ion channels.This makes animal venoms an important source for developing ion channels related diseases drugs.Raventoxin-? is a 35-residue peptide from the venom of the spider Macrothele raveni.The molecular weight of raventoxin-? was determined to be 3965.202 Da by MALDI-TOF mass spectrometry.Considering the difference between the actual and theoretical molecular weight,we speculated that the toxin forms three disulfide bonds.The full amino acid sequences of raventoxin-? is TEESACGGFNARCPLHKCCPQYICKGWRTKRCLNP as established by a combination of Edman degradation and cDNA sequences.Under the whole-cell patch-clamp,the activity of raventoxin-? was examined on ten voltage-gated potassium channel subtypes expressed in HEK293 cells.Data showed raventoxin-? could selectively inhibit Kv1.3 currents with an IC50 value of 36.3nM.But the treatment of 1?M raventoxin?,caused no obvious effects on Kv1.2?Kv2.1?Kv2.2?Kv3.1?Kv4.1?Kv4.2 and Kv4.3.It also blocked the Kv1.1 and Kv1.4 channels with an IC50 value of 2.49 ?M and 1.41 ?M,38-and 68-fold higher than that on Kv1.3,respectively.Furthermore,we investigated the kinetic characteristics of the interaction between raventoxin-? and Kv1.3,including activation and inhibition kinetics.According to the fast inhibition kinetics and no change of the activation kinetics,we hypothesized that raventoxin-? should be a pore-blocking toxins.Basing on the binding sites of between pore blockers and voltage-dependent K+ channels,we proposed that the interaction between raventoxin-? and Kv1.3 was probably located in Kv1.3 S5-S6 linker.According to the sequence differences in the S5-S6 linker of Kv1.1,Kv1.2,Kv1.3 and Kv1.4,individual amino acid residue in the S5-S6 linker of Kv1.3 was mutated to alanine or other amino acid residue.Site-directed mutagenesis analysis demonstrated that four residues(D386?T394?Y400 and D402)in the S5-S6 linker contributed to the formation of raventoxin-? binding receptor site.Alanine replacement of residue T394?Y400 or D402 could decrease raventoxin-? affinity by>68-fold.The mutation D386A reduced toxin binding affinity by 10.23-fold.Scolopendra subspinipes mutilans,also known as Chinese red-headed centipede,is a venomous centipede from East Asia and Australasia.Venom from this animal has not been researched as thoroughly as venom from snakes,scorpions,and spiders.In this study,we isolated and characterized SsmTx-I,a novel neurotoxin from the venom of S.subspinipes mutilans.SsmTx-I contains 36 residues with four cysteines forming two disulfide bonds.It had low sequence similarity(<10%)with other identified peptide toxins.By whole-cell recording,SsmTx-I significantly blocked voltage-gated K+ channels in dorsal root ganglion neurons with an IC50 value of 200 nM,but it had no effect on voltage-gated Na+ channels.Among the nine K+ channel subtypes expressed in human embryonic kidney 293 cells,SsmTx-I selectively blocked the Kv2.1 current with an IC50 value of 41.7 nM,but it had little effect on currents mediated by other K+ channel subtypes.Blockage of Kv2.1 by SsmTx-I was not associated with significant alteration of steady-state activation,suggesting that SsmTx-I might act as a simple inhibitor or channel blocker rather than a gating modifier.Our study reported a specific Kv2.1-blocker from centipede venom and provided a basis for future investigations of SsmTx-I,for example on structure-function relationships,mechanism of action,and pharmacological potential.