The Structure And Functional Study Of Intermediate Conductance Calcium Activated Potassium Channels Activator HNTX-I

Hainan Tarantulas is one of the tarantulas originating from Hainan and Guangxi.We found a variety of polypeptide components and purified a neurotoxin,named Hainantoxin-I(HNTX-I),from Hainan Tarantulas through RP-HPLC/MALDI-TOF-MS analysis.Venom was collected using an electropulse stimulator contacting cheliceral claws and pure HNTX-I was used cation exchange and reversed-phase high-performance liquid chromatography.HNTX-I had a relative molecular weight(MW)of 3601.8 Da,and its amino acid sequence was ECKGFGKSCVPGKNECCSGYACNSRDKWCKVLL.HNTX-I contained six cysteines(Cys)which had three disulfide bonds,and a primary sequence was consisted of two adjacent cysteines.The disulfide paring modes were ?-?,?-?,?-?.The IK channel(intermediate-conductance-calcium activated potassium channel)played an important role in the relaxation process of smooth muscle.Activation of the IK channel was capable of reducing blood pressure and relieving asthma.Previous patch clamp experiments showed that high concentration of HNTX-I had no significant effect on tetrodotoxin-sensitive sodium channel and tetrodotoxin-resistance sodium channel.And 100 ?M HNTX-I also had no effect on expression of hERG potassium channel in HEK293T cells,expression of L-type Ca2+ channel,T-type Ca2+ channel and hERG potassium channel in rat dorsal root ganglia(dorsal root ganglion).For researching 100 ?M HNTX-I had effect on BK channel,IK channel and SK channel,we found that the toxin had the strongest effect on IK channel and its EC50 value is about 26?M,about 30%activation for SK channel and had no significant role on the BK channel.Further research found that the activation of IK channel was not obvious.Gene coding HNTX-I was inserted into prokaryotic expression plasmid and expressed in E.coli.We constructed a plasmid expression vector and successfully expressed HNTX-I.There were not much difference from the IK channel EC50 of wild HNTX-I by mass spectrometry analysis and patch-clamp testing.Fangchenggang toxin-I(FCGTX-I)and HNTX-I had 97%sequence homology and only the fifteenth amino acid residues are different.High concentrations of FCGTX-I has no effect on IK channels.Fifteenth amino acid residues of IK channel maybe a key residues of active role.Then we constructed five IK channel mutants(W54,L56,F59,K62,R123,E149)by molecular modeling and docking.After patch clamp detection,the EC50 values of L56,F59,R123,E149 and wild IK channel were almost about 25 ?M-29?M.But HNTX-I had no effect on both W54 and K62.Therefore,we could speculate W54 and K62 are key residues in IK channels.Then we constructed three HNTX-I toxin mutant E1F-HNTX-I,K13D-HNTX-I,N14F-HNTX-I by molecular modeling and docking.Patch clamp experiments indicated that the activation of E1F-HNTX-I increased about 29 fold than wild HNTX-I to IK channels.Meanwhile,we succeed in expressing HNTX-I and detected the mutants of IK channel using a small molecule activators NS309.It had a strong activation on IK channel.We used it to detect W54 and K62,still found a strong activation,showed the activation site of HNTX-I and NS309 to IK channels are not the same.