East Asia Markov Scorpion In A New Active Peptide Purification, Cloning And Functional Identification

As one of the most fundamental proteins in organisms, K+ channels play key roles in many crucial physiological processes. So far, more than 80 K+ channels have been identified but many of them are poorly characterized. Scorpions have survived more than 350 million years with no detectable changes in their anatomy due to their efficient, dynamic machinery production of bioactive peptides mainly blocking Na+ channels and K+ channels. Scorpion peptides have been proven to be powerful tools for testing the pharmacological, physiological, structural and functional characteristics of K+ channels. To get more tools for K+ channel studies, it is important to keep searching for novel bioactive peptides from scorpion venom.In the present study, five novel K+ channel blockers, namely BmBKTxl, BmTx3, BmSKTxl, BmSKTx2 and BmK38, have been purified from the crude venom of Scorpion Buthus martensi Karsh (BmK). Based on their determined protein sequences, gene special primers were designed and the cDNA genes of BmSKTxl, BmSKTx2 and BmK38 were cloned by Rapid Amplification of cDNA Ends (RACE). Also, the genomic gene of BmSKTxl was cloned by PCR. After screened on various channels, BmBKTxl was turned out to be a selective blocker for Ca2+-activated K+ channels, with the activity both on BK-type (big conductance) and SK-type (small conductance) KCa channels. Interestingly, BmBKTxl tends to have the insect specificity in respect of the BK channel blocking. BmTx3 is a peptide with two different functions: HERG channel and A-type K+ current blocking activities. There are two separate functional faces locating on the N-terminal region and the C-terminal region of the BmTx3 molecule, respectively corresponding to its two different K+ currents blocking abilities. BmSKTxl and BmSKTx2 are selective blockers for SK channels. Although the function of BmK38 has not been clearly demonstrated, it has the same cysteine scaffold shared by other K+ channel blockers, indicating that it should target K+ channels also. However, there is a unique turn sticking out from the whole molecule surface of BmK38, implying that this toxin might have a unique function.A chlorotoxin analogue, designated BmClTx, was also purified from scorpion BmK. Chlorotoxin is a selective drug to treat gliomas and has recently won the FDA approval for use in a phase I/II clinical trial. Given the high sequence homology between BmClTxl and Chlorotoxin, BmClTx also holds the potential to be developed to an anti-cancer drug.Besides various experimental methods, a computational method, Evolutionary trace (ET) analysis, was performed to study the evolutionary convergence and divergence among different scorpion K+ channels blockers. The functional face of y-KTx active on HERG channel was predicted by ET analysis and the differences between two block modes - turret block and pore block- were fully discussed in this study.