Recombinant expression and functional characterization of martentoxin: a selective inhibitor for BK channel (α + β4).
Martentoxin (MarTX), a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch), was capable of blocking large-conductance Ca2+-activated K+ (BK) channels. Here, we report an effective expression and purification approach for this toxin. The cDNA encoding martentoxin was expressed by the prokaryotic expression system pGEX-4T-3 which was added an enterokinase cleavage site by PCR. The fusion protein (GST-rMarTX) was digested by enterokinase to release hetero-expressed toxin and further purified via reverse-phase HPLC. The molecular weight of the hetero-expressed rMarTX was 4059.06 Da, which is identical to that of the natural peptide isolated from scorpion venom. Functional characterization through whole-cell patch clamp showed that rMarTX selectively and potently inhibited the currents of neuronal BK channels (α + β4) (IC50 = 186 nM), partly inhibited mKv1.3, but hardly having any significant effect on hKv4.2 and hKv3.1a even at 10 μM. Successful expression of martentoxin lays basis for further studies of structure-function relationship underlying martentoxin or other potassium-channel specific blockers.
Lab of Neuropharmacology and Neurotoxicology, Shanghai University, Nanchen Road 333, Shanghai 200444, China. jietao@shu.edu.cn.
Jie
J
Tao
Lab of Neuropharmacology and Neurotoxicology, Shanghai University, Nanchen Road 333, Shanghai 200444, China. shupenguin@shu.edu.cn.
Zhi Lei
ZL
Zhou
Lab of Neuropharmacology and Neurotoxicology, Shanghai University, Nanchen Road 333, Shanghai 200444, China. colabin@shu.edu.cn.
Bin
B
Wu
Lab of Neuropharmacology and Neurotoxicology, Shanghai University, Nanchen Road 333, Shanghai 200444, China. jshi@shu.edu.cn.
Jian
J
Shi
Lab of Neuropharmacology and Neurotoxicology, Shanghai University, Nanchen Road 333, Shanghai 200444, China. ming0911@shu.edu.cn.
Xiao Ming
XM
Chen
Lab of Neuropharmacology and Neurotoxicology, Shanghai University, Nanchen Road 333, Shanghai 200444, China. yhji@staff.shu.edu.cn.
Yong Hua
YH
Ji
D000595:Amino Acid Sequence; D057809:HEK293 Cells; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D012604:Scorpion Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
C494841:KCNMA1 protein, human; C410183:KCNMB4 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D009419:Nerve Tissue Proteins; D026902:Potassium Channel Blockers; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C471035:martentoxin, Buthus martensi