Gene expression, mutation, and structure-function relationship of scorpion toxin BmP05 active on SK(Ca) channels.
Four peptide inhibitors of small-conductance Ca(2+)-activated, apamin-sensitive K(+) channels (SK(Ca)) have been isolated from the venom of the Chinese scorpion Buthus martensi, named BmP01, BmP02, BmP03, and BmP05, respectively [Romi-Lebrun, R. (1997) Eur. J. Biochem. 245, 457-464]. Among them BmP05 with 31 amino acid residues has been intensively studied due to its most potent toxicity. To investigate the structure-function relationship of BmP05, its wild type and seven mutants (their C-termini unamidated) were successfully expressed in the yeast secretion system and purified with a high yield over 8 mg/L. Their toxicity to mice and electrophysiological activity on the K(+) currents (SK(Ca) and Kv) in rat adrenal chromaffin cells were measured and compared. The results indicated the following: (1) As a selective antagonist against SK(Ca), 1 microM rBmP05 is equivalent to 0.2 microM apamin, and its IC(50) is 0.92 microM. (2) The basic residues Lys and Arg located at positions 6 and 13 in the N-terminal alpha-helix region are essential and synergetic in the interaction of the toxin with SK(Ca). (3) Disruption of the alpha-helix by mutation of Gln at position 9 with Pro results in almost total loss of toxicity. (4) The C-terminal residue His31 plays an auxiliary role in the interaction of the toxin with SK(Ca). (5) The beta-turn connecting two beta-sheets near the C-terminal part is responsible for the specificity of the toxin to the different subtypes of K(+) channels.
Wu
Jing-Jiang
JJ
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, and Institute of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China.
He
Lin-Lin
LL
Zhou
Zhuan
Z
Chi
Cheng-Wu
CW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002942:Circular Dichroism; D017931:DNA Primers; D015870:Gene Expression; D007928:Lethal Dose 50; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D015221:Potassium Channels / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012604:Scorpion Venoms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
C106380:BmP05 toxin; D017931:DNA Primers; D015221:Potassium Channels; D011994:Recombinant Proteins; D012604:Scorpion Venoms