A novel conotoxin, -conotoxin (-BtX), has been purified and characterized from the venom of a wormhunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are ␥-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, -BtX is a specific modulator of K ؉ channels; 2) among the K channels, -BtX specifically up-modulates the Ca 2؉ -and voltage-sensitive BK channels (252 ؎ 47%); 3) its EC 50 is 0.7 nM with a single binding site (Hill ‫؍‬ 0.88); 4) the time constant of wash-out is 8.3 s; and 5) -BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that -BtX is a novel specific biotoxin against BK channels.

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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 µM rBmP05 is equivalent to 0.2 µM apamin, and its IC 50 is 0.92 µM. (2) The basic residues Lys and Arg located at positions 6 and 13 in the N-terminal R-helix region are essential and synergetic in the interaction of the toxin with SK Ca . (3) Disruption of the R-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 -turn connecting two -sheets near the C-terminal part is responsible for the specificity of the toxin to the different subtypes of K + channels.