A novel HERG channel blocker was isolated from the venom of the scorpion Buthus martensi Karsch, sequenced and characterized at the pharmacological level after chemical synthesis. According to the determined amino acid sequence, the cDNA and genomic genes were then cloned. The genomic gene consists of two exons interrupted by an intron of 65 bp at position −6 upstream from the mature toxin. The protein sequence of this toxin was completely identical with that of a known A-type K+ current blocker BmTx3, belonging to scorpion α-KTx subfamily 15. Thus BmTx3 is the first reported α-KTx peptide also showing HERG-blocking activity, like γ-KTx peptides. Moreover, different from classical α-KTx peptides, such as charybdotoxin, BmTx3 cannot block Shaker-type K+ channels. Phylogenetic tree analysis reveals that this toxin takes an intermediate position between classical α-KTx and γ-KTx toxins. From a structural point of view, we propose that two separate functional faces might exist on the BmTx3 molecule, responsible for the two different K+-current-blocking functions. Face A, composed of Arg18 and Lys19 in the α-helix side, might correspond to HERG blocking activity, whereas Face B, containing a putative functional dyad (Lys27 and Tyr36) in the β-sheet side, might correspond to A-type blocking activity. A specific deletion mutant with the disrupted Face B, BmTx3-Y36P37del, loses the A-type current-blocking activity, but keeps a similar HERG-blocking activity, as seen with the wild-type toxin.
Abbreviations used: BmK, Buthus martensi Karsch; ChTx, charybdotoxin; GSP, gene-specific primer; HERG, human ether-a-go-go-related gene; Ifully activated, inward current; It, small outward current; Itail, large outward tail current; KTx, K+ channel toxin; RACE, rapid amplification of cDNA ends; RP-HPLC, reverse-phase HPLC; sBmTx3, synthetic BmTx3; TFA, trifluoroacetic acid; UTR, untranslated region; V1/2, half maximal potential; Vt, test potential.
These authors contributed equally to this work.
The nucleotide sequences for cDNA and genomic gene of BmTx3 has been deposited in the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AF541980 and AY156725 respectively.