Molecular Cloning, Expression, Structure-function Relationship Of K～+ Channel Scorpion Toxin From Buthus Martensii Karsch

Scorpion venom contains many kinds of bioactive polypeptides composed of 20-90 amino acid residues with 3 or 4 disulfide bridges. Almost all scorpion toxins have a highly conserved dense core formed by an a -helix and two or three P -sheets, and this core is maintained by disulfide bonds. Scorpion toxins are able to specially and selectively act on K+, Na+, Cl- and Ca2+ channel on cell membrane, which are known as modulators of ionic channels and useful tools for investigating the mechanism of ion conductance and channel selectivity. Therefore, scorpion toxins are of important value in the theoretical and applied research.Based on the conserved region of two long chain potassium channel toxins TsTXKB and AaTXKB , a new long chain potassium channel toxin BmTXK B was isolated and identified from the constructed cDNA library of Chinese scorpion{Buthus martensii Karsch,BmK) venom gland using PCR screening strategy. Sequence analysis showed that the full length cDNA of BmTXK B is 348 nucleotides including 50nt 5'UTR, 30nt 3'UTR and 273nt open reading frame. A tailing signal ATTAA was found at the location of 14nt forward to polyA site. The precursor of BmTXKB encodes 90 amino acids with a putative signal peptide of 22 residues, a propeptide of 7 residues and a mature toxin of 61 residues. BmTXKB shared a low similarity with TsTXK B AaTXK B and BmTXKB2 at the nucleotide and amino acid level, but the organization of cDNA and the spacing pattern of six cysteines were consistent withthem. Therefore, BmTXKB should represent a group of scorpion toxins.Genomic region corresponding to BmTXKB mature peptide was cloned and characterized by traditional PCR. Analysis of nucleotide sequence showed that the region encoding mature peptide of BmTXK B was disrupted by an intron with 886bp, which was different from any other scorpion toxin gene with their intron in the signal peptide coding region. Southern hybridization of the scorpion genome total DNA was performed using the full-length cDNA sequence of BmTXK P as probe by a random priming radiolabelled procedure. Southern hybridization analysis showed that there were two specific hybridization signals. With primers designed according to known sequence of BmTXKB and southern hybridization result, its 5' and 3' flanking region were cloned by the Vecttorette Pack method and sequenced. Another intron longer than 997bp was found at the location of the signal peptide region of BmTXK B , which makes BmTXK B completely different from all the other scorpion toxins. The special genomic organization of BmTXKB indicates that BmTXK B is a new membership of long chain potassium channel toxin.BmTXKB was expressed as a GST fusion protein in BL21(DE3) strain. The recombinant GST-BmTXKB protein was purified by affinity chromatography. When treated with enterokinase, the recombinant BmTXKB protein (rBmTXKB) was obtained from GST-BmTXKB . The function of rBmTXKB was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKB inhibited the transient outward current (Ito) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKB prolonged action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.According to the protein and nucleotide sequence of small-conductance Ca2+ activated potassium channel toxin BmP05, a short chain potassium channel toxin BmP05' high homology with BmP05 was successfully cloned from Buthus martensii Karsch venom by 5' and 3' rapid amplify cDNA end(RACE). Full-length cDNA sequence of BmP05' is composed of 274 nt: 39 nt 5'UTR, 49 nt 3'UTR and a 186 nt ORE This ORF encodes 61 amino acid residues including 28 amino acid residues. 31 ammo acid residues mature peptide and 2 extra amino acids residues(Gly-Lys). Atailing signal AATAAA is located at the backward 32 nt of terminal TAA. BmP05' shares 98% homology with BmP05 at the nucleotide level, but their amino acid residues a...