Cloning, Genomic Organization And Functional Characterization Of A New Short-chain Potassium Channel Toxin-like Peptide BmTxKS4 From Buthus Martensii Karsch(BmK)

Scorpion venom glands synthesize and secrete a great number of low molecular mass toxic peptides for prey and defense, which can modulate N^a+, K⁺, Cl^-, and Ca²⁺ ion-channel conductance in the cell membrane. Up to now, the SCORPION database (the Web site is http://sdmc.i2r.a-star.edu.sg/scorpion) has represented 277 scorpion toxin genes. The venoms of only 30 species of scorpions have been analyzed in detail, out of a roughly estimated 1500 distinct species around the world including the old and new world scorpion species. It is surmised about 100,000 different peptides exist in the venoms, only 0.02% are known. The Buthus martensii Karsch (BmK) scorpion from Old world is a famous Chinese herbal medicinal material and not dangerously venomous for mammals. In fact, many components from BmK are active and demonstrated to be functional. So far, 74 different potassium toxins from scorpion venom have been identified in amino acid residue sequences, physiological or pharmacological functions and their corresponding receptors. Since each K⁺ channel toxin shows an individual pattern of selectivity and specificity towards the vast array of existing K⁺-channels, the discovery of new K⁺-channel toxins may provide valuable tools for the further study. Moreover, K⁺-channel toxins are also useful in drug search and discovery because they can be used as pharmacological tools for understanding many physiological processes and uncovering potential therapeutic targets.Based on the constructed scorpion venom glands cDNA library, a full-length cDNA sequence encoding a novel type of K⁺-channel toxin (named BmTxKS4) was first isolated and identified from the cDNA library of BmK. The precursor nucleotide sequence encoding BmTxKS4 is 537bp long, which is composed of three parts: 5'UTR, ORF and 3'UTR. The 5'UTR and 3'UTR regions of BmTxKS4 cDNA sequence are 111 bp and 189 bp, respectively. The 5'UTR nucleotide sequence contains high A+T content (82.9%), two A_n(n>3) and seven TTAA/AATT elements, which may have relevance to the regulation of toxin gene expression by supporting the stable bending of their promoters.An ORF of 234 bp encodes a precursor of 78 amino acid residues. The flanking region of initiation codon ATG is AAAGCATGAA, which is highly similar with the others of scorpion toxins acting on Na+, K+, Cl" ion-channels, but different from vertebrates (CC(A/G)CCATGG)]. 3'UTR is also rich in A+T (82.2%). The 3'-end of the cDNA, two putative poly (A) signals (AATAAA) were identified at 37 bp and 14 bp upstream of the poly(A) tail. In addition, 3'UTR contains four copies of the ATTTA motif. The result by Signal P V2.0 analysis showed that the precursor of BmTxKS4 contains a putative signal peptide of 21 residues and a putative propeptide of 11 residues, following a mature toxin of 46 residues cross-linked by three disulfide bridges. BmTxKS4 primary structure shares low similarity with the other scorpion toxins. The highest homology between BmTxKS4 and the other scorpion toxins is less than 36%. But BmTxKS4 shares the same disulfide bridge pattern as most short-chain three disulfide-bridges K+-channel toxins.Analysis of this sequence showed that the genomic organization of BmTxKS4 is highly similar to that of the scorpion K+-channel toxins. BmTxKS4 contains two exons disrupted by a single intron of 87 bp. The intron located after the first base (T) of Phe codon in the end of the signal peptide encoding region, similar with the most other introns of Na+, K+ and Cl-channel scorpion toxin genes, and. The A+T content of the intron is 88.5% (39.1% A, 49.4% T, 8.1% G, 3.4% C) and significantly more than exon encoding regional flanking (65.4%).The GST-BmTxKS4 fusion protein was successfully expressed in BL21 (DE3) and purified with affinity chromatography. About 2.5 mg purified recombinant BmTxKS4 (rBmTxKS4) protein was achieved by treated GST-BmTxKS4 with enterokinase and sephadex G50 and G10 chromatography from 1 L bacterial culture.The electrophysiological activity of 1.0 P M rBmTxKS4 was measured and compared by whole-cell patch-clamp technique. The recording results indicated that addition of 1.0 fiM rBmTxKS4 to the external solution leaded to a reduction of the current of /to and /ki at all voltage. We can considered that rBmTxKS4 reversiblyinhibited /to and delayed fa and prolonged the action potential duration of the rabbit ventricular myocyte, but it has no effect on the action potential amplitude.By the similar of protocal whole-cell patch-clamp, the blocking results of rBmTxKS4 on the K+-channel through rat hippocampal neuon myocytes have been confinned. The results showed that rBmTxKS4 inhibited the total potassium current(TK) and delayed rectifier potassium current (.10 by about 30%.The toxicity of rBmTxKS4 also been assayed with the mouse model and the Hela myocyte. The results showed that the l⁰.1ug/g (rBmTxKS4/mouse weight) rBmTxKS4 should lead the Kunming mouse to dead and 50 ng/ u 1-100 ng/ u 1 rBmTxKS4 inhibited divided of the cultured Hela.In conclusion, we suggested that BmTxKS4 should be a member of a new subfamily member of K+-channel toxins, which will provide a new material for further studying oftheir receptors and the structure-function relationship of these peptides.BmKBT is also a toxic component purified from the venom of Chinese scorpionBmK, acting on Na+ channel specially. Although it is structurally similar to P -type Na+ channel toxins, BmKBT is functionally similar to a -type Na+ channel toxins. Base on the known sequence of BmKBT isolated from the cDNA library of the venom gland, genomic BmKBT was cloned by PCR method. Sequence analysis of genomic BmKBT shows that BmKBT has an intron of 225 bp located in the end of the region encoding of signal peptide. The intron of BmKBT is similar to the a -type sodium ion-channel toxins in the size, the location, the sequence consensus of the 5'splice donor, putative branch point distance 47 bp upstream of the 3'splice receptor. Combining with the function of BmKBT, the genomic organization of BmKBT indicates that it is a membership between a - and ^ -type sodium ion-channel toxin, which might serve as a useful matter to research evolution of the scorpion venom gene.