Molecular cloning, genomic organization and functional characterization of a new short‐chain potassium channel toxin‐like peptide BmTxKS4 from Buthus martensii Karsch(BmK)

Abstract

Scorpion venom contains many small polypeptide toxins, which can modulate Na(+), K(+), Cl(-), and Ca(2+) ion-channel conductance in the cell membrane. A full-length cDNA sequence encoding a novel type of K(+)-channel toxin (named BmTxKS4) was first isolated and identified from a venom gland cDNA library of Buthus martensii Karsch (BmK). The encoded precursor contains 78 amino acid residues including a putative signal peptide of 21 residues, propeptide of 11 residues, and a mature peptide of 43 residues with three disulfide bridges. BmTxKS4 shares the identical organization of disulfide bridges with all the other short-chain K(+)-channel scorpion toxins. By PCR amplification of the genomic region encoding BmTxKS4, it was shown that BmTxKS4 composed of two exons is disrupted by an intron of 87 bp inserted between the first and the second codes of Phe (F) in the encoding signal peptide region, which is completely identical with that of the characterized scorpion K(+)-channel ligands in the size, position, consensus junctions, putative branch point, and A+T content. 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 obtained by treating GST-BmTxKS4 with enterokinase and sephadex chromatography from 1 L bacterial culture. The electrophysiological activity of 1.0 microM rBmTxKS4 was measured and compared by whole cell patch-clamp technique. The results indicated that rBmTxKS4 reversibly inhibited the transient outward K(+) current (I(to)), delayed inward rectifier K(+) current (I(k1)), and prolonged the action potential duration of ventricular myocyte, but it has no effect on the action potential amplitude. Taken together, BmTxKS4 is a novel subfamily member of short-strain K(+)-channel scorpion toxin.

Overview of Scorpion Species from China and Their Toxins

“…From the M . martensii library, a large number of putative toxins have been described, including NaTx (toxins specific for the sodium channels) [51,52,53,54,55,56], KTx (toxins specific for the potassium channels) [57,58,59,60,61,62,63], ClTx (toxins specific for chloride channels) [64], CaTx (toxins for ryanodine receptors) [65], Bpp-like (bradykinin-potentiating peptide-like) peptide [66], cytolytic peptide [67,68], and other functional peptides [69,70,71]. In 2009, Ma et al .…”

Molecular Toxinology – Cloning Toxin Genes for Addressing Functional Analysis and Disclosure Drug Leads

“…Additional information regarding the cloning of large genomic DNA fragments for preparation of libraries can be obtained from dedicated companies such as Agilent Technologies, Inc. (Santa Clara, CA-U.S.A.). In addition to the construction of genomic DNA libraries, there are other simpler techniques for cloning toxin genes and examining their structural organization, such as direct clone by PCR-mediated amplification based on specific primers probes for cDNA sequences (Jiqun et al, 2004;Zhijian et al, 2006, Mao et al, 2007 and 'genome (DNA) walking' (Gendeh et al, 1997;Legros et al, 1997;Afifiyan et al, 1999;Jeyaseelan et al, 2003).…”