A new potassium channel toxin from the sea anemone Heteractis magnifica : isolation, cDNA cloning, and functional expression

Gendeh, G S and Young, L C and de Medeiros, C L and Jeyaseelan, K and Harvey, A L and Chung, M C (1997) A new potassium channel toxin from the sea anemone Heteractis magnifica : isolation, cDNA cloning, and functional expression. BMC Biochemistry, 36 (38). pp. 11461-11471. ISSN 1471-2091 (https://doi.org/10.1021/bi970253d)

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Abstract

A new potassium channel toxin, HmK, has been isolated from the sea anemone Heteractis magnifica. It inhibits the binding of [125I]-alpha-dendrotoxin (a ligand for voltage-gated K channels) to rat brain synaptosomal membranes with a Ki of about 1 nM, blocks K+ currents through Kv 1.2 channels expressed in a mammalian cell line, and facilitates acetylcholine release at the avian neuromuscular junction. HmK comprises of 35 amino acids (Mr 4055) with the sequence R1TCKDLIPVS10ECTDIRCRTS20MKYRLNLCRK30TCGSC35. A full assignment of the disulfide linkages was made by using partial reduction with tri(2-carboxyethyl)phosphine (TCEP) at acid pH and rapid alkylation with iodoacetamide. The disulfide bridges were identified as Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. A cDNA clone encoding HmK was isolated using RT-PCR from the total RNA obtained from sea anemone tentacles, while the 5'- and 3'-flanking regions of the cDNA were amplified by RACE. The full-length cDNA was 563 bp long and contained a sequence encoding a signal peptide of 39 amino acids. The coding region for matured HmK toxin was cloned and expressed as a glutathione S-transferase (GST) fusion product in the cytoplasm of Escherichia coli. After affinity purification and cleavage, the recombinant toxin was shown to be identical to native HmK in its N-terminal sequence, chromatographic behavior, and binding to dendrotoxin binding sites on rat brain membranes.

  • Item type:Article
    ID code:31625
    Dates:
    Date
    Event
    23 September 1997
    Published
    Keywords:amino acid sequence, animals, base sequence, competitive binding, brain, molecular cloning, cnidarian venoms, complementary DNA, elapid venoms, escherichia coli, membranes, molecular sequence data, neuromuscular junction, neurotoxins, potassium channel blockers, protein binding, recombinant fusion proteins, sea anemones, sequence analysis, amino acid sequence homology, Pharmacy and materia medica, Biochemistry, Molecular Biology
    Subjects:Medicine > Pharmacy and materia medica
    Department:Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences
    Depositing user: Pure Administrator
    Date deposited:13 Jul 2011 08:56
    Last modified:20 May 2023 03:50
    URI:https://strathprints.strath.ac.uk/id/eprint/31625
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