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

G S Gendeh, L C Young, C L de Medeiros, K Jeyaseelan, A L Harvey, M C Chung

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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.
Original languageEnglish
Pages (from-to)11461-11471
Number of pages11
JournalBMC Biochemistry
Volume36
Issue number38
DOIs
Publication statusPublished - 23 Sep 1997

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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

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