A new potassium channel toxin from the sea anemone Heteractis magnifica: isolation, cDNA cloning, and functional expression.
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.
Gendeh
G S
GS
Department of Biochemistry, Bioprocessing Technology Center, and Bioscience Center, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260.
Young
L C
LC
de Medeiros
C L
CL
Jeyaseelan
K
K
Harvey
A L
AL
Chung
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D003064:Cnidarian Venoms / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004546:Elapid Venoms / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000187:drug effects; D009498:Neurotoxins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D026902:Potassium Channel Blockers; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012615:Sea Anemones / Q000235:genetics*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D003064:Cnidarian Venoms; D018076:DNA, Complementary; D004546:Elapid Venoms; C108359:HmK toxin, sea anemone; D009498:Neurotoxins; D026902:Potassium Channel Blockers; D011993:Recombinant Fusion Proteins; C020273:dendrotoxin