A New Potassium Channel Toxin from the Sea Anemone Heteractis magnifica : Isolation, cDNA Cloning, and Functional Expression ,
1997; American Chemical Society; Volume: 36; Issue: 38 Linguagem: Inglês
10.1021/bi970253d
ISSN1943-295X
AutoresG. Gendeh, Louise Young, C. L. C. de Medeiros, Kandiah Jeyaseelan, Alan L. Harvey, M.C.M. Chung,
Tópico(s)Marine Toxins and Detection Methods
ResumoA new potassium channel toxin, HmK, has been isolated from the sea anemone Heteractis magnifica. It inhibits the binding of [125I]-α-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.
Referência(s)