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 [I-125]-alpha-dendrotoxin (a ligand for voltage-gated K channels) to rat brain synaptosomal membranes with a K-i 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 (M-r 4055) with the sequence R(1)TCKDLIPVS(10)ECTDIRCRTS(20)MKYRLNLCRK(30)TCGSC(35). 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 Cys(3)-Cys(35), Cys(12)-Cys(28), and Cys(17)-Cys(32). 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.