ON THE SITE BY WHICH ALPHA-DENDROTOXIN BINDS TO VOLTAGE-DEPENDENT POTASSIUM CHANNELS - SITE-DIRECTED MUTAGENESIS REVEALS THAT THE LYSINE-TRIPLET-28-30 IS NOT ESSENTIAL FOR BINDING

We constructed a synthetic gene encoding the published amino acid sequ ence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependen t postassium channels that facilitates neurotransmitter release. We ex pressed it in Escherichia coli as a fusion protein secreted in the cul ture medium. The recombinant DTx was generated in vitro by chemical tr eatment and recovered as two isoforms. One of them (rDTx), like the ve nom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) h as a free N-terminal glutamine. Chromatographic differences between rD Tx and natural DTx led us to re-examine the amino acid sequence of nat ural DTx. In contrast to what was previously published, position 12 wa s an Asp and not Asn. Despite this difference, rDTx and DTx had simila r toxicity in mice and binding affinity to synaptosomes, suggesting th at residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had simi lar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala -Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the p ositively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.