SITE-DIRECTED MUTAGENESIS OF DENDROTOXIN-K REVEALS AMINO-ACIDS CRITICAL FOR ITS INTERACTION WITH NEURONAL K+ CHANNELS

Dendrotoxin K (DTXK) is a 57-residue protein from mamba venom that blo cks certain non-inactivating, voltage-activated K+ currents in neurone s. In order to pinpoint the residues responsible for its specificity, structure-activity relations of DTXK were investigated by mutagenesis. A previously cloned gene encoding this toxin [Smith et al. (1993) Bio chemistry 32, 5692-5697] was used to make single mutations; after expr ession in Escherichia coli as fusion proteins and enzymatic cleavage o f the conjugates isolated from the periplasmic space, nine toxins were purified. Structural analysis of the native DTXK and representative m utants by circular dichroism showed that no significant differences we re detectable in their folded structures. The biological activity of t he mutants, which contained alterations of positively charged and othe r amino acids, was determined from their abilities to compete for the binding of I-125-labeled DTXK to K+ channels in synaptic plasma membra nes from rat cerebral cortex. Mutants with residues substituted in the alpha-helix near the C-terminus (R52A or R53A) yielded binding parame ters similar to those of wild-type and native DTXK. In the case of the beta-turn (residues 24-28), however, altering single amino acids redu ced binding to the high-affinity site of K+ channels, with the rank or der of decreases being K26A much greater than W25A > K24A = K28A. Also , substitutions made in the 3(10)-helix (residues 3-7), a region locat ed close to the beta-turn, produced equivalent effects (K3A > K6A). Th us, it is deduced that residues in the distorted beta-turn and neighbo ring 3(10)-helix of DTXk are critical for its interaction with neurona l K+ channels.