Site-Directed Mutagenesis of Dendrotoxin K Reveals Amino Acids Critical for Its Interaction with Neuronal K+ Channels

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Abstract
Dendrotoxin K (DTXK) is a 57-residue protein from mamba venom that blocks certain non-inactivating, voltage-activated K+ currents in neurones. 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) Biochemistry 32, 5692−5697] was used to make single mutations; after expression in Escherichia coli as fusion proteins and enzymatic cleavage of the conjugates isolated from the periplasmic space, nine toxins were purified. Structural analysis of the native DTXK and representative mutants by circular dichroism showed that no significant differences were detectable in their folded structures. The biological activity of the mutants, which contained alterations of positively charged and other amino acids, was determined from their abilities to compete for the binding of 125I-labeled DTXK to K+ channels in synaptic plasma membranes from rat cerebral cortex. Mutants with residues substituted in the α-helix near the C-terminus (R52A or R53A) yielded binding parameters similar to those of wild-type and native DTXK. In the case of the β-turn (residues 24−28), however, altering single amino acids reduced binding to the high-affinity site of K+ channels, with the rank order of decreases being K26A ≫ W25A > K24A = K28A. Also, substitutions made in the 310-helix (residues 3−7), a region located close to the β-turn, produced equivalent effects (K3A > K6A). Thus, it is deduced that residues in the distorted β-turn and neighboring 310-helix of DTXk are critical for its interaction with neuronal K+ channels.