Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K+ channels containing Kv1.1 and 1.2 alpha subunits

Dendrodoxin (DTX) homologues are powerful blockers of K+ channels that cont ain certain subfamily Kv1 (1.1-1.6) alpha- and beta-subunits, in (alpha)(4) (beta)(4) stoichiometry. DTXk inhibits potently Kv1.1-containing channels o nly, whereas alpha DTX is less discriminating, hut exhibits highest affinit y for Kv1.2. Herein. the nature of interactions of DTXk with native K+ chan nels composed of Kv1.1 and 1.2 (plus other) subunits were examined, using 1 5 site-directed mutants in which amino acids were altered in the 3(10)-heli x, beta-turn, alpha-helix and random-coil regions. The mutants' antagonism of high-affinity [I-125]DTXk binding to Kv1.1-possessing channels in rat br ain membranes and blockade of the Kv1.1 current expressed in oocytes were q uantified. Also, the levels of inhibition of [I-125]alpha DTX binding to br ain membranes by the DTXk mutants were used to measure their high- and low- affinity interactions, respectively, with neuronal Kv1.2-containing channel s that possess Kv1.1 as a major or minor constituent. Displacement of toxin binding to either of these subtypes was not altered by single substitution with alanine of three basic residues in the random-coil region, or R52 or R53 in the alpha-helix: accordingly, representative mutants (K17A, R53A) bl ocked the Kv1.1 current with the same potency as the natural toxin. In cont rast, competition of the binding of the radiolabelled alpha DTX or DTXk was dramatically reduced by alanine substitution of K26 or W25 in the beta-tur n whereas changing nearby residues caused negligible alterations. Consisten tly, W25A and K26A exhibited diminished functional blockade of the Kv1.1 ho mo-oligomer. The 3(10)-helical N-terminal region of DTXk was found to be re sponsible for recognition of Kv1.1 channels because mutation of K3A led to approximate to 1246-fold reduction in the inhibitory potency for [I-125]DTX k binding and a large decrease in its ability to block the Kv1.1 current, t he effect of this substitution on the affinity of DTXk for Kv1.2-possessing oligomers was much less dramatic (approximate to 16-fold).