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

1999; Wiley; Volume: 263; Issue: 1 Linguagem: Inglês

10.1046/j.1432-1327.1999.00494.x

1432-1033

Fan C. Wang, Natalie Victoria Bell, Paul F. Reid, Leonard A. Smith, Paul McIntosh, Brian D. Robertson, J. Oliver Dolly,

Nicotinic Acetylcholine Receptors Study

Dendrotoxin (DTX) homologues are powerful blockers of K + channels that contain certain subfamily Kv1 (1.1–1.6) α‐ and β‐subunits, in (α) 4 (β) 4 stoichiometry. DTX k inhibits potently Kv1.1‐containing channels only, whereas αDTX is less discriminating, but exhibits highest affinity for Kv1.2. Herein, the nature of interactions of DTX k with native K + channels composed of Kv1.1 and 1.2 (plus other) subunits were examined, using 15 site‐directed mutants in which amino acids were altered in the 3 10 ‐helix, β‐turn, α‐helix and random‐coil regions. The mutants’ antagonism of high‐affinity [ 125 I]DTX k binding to Kv1.1‐possessing channels in rat brain membranes and blockade of the Kv1.1 current expressed in oocytes were quantified. Also, the levels of inhibition of [ 125 I]αDTX binding to brain membranes by the DTX k mutants were used to measure their high‐ and low‐affinity interactions, respectively, with neuronal Kv1.2‐containing channels 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 α‐helix; accordingly, representative mutants (K17A, R53A) blocked the Kv1.1 current with the same potency as the natural toxin. In contrast, competition of the binding of the radiolabelled αDTX or DTX k was dramatically reduced by alanine substitution of K26 or W25 in the β‐turn whereas changing nearby residues caused negligible alterations. Consistently, W25A and K26A exhibited diminished functional blockade of the Kv1.1 homo‐oligomer. The 3 10 ‐helical N‐terminal region of DTX k was found to be responsible for recognition of Kv1.1 channels because mutation of K3A led to ≈1246‐fold reduction in the inhibitory potency for [ 125 I]DTX k binding and a large decrease in its ability to block the Kv1.1 current; the effect of this substitution on the affinity of DTX k for Kv1.2‐possessing oligomers was much less dramatic (≈16‐fold).