Wang F C, Bell N, Reid P, Smith L A, McIntosh P, Robertson B, Dolly J O
Department of Biochemistry, Imperial College, London, UK.
Eur J Biochem. 1999 Jul;263(1):222-9. doi: 10.1046/j.1432-1327.1999.00494.x.
Dendrotoxin (DTX) homologues are powerful blockers of K+ channels that contain certain subfamily Kv1 (1.1-1.6) alpha- and beta-subunits, in (alpha)4(beta)4 stoichiometry. DTXk inhibits potently Kv1.1-containing channels only, whereas alphaDTX is less discriminating, but exhibits highest affinity for Kv1.2. Herein, the nature of interactions of DTXk 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 310-helix, beta-turn, alpha-helix and random-coil regions. The mutants' antagonism of high-affinity [125I]DTXk 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 [125I]alphaDTX binding to brain membranes by the DTXk 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 alpha-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 alphaDTX or DTXk was dramatically reduced by alanine substitution of K26 or W25 in the beta-turn whereas changing nearby residues caused negligible alterations. Consistently, W25A and K26A exhibited diminished functional blockade of the Kv1.1 homo-oligomer. The 310-helical N-terminal region of DTXk was found to be responsible for recognition of Kv1.1 channels because mutation of K3A led to approximately 1246-fold reduction in the inhibitory potency for [125I]DTXk binding and a large decrease in its ability to block the Kv1.1 current; the effect of this substitution on the affinity of DTXk for Kv1.2-possessing oligomers was much less dramatic (approximately 16-fold).
树突毒素（DTX）同系物是钾离子通道的强效阻滞剂，这些通道包含特定的Kv1亚家族（1.1 - 1.6）的α和β亚基，其化学计量比为（α）4（β）4。DTXk仅能有效抑制包含Kv1.1的通道，而αDTX的选择性较低，但对Kv1.2表现出最高亲和力。在此，利用15个定点突变体研究了DTXk与由Kv1.1和1.2（以及其他）亚基组成的天然钾离子通道之间相互作用的性质，这些突变体中310螺旋、β转角、α螺旋和无规卷曲区域的氨基酸发生了改变。对突变体拮抗高亲和力[125I]DTXk与大鼠脑膜中含有Kv1.1的通道结合以及阻断卵母细胞中表达的Kv1.1电流的情况进行了定量分析。此外，DTXk突变体对[125I]αDTX与脑膜结合的抑制水平分别用于测量它们与以Kv1.1为主要或次要成分的含神经元Kv1.2通道的高亲和力和低亲和力相互作用。用丙氨酸单取代无规卷曲区域的三个碱性残基或α螺旋中的R52或R53，均不会改变毒素与这两种亚型中任何一种的结合置换情况；因此，代表性突变体（K17A、R53A）阻断Kv1.1电流的效力与天然毒素相同。相比之下，β转角中K26或W25被丙氨酸取代会显著降低放射性标记的αDTX或DTXk的结合竞争，而改变附近残基则只会引起微不足道的变化。同样，W25A和K26A对Kv1.1同型寡聚体的功能阻断作用减弱。发现DTXk的310螺旋N端区域负责识别Kv1.1通道，因为K3A突变导致[125I]DTXk结合的抑制效力降低约1246倍，并且其阻断Kv1.1电流的能力大幅下降；该取代对DTXk与含有Kv1.2的寡聚体亲和力的影响则小得多（约16倍）。
