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首页> 外文期刊>Biochemistry >125I)Iberiotoxin-D19Y/Y36F, the first selective, high specific activity radioligand for high-conductance calcium-activated potassium channels.
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125I)Iberiotoxin-D19Y/Y36F, the first selective, high specific activity radioligand for high-conductance calcium-activated potassium channels.

机译：125I）Iberiotoxin-D19Y / Y36F，第一个选择性，高比活度放射性配体，用于高电导钙激活钾通道。

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摘要

Iberiotoxin (IbTX), a selective peptidyl ligand for high-conductance Ca2(+)-activated K+ (maxi-K) channels cannot be radioiodinated in biologically active form due to the importance of Y36 in interacting with the channel pore. Therefore, an IbTX double mutant (IbTX-D19Y/Y36F) was engineered, expressed in Escherichia coli, purified to homogeneity, and radiolabeled to high specific activity with 125I. IbTX-D19Y/Y36F and [127I]IbTX-D19Y/Y36F block maxi-K channels expressed in Xenopus laevis oocytes with equal potency as wild-type IbTX (Kd approximately 1 nM). Under low ionic strength conditions, [125I]IbTX-D19Y/Y36F binds with high affinity to smooth muscle sarcolemmal maxi-K channels (Kd of 5 pM as determined by either equilibrium binding or kinetic binding analysis), and with a binding site density of 0.45 pmol/mg of protein. Competition studies with wild-type IbTX, IbTX-D19Y/Y36F or charybdotoxin (ChTX) result in complete inhibition of binding whereas toxins selective for voltage-gated K+ channels (margatoxin (MgTX) or alpha-dendrotoxin (alpha-DaTX) do not have any effect on IbTX binding. Indole diterpene alkaloids, which are selective inhibitors of maxi-K channels, and potassium ions both modulate [125I]IbTX-D19Y/Y36F binding in a complex manner. This pattern is also reflected during covalent incorporation of the radiolabeled toxin into the 31 kDa beta-subunit of the maxi-K channel in the presence of a bifunctional cross-linking reagent. In rat brain membranes, IbTX-D19Y/Y36F does not displace binding of [125I]MgTX or [125I]-alpha-DaTX to sites associated with voltage-gated K+ channels, nor do these latter toxins inhibit [125I]IbTX-D19Y/Y36F binding. Taken together, these results demonstrate that [125I]IbTX-D19Y/Y36F is the first selective radioligand for maxi-K channels with high specific activity.
机译：伊比利亚毒素（IbTX），高传导性Ca2（+）激活的K +（maxi-K）通道的选择性肽基配体，由于Y36在与通道孔相互作用中的重要性，因此无法以生物活性形式进行放射性碘标记。因此，工程改造了一个IbTX双突变体（IbTX-D19Y / Y36F），在大肠杆菌中表达，纯化至均一，并用125 I放射性标记为高比活。 IbTX-D19Y / Y36F和[127I] IbTX-D19Y / Y36F阻断非洲爪蟾卵母细胞中表达的maxi-K通道，其效力与野生型IbTX（Kd约1 nM）相同。在低离子强度条件下，[125I] IbTX-D19Y / Y36F与平滑肌肌膜最大k通道具有高亲和力（通过平衡结合或动力学结合分析确定，Kd为5 pM），且结合位点密度为0.45 pmol / mg的蛋白质。与野生型IbTX，IbTX-D19Y / Y36F或Charybdotoxin（ChTX）的竞争研究可完全抑制结合，而对电压门控K +通道具有选择性的毒素（玛格毒素（MgTX）或α-树突毒素（alpha-DaTX）吲哚二萜生物碱（maxi-K通道的选择性抑制剂）和钾离子均以复杂的方式调节[125I] IbTX-D19Y / Y36F结合，这种方式也反映在共价掺入放射性标记的过程中在双功能交联剂存在下，将毒素转化为maxi-K通道的31 kDaβ-亚基在大鼠脑膜中，IbTX-D19Y / Y36F不会取代[125I] MgTX或[125I]-α的结合-DaTX与与电压门控的K +通道相关的位点，这些毒素也不会抑制[125I] IbTX-D19Y / Y36F结合，总的来说，这些结果表明[125I] IbTX-D19Y / Y36F是第一个最大的选择性放射性配体-K通道具有较高的比活度。

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