A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors.
A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors.
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DOI：
10.1097/j.pain.0000000000000147
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发表时间：
2015-07
期刊：
影响因子：
7.4
通讯作者：
Khanna R
中科院分区：
文献类型：
--
作者：
François-Moutal L;Wang Y;Moutal A;Cottier KE;Melemedjian OK;Yang X;Wang Y;Ju W;Largent-Milnes TM;Khanna M;Vanderah TW;Khanna R
靶向N型电压门控钙通道（CaV2.2）复合物内的蛋白质已被证明是开发新型疼痛治疗剂的有效策略。我们描述了一种新的肽适体来自于钙调素反应介体蛋白2（CRMP 2），CaV2.2调节蛋白。在肽（myr-tat-CBD 3）中添加14碳肉豆蔻酸酯基团将其拴系到靠近表面CaV2.2的初级感觉神经元的膜上。Pull-down研究表明myr-tat-CBD 3肽干扰CRMP 2-CaV2.2相互作用。定量共聚焦免疫荧光显示，与肽tat-CBD 3相比，myr-tat-CBD 3处理后CaV 2.2运输显著减少，并且破坏CRMP 2-CaV 2.2共定位的效率增加。因此，myr-tat-CBD 3抑制感觉神经元中去极化诱导的钙内流。电压钳电生理学实验显示，myr-tat-CBD 3暴露后感觉神经元中的Ca 2+电流减少，但Na+电流没有减少。电流钳电生理学实验表明，暴露于myr-tat-CBD 3后，小直径背根神经节神经元的兴奋性降低。Myr-tat-CBD 3在显著减弱角叉菜胶诱导的热超敏反应和逆转由大鼠后爪的足底表面的手术切口（手术后疼痛模型）诱导的热超敏反应中有效。将这些作用与tat-CBD 3-非豆蔻酰化的tat缀合的CRMP 2肽以及CBD 3和缺乏CBD 3的对照肽的乱序形式的那些作用进行比较。我们的结果表明，肉豆蔻酰标签增强了CRMP 2肽适体在膜界定的钙通道附近的细胞内递送和局部浓度，导致对钙通道复合物的显著干扰、对钙内流的上级抑制和更好的抗伤害感受潜力。
Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2–CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca2+, but not Na+, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3—the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.
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