Cross-Modulation and Molecular Interaction at the Cav3.3 Protein between the Endogenous Lipids and the T-Type Calcium Channel Antagonist TTA-A2.
Cross-Modulation and Molecular Interaction at the Cav3.3 Protein between the Endogenous Lipids and the T-Type Calcium Channel Antagonist TTA-A2.
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DOI：
10.1124/mol.113.089581
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发表时间：
2014-02-01
影响因子：
3.6
通讯作者：
Chemin, Jean
中科院分区：
文献类型：
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作者：
Cazade, Magali;Nuss, Cindy E.;Chemin, Jean
T型钙通道（T/Ca(v)3通道）参与多种生理和病理生理过程，如癫痫、睡眠障碍、高血压和癌症。t通道是内源性信号脂质的靶标，包括内源性大麻素、ω - 3脂肪酸和脂胺酸。然而，这些分子抑制t电流的确切分子机制尚不清楚。在这项研究中，我们提供了详细的电生理和药理学分析，表明主要的n-酰基衍生物对Ca(v)3.3电流的影响与合成t通道抑制剂TTA-A2 [(R)-2-（4-环丙基苯基）- n-（1-（5-（2,2,2-三氟乙氧基）吡啶-2-基）乙基）乙酰胺]有许多相似之处。通过与TTA-A2衍生物[H-3] TTA-A1 [(R)-2-（4-（叔丁基）苯基）- n-（1（5-甲氧基吡啶-2-基）乙基）乙酰胺的放射性结合试验，我们证明了抑制Ca(v)3.3电流的多不饱和脂质为NAGly （n-花生四烯酰基甘氨酸）、NASer （n-花生四烯酰基l丝氨酸）、anandamide、NADA （n-花生四烯酰基多巴胺）、NATau （n-花生四烯酰基牛磺酸）和NA-5HT （n-花生四烯酰基血清素）。所有移位的[3H] TTA-A1结合到由表达Ca(v)3.3的细胞制备的膜上，K-i在微摩尔或亚微摩尔范围内。相比之下，具有饱和烷基链的脂质，如n -花生四烯酰甘氨酸和n -花生四烯酰乙醇胺，对Ca(v)3.3电流没有抑制作用，对[3H] TTA-A1结合没有影响。因此，生物活性脂类阻断了TTA-A2对Ca(v)3.3电流的影响。此外，TTA-Q4 [(S)-4-（6-氯-4-环丙基-3-（2,2-二氟乙基）-2oxo- 1,2,3,4-四氢喹唑啉-4-基）苯腈]是一种[3H] TTA-A1结合和TTA-A2功能抑制的正变构调节剂，协同作用增强脂质诱导的Cav3.3电流抑制。总之，我们的研究结果证明了合成t通道抑制剂和内源性脂质的共同分子机制，并表明TTA-A2和TTA-Q4可能是解析t电流参与内源性脂质的生理效应的重要药理学工具。
T-type calcium channels (T/Ca(v)3-channels) are implicated in various physiologic and pathophysiologic processes such as epilepsy, sleep disorders, hypertension, and cancer. T-channels are the target of endogenous signaling lipids including the endocannabinoid anandamide, the omega 3-fatty acids, and the lipoamino-acids. However, the precise molecular mechanism by which these molecules inhibit T-current is unknown. In this study, we provided a detailed electrophysiologic and pharmacologic analysis indicating that the effects of the major N-acyl derivatives on the Ca(v)3.3 current share many similarities with those of TTA-A2 [(R)-2-(4-cyclopropylphenyl)-N-(1-(5-(2,2,2-trifluoroethoxy) pyridin-2-yl) ethyl) acetamide], a synthetic T-channel inhibitor. Using radioactive binding assays with the TTA-A2 derivative [H-3] TTA-A1 [(R)-2-(4-(tert-butyl) phenyl)-N-(1( 5-methoxypyridin-2-yl) ethyl) acetamide], we demonstrated that polyunsaturated lipids, which inhibit the Ca(v)3.3 current, as NAGly (N-arachidonoyl glycine), NASer (N-arachidonoyl-Lserine), anandamide, NADA (N-arachidonoyl dopamine), NATau (N-arachidonoyl taurine), and NA-5HT (N-arachidonoyl serotonin), all displaced [3H] TTA-A1 binding to membranes prepared from cells expressing Ca(v)3.3, with K-i in a micromolar or submicromolar range. In contrast, lipids with a saturated alkyl chain, as N-arachidoyl glycine and N-arachidoyl ethanolamine, which did not inhibit the Ca(v)3.3 current, had no effect on [3H] TTA-A1 binding. Accordingly, bio-active lipids occluded TTA-A2 effect on Ca(v)3.3 current. In addition, TTA-Q4 [(S)-4-(6-chloro-4-cyclopropyl-3-(2,2-difluoroethyl)-2oxo- 1,2,3,4-tetrahydroquinazolin-4-yl) benzonitrile], a positive allosteric modulator of [3H] TTA-A1 binding and TTA-A2 functional inhibition, acted in a synergistic manner to increase lipid-induced inhibition of the Cav3.3 current. Overall, our results demonstrate a common molecular mechanism for the synthetic T-channel inhibitors and the endogenous lipids, and indicate that TTA-A2 and TTA-Q4 could be important pharmacologic tools to dissect the involvement of T-current in the physiologic effects of endogenous lipids.