Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, 211198, China.
Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, 211198, China.
Neuropharmacology. 2020 Dec 1;180:108291. doi: 10.1016/j.neuropharm.2020.108291. Epub 2020 Sep 12.
Voltage-gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in excitable cells and are the molecular targets of an array of neurotoxins. BmK NT1, an α-scorpion toxin obtained from the scorpion Buthus martensii Karsch (BmK), produces neurotoxicity that is associated with extracellular Ca influx through Na-Ca exchangers, N-methyl-d-aspartic acid (NMDA) receptors, and L-type Ca channels in cultured cerebellar granule cells (CGCs). In the present study, we demonstrated that BmK NT1 triggered concentration-dependent release of excitatory neurotransmitters, glutamate and aspartate; both effects were eliminated by VGSC blocker, tetrodotoxin. More importantly, we demonstrated that a threshold concentration of BmK NT1 that produced marginal Ca influx and neuronal death augmented glutamate-induced Ca elevation and neuronal death in CGCs. BmK NT1-augmented glutamate-induced Ca influx and neuronal death were suppressed by tetrodotoxin and MK-801 suggesting that the augmentation was through activation of VGSCs and NMDA receptors. Consistently, BmK NT1 also enhanced NMDA-induced Ca influx. Further mechanistic investigations demonstrated that BmK NT1 increased the expression level of NMDA receptors on the plasma membrane and increased the phosphorylation level of NR2B at Tyr1472. Src family kinase inhibitor, 1-tert-butyl-3-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidin-4-yl]amine (PP2), but not the inactive analogue, 4-amino-1-phenylpyrazolo[3,4-d]pyrimidine (PP3), eliminated BmK NT1-triggered NR2B phosphorylation, NMDA receptor trafficking, as well as BmK NT1-augmented NMDA Ca response and neuronal death. Considered together, these data demonstrated that both presynaptic (excitatory amino acid release) and postsynaptic mechanisms (augmentation of NMDA receptor function) are critical for VGSC activation-induced neurotoxicity in primary CGC cultures.
电压门控钠离子通道（VGSCs）负责产生和传播兴奋细胞中的动作电位，是一系列神经毒素的分子靶点。BmK NT1 是一种从蝎子 Buthus martensii Karsch（BmK）中获得的α-蝎毒素，它产生的神经毒性与通过 Na-Ca 交换器、N-甲基-D-天冬氨酸（NMDA）受体和 L 型钙通道的细胞外 Ca 内流有关在培养的小脑颗粒细胞（CGC）中。在本研究中，我们证明 BmK NT1 触发了兴奋性神经递质谷氨酸和天冬氨酸的浓度依赖性释放；这两种作用都被 VGSC 阻断剂河豚毒素消除。更重要的是，我们证明了产生微小 Ca 内流和神经元死亡的 BmK NT1 阈浓度增强了 CGC 中谷氨酸诱导的 Ca 升高和神经元死亡。河豚毒素和 MK-801 抑制了 BmK NT1 增强的谷氨酸诱导的 Ca 内流和神经元死亡，表明这种增强是通过 VGSCs 和 NMDA 受体的激活。一致地，BmK NT1 也增强了 NMDA 诱导的 Ca 内流。进一步的机制研究表明，BmK NT1 增加了质膜上 NMDA 受体的表达水平，并增加了 Tyr1472 处的 NR2B 磷酸化水平。Src 家族激酶抑制剂 1-叔丁基-3-(4-氯苯基)吡唑并[3,4-d]嘧啶-4-基]胺（PP2），而不是无活性类似物 4-氨基-1-苯基吡唑并[3,4-d]嘧啶（PP3），消除了 BmK NT1 触发的 NR2B 磷酸化、NMDA 受体转运以及 BmK NT1 增强的 NMDA Ca 反应和神经元死亡。综上所述，这些数据表明，在原代 CGC 培养物中，VGSC 激活诱导的神经毒性既需要突触前（兴奋性氨基酸释放）机制，也需要突触后（NMDA 受体功能增强）机制。
