PDF(Pubmed)
Abstract：
Excessive Ca2+ influx through N-methyl-D-aspartate type glutamate receptors (NMDAR) is associated with excitotoxicity and neuronal death, but the inhibition of this receptor-channel causes severe adverse effects. Thus, a selective reduction of NMDA-mediated Ca2+ entry, leaving unaltered the Na+ current, could represent a valid neuroprotective strategy. We developed a new two-fluorophore approach to efficiently assess the Ca2+ permeability of ligand-gated ion channels, including NMDARs, in different conditions. This technique was able to discriminate differential Ca2+/Na+ permeation ratio through different receptor channels, and through the same channel in different conditions. With this method, we confirmed that EU1794-4, a negative allosteric modulator of NMDARs, decreased their Ca2+ permeability. Furthermore, we measured for the first time the fractional Ca2+ current (Pf, i.e. the percentage of the total current carried by Ca2+ ions) of human NMDARs in the presence of EU1794-4, exhibiting a 40% reduction in comparison to control conditions. EU1794-4 was also able to reduce NMDA-mediated Ca2+ entry in human neurons derived from induced pluripotent stem cells. This last effect was stronger in the absence of extracellular Mg2+, but still significant in its presence, supporting the hypothesis to use NMDA-selective allosteric modulators to lower Ca2+ influx in human neurons, to prevent Ca2+-dependent excitotoxicity and consequent neurodegeneration.
摘要：
通过N-甲基-D-天冬氨酸型谷氨酸受体（NMDAR）过量的Ca2流入与兴奋性毒性和神经元死亡有关，但是这种受体通道的抑制会导致严重的不良反应。因此，选择性减少NMDA介导的Ca2+进入，保持Na+电流不变，可以代表一种有效的神经保护策略。我们开发了一种新的双荧光团方法来有效评估配体门控离子通道的Ca2+渗透性，包括NMDAR，在不同的条件下。该技术能够区分通过不同受体通道的不同Ca2/Na渗透比，在不同的条件下通过相同的通道。使用这种方法，我们证实了EU1794-4，NMDAR的负变构调节剂，降低了它们的Ca2+渗透性。此外，我们首次测量了分数Ca2+电流(Pf，即在EU1794-4的存在下，人NMDAR的Ca2离子）所携带的总电流的百分比，与对照条件相比表现出40％的减少。EU1794-4还能够减少源自诱导多能干细胞的人神经元中NMDA介导的Ca2+进入。在没有细胞外Mg2+的情况下，最后的作用更强，但在它的存在下仍然很重要，支持使用NMDA选择性变构调节剂降低人类神经元中Ca2流入的假设，防止Ca2+依赖性兴奋性毒性和随之而来的神经变性。
