Institute of Experimental Medicine of the Czech Academy of Sciences, Videnska 1083, 14220, Prague 4, Czech Republic; Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220, Prague 4, Czech Republic.
Institute of Experimental Medicine of the Czech Academy of Sciences, Videnska 1083, 14220, Prague 4, Czech Republic; Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220, Prague 4, Czech Republic; Department of Physiology, Faculty of Science, Charles University in Prague, Albertov 6, 12843, Prague 2, Czech Republic.
Neuropharmacology. 2019 Oct;157:107671. doi: 10.1016/j.neuropharm.2019.107671. Epub 2019 Jun 13.
N-methyl-d-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission within the mammalian central nervous system (CNS). NMDARs are heteromultimers containing GluN1, GluN2, and/or GluN3 subunits, thus giving rise to a wide variety of subunit combinations, each with unique functional and pharmacological properties. Importantly, GluN1/GluN3A and GluN1/GluN3B receptors form glycine-gated receptors. Here, we combined electrophysiology with rapid solution exchange in order to determine whether the presence of specific N-glycans and/or interactions with specific lectins regulates the functional properties of GluN1/GluN3A and GluN1/GluN3B receptors expressed in human embryonic kidney 293 (HEK293) cells. We found that removing putative N-glycosylation sites alters the functional properties of GluN1/GluN3B receptors, but has no effect on GluN1/GluN3A receptors. Moreover, we found that the functional properties of both GluN1/GluN3A and GluN1/GluN3B receptors are modulated by a variety of lectins, including Concanavalin A (ConA), Wheat Germ Agglutinin (WGA), and Aleuria Aurantia Lectin (AAL), and this effect is likely mediated by a reduction in GluN1 subunit-mediated desensitization. We also found that AAL has the most profound effect on GluN1/GluN3 receptors, and this effect is mediated partly by a single N-glycosylation site on the GluN3 subunit (specifically, N565 on GluN3A and N465 on GluN3B). Finally, we found that lectins mediate their effect only when applied to non-activated receptors and have no effect when applied in the continuous presence of glycine. These findings provide further evidence to distinguish GluN1/GluN3 receptors from the canonical GluN1/GluN2 receptors and offer insight into how GluN1/GluN3 receptors may be regulated in the mammalian CNS.
N-甲基-D-天冬氨酸受体（NMDARs）在哺乳动物中枢神经系统（CNS）的兴奋性神经传递中发挥着重要作用。NMDAR 是由 GluN1、GluN2 和/或 GluN3 亚基组成的异源多聚体，从而产生各种亚基组合，每种组合都具有独特的功能和药理学特性。重要的是，GluN1/GluN3A 和 GluN1/GluN3B 受体形成甘氨酸门控受体。在这里，我们结合了电生理学和快速溶液交换，以确定特定的 N-糖基化位点的存在和/或与特定凝集素的相互作用是否调节在人胚肾 293（HEK293）细胞中表达的 GluN1/GluN3A 和 GluN1/GluN3B 受体的功能特性。我们发现，去除假定的 N-糖基化位点会改变 GluN1/GluN3B 受体的功能特性，但对 GluN1/GluN3A 受体没有影响。此外，我们发现，多种凝集素，包括刀豆球蛋白 A（ConA）、麦胚凝集素（WGA）和金耳凝集素（AAL），都可以调节 GluN1/GluN3A 和 GluN1/GluN3B 受体的功能特性，这种作用可能是通过减少 GluN1 亚基介导的脱敏来介导的。我们还发现，AAL 对 GluN1/GluN3 受体的影响最为显著，这种作用部分是由 GluN3 亚基上的单个 N-糖基化位点（具体为 GluN3A 上的 N565 和 GluN3B 上的 N465）介导的。最后，我们发现，凝集素只有在应用于非激活受体时才会发挥作用，而在持续存在甘氨酸的情况下应用则没有效果。这些发现为区分 GluN1/GluN3 受体与典型的 GluN1/GluN2 受体提供了进一步的证据，并深入了解哺乳动物中枢神经系统中 GluN1/GluN3 受体可能如何受到调节。
