Netolicky Jakub, Zahumenska Petra, Misiachna Anna, Kolcheva Marharyta, Rehakova Kristyna, Hemelikova Katarina, Kortus Stepan, Langore Emily, Doderovic Jovana, Ladislav Marek, Korabecny Jan, Otyepka Michal, Srejber Martin, Horak Martin
Department of Neurochemistry, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague 14220, Czech Republic.
Department of Physiology, Faculty of Science, Charles University in Prague, Prague 12843, Czech Republic.
J Neurosci. 2025 Jun 25;45(26):e0226242025. doi: 10.1523/JNEUROSCI.0226-24.2025.
-Methyl-d-aspartate receptors (NMDARs) play a crucial role in excitatory neurotransmission, with numerous pathogenic variants identified in the GluN subunits, including their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs, but this was tested in a limited number of studies. Using microscopy and electrophysiology in the human embryonic kidney 293 (HEK293) cells, we found that surface expression of GluN1/GluN2A receptors containing a set of alanine substitutions within the LBDs correlated with the measured EC values for glycine (GluN1 subunit mutations) while not correlating with the measured EC values for l-glutamate (GluN2A subunit mutations). The mutant cycle of GluN1-S688 residue, including the pathogenic GluN1-S688Y and GluN1-S688P variants, showed a correlation between relative surface expression of the GluN1/GluN2A receptors and the measured EC values for glycine, as well as with the calculated Δ values for glycine obtained from molecular dynamics simulations. In contrast, the mutant cycle of GluN2A-S511 residue did not show any correlation between the relative surface expression of the GluN1/GluN2A receptors and the measured EC values for l-glutamate or calculated Δ values for l-glutamate. Coexpression of both mutated GluN1 and GluN2A subunits led to additive or synergistic alterations in the surface number of GluN1/GluN2A receptors. The synchronized ER release by ARIAD technology confirmed the altered early trafficking of GluN1/GluN2A receptors containing the mutated LBDs. The microscopical analysis from embryonal rat hippocampal neurons (both sexes) corroborated our conclusions from the HEK293 cells.
N-甲基-D-天冬氨酸受体（NMDARs）在兴奋性神经传递中起关键作用，在GluN亚基中鉴定出许多致病变体，包括它们的配体结合域（LBDs）。普遍的假设认为内质网（ER）质量控制机制会验证NMDARs的激动剂占有率，但这仅在有限的研究中得到检验。通过在人胚肾293（HEK293）细胞中使用显微镜和电生理学方法，我们发现，在LBDs内含有一组丙氨酸替代的GluN1/GluN2A受体的表面表达与甘氨酸的测量EC值（GluN1亚基突变）相关，而与L-谷氨酸的测量EC值（GluN2A亚基突变）不相关。GluN1-S688残基的突变循环，包括致病性的GluN1-S688Y和GluN1-S688P变体，显示GluN1/GluN2A受体的相对表面表达与甘氨酸的测量EC值之间存在相关性，也与从分子动力学模拟获得的甘氨酸的计算Δ值相关。相比之下，GluN2A-S511残基的突变循环在GluN1/GluN2A受体的相对表面表达与L-谷氨酸的测量EC值或L-谷氨酸的计算Δ值之间未显示任何相关性。两种突变的GluN1和GluN2A亚基的共表达导致GluN1/GluN2A受体表面数量的加性或协同性改变。ARIAD技术的同步内质网释放证实了含有突变LBDs的GluN1/GluN2A受体早期运输的改变。来自胚胎大鼠海马神经元（雌雄均有）的显微镜分析证实了我们从HEK293细胞得出的结论。
