Direct and acute effects of human monoclonal anti-GluN1 autoantibodies on NMDA receptor channel function

NMDA受体（NMDAR）GluN1亚基氨基末端结构域（ATD）的特异性抗体可诱导NMDAR脑炎症状。已经描述了NMDAR抗体对受体内化的长期作用，但尚未探索GluN1 ATD抗体对NMDAR通道动力学和调节的直接和急性作用。在过去的资助期间，我们发现一些患者来源的单克隆人IgG自身抗体对GluN1亚基下调离子通道活性和突触NMDAR电流，而人Fab片段不影响离子通道活性。此外，结构分析表明，不同的单克隆抗体可能会覆盖不同的非重叠表位的GluN1 ATD。在这里，我们继续研究可能导致疾病相关受体和神经元功能障碍的特异性人NMDAR抗体的直接作用。我们将通过细胞贴附的单通道记录来阐明NMDAR通道动力学的通道开放概率和精细结构。我们将在转染的HEK细胞和神经元中测试不同的患者来源的和遗传修饰的重组单克隆IgG抗体和Fab，以区分交联和对NMDAR通道功能的内在影响。此外，我们将测试GluN1 IgG介导的对NMDAR配体、pH和Zn2+依赖性调节以及对NMDAR纳米结构域与大电导钙和电压激活的K+（BK）通道偶联的影响。将评价非功能性GluN1 IgG抗体和Fab片段作为可能的治疗选择。

Specific antibodies to the NMDA receptor (NMDAR) GluN1 subunit aminoterminal domain (ATD) induce symptoms of NMDAR encephalitis. Long-term effects of NMDAR antibodies on receptor internalization have been described but direct and acute effects of antibodies to the GluN1 ATD on NMDAR channel kinetics and modulation are unexplored. In the past funding period, we found that some patient-derived monoclonal human IgG autoantibodies against the GluN1 subunit downregulate ion channel activity and synaptic NMDAR currents whereas human Fab fragments do not affect ion channel activity. Furthermore, structural analyses revealed that different monoclonal antibodies may cover distinct non-overlapping epitopes on the GluN1 ATD. Here, we continue on investigating direct effects of specific human NMDAR antibodies possibly contributing to disease-relevant receptor and neuronal dysfunction. We will unravel effects channel open probabilities and fine structure of NMDAR channel kinetics by cell-attached single channel recordings. We will test different patient-derived and genetically modified recombinant monoclonal IgG antibodies and Fabs in transfected HEK cells and neurons to differentiate between crosslinking and intrinsic effects on NMDAR channel function. Furthermore, we will test GluN1 IgG-mediated effects on NMDAR ligand, pH, and Zn2+ dependent modulation and on NMDAR nanodomain coupling with large-conductance calcium- and voltage-activated K+ (BK) channels. Non-functional GluN1 IgG antibodies and Fab fragments will be evaluated as possible therapeutic options.