Electrostatic Interactions in the Transduction Pathway Alter NMDAR Gating

哺乳动物中枢神经系统中的大部分兴奋性突触传递是由神经递质谷氨酸介导的。谷氨酸能信号对细胞间的快速传递、正常的大脑发育以及学习和记忆至关重要。谷氨酸能信号转导功能障碍与许多急、慢性神经系统疾病以及许多精神疾病有关。NMDA受体(NMDAR)是谷氨酸激活的离子通道(IGluR)，是这种快速信号的组成部分。NMDARs的一个关键功能特征是门控--配体结合/解结合的过程，导致孔的打开/关闭。门控涉及配体诱导的配基结合域的构象变化，这些构象变化转移到形成孔的跨膜域，增加通道开放的可能性。这种门控过程是药物干预的一个很有前途的靶点。我将解决这个新的假设，即连接配体结合域和跨膜域的连接体(特别是M3-S2和S2-M4)中的静电相互作用强烈影响NMDARs中的门控能量。在GluN2A亚基中，M3-S2和S2-M4连接子有许多带电残基，我有初步数据表明，这些连接子是近端的，如果电荷突变，门控动力学会显著改变。目标1将重点介绍M3-S2和S2-M4连接子中带电残基之间的静电相互作用如何影响门控的详细机制。我将使用单通道分析、免疫印迹和取代半胱氨酸来确定这些带电残基如何相互作用来调节门控能量。在目标2中，我将探索连接子中的静电相互作用如何有助于亚单位特异性的门控机制。GluN2和GluN3亚基赋予NMDAR不同的门控特性，并定义NMDAR在自然突触中的功能。我假设，亚基内连接子静电相互作用的差异是亚基特异性门控的潜在机制的一部分。我将利用从Aim 1和单通道录音中获得的见解来测试连接子中的静电相互作用可能是如何亚基特有的。总体而言，从这些研究中获得的知识将为NMDAR门控提供重要的洞察力，并为亚型和亚单位特异性的潜在药物干预部位开辟道路。

Most excitatory synaptic transmission in the mammalian central nervous system is mediated by the neurotransmitter glutamate. Glutamatergic signaling is critical to fast cell-to-cell transmission, normal brain development, and learning and memory. Dysfunctional glutamatergic signaling is implicated in numerous acute and chronic neurological diseases as well as many psychiatric disorders. NMDA receptors (NMDARs) are glutamate activated ion channels (iGluRs) that are integral to this fast signaling. A key functional feature of NMDARs is gating - the process of ligand binding/unbinding resulting in pore opening/closing. Gating involves ligand induced conformational changes in the ligand binding domain that are transferred to the pore forming transmembrane domain, increasing the likelihood of channel opening. This gating process is a promising target for pharmacological intervention. I will address the novel hypothesis that electrostatic interactions in the linkers connecting the ligand binding domain to the transmembrane domain (specifically, M3-S2 and S2-M4) strongly influence the energetics of gating in NMDARs. In GluN2A subunits, the M3-S2 and S2-M4 linkers have numerous charged residues and I have preliminary data suggesting that these linkers are proximal and that gating kinetics are significantly altered if charges are mutated. Aim 1 will focus on the detailed mechanisms of how electrostatic interactions between charged residues in the M3-S2 and S2- M4 linkers affect gating. I will use single channel analysis, immunoblots, and substituted cysteines to determine how these charged residues interact to modulate gating energetics. In Aim 2, I will explore how electrostatic interactions in the linkers may contribute to subunit- specific gating mechanisms. The GluN2 and GluN3 subunits confer distinct gating properties onto NMDARs and define how NMDARs function at native synapses. I hypothesize that differences in intrasubunit linker electrostatic interactions are part of the underlying mechanism of subunit-specific gating. I will take advantage of insights gained from Aim 1 and single-channel recordings to test how electrostatic interactions in the linkers might be subunit-specific. Overall, the knowledge gained from these studies will provide significant insight into NMDAR gating and open avenues for potential sites of pharmacological intervention that are both subtype and subunit specific.