Electrostatic Interactions in the Transduction Pathway Alter NMDAR Gating

哺乳动物中枢神经系统中的大多数兴奋性突触传递是由神经递质谷氨酸介导的。谷氨酸能信号对快速细胞间传递、正常大脑发育以及学习和记忆至关重要。功能障碍性神经元能信号传导与许多急性和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），是这种快速信号传导的组成部分。NMDAR的一个关键功能特征是门控-配体结合/解结合导致孔打开/关闭的过程。门控涉及配体结合结构域中的配体诱导的构象变化，其被转移到孔形成跨膜结构域，增加通道开放的可能性。该门控过程是药理学干预的有前景的靶点。我将解决新的假设，连接配体结合结构域的跨膜结构域（特别是，M3-S2和S2-M4）的连接器中的静电相互作用强烈影响NMDAR门控的能量学。在GluN 2A亚基中，M3-S2和S2-M4连接体具有许多带电残基，我有初步数据表明，这些连接体是近端的，如果电荷突变，门控动力学会显著改变。目标1将集中于M3-S2和S2- M4连接体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，和取代的半胱氨酸，以确定这些带电残基如何相互作用，以调节门控能量。在目标2中，我将探讨如何在连接静电相互作用可能有助于亚基特异性门控机制。GluN 2和GluN 3亚基赋予NMDAR不同的门控特性，并定义NMDAR如何在天然突触中发挥作用。我假设亚基内连接器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和单通道记录中获得的见解来测试连接器中的静电相互作用如何具有亚基特异性。总体而言，从这些研究中获得的知识将为NMDAR门控提供重要见解，并为亚型和亚基特异性的潜在药理学干预部位开辟途径。

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.