Electrostatic Interactions in the Transduction Pathway Alter NMDAR Gating

哺乳动物中枢神经系统中大多数兴奋性突触传递是由神经递质谷氨酸介导的。谷氨酸能信号对于细胞间的快速传递、正常的大脑发育以及学习和记忆至关重要。功能失调的谷氨酸能信号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（NMDARs）是谷氨酸激活离子通道（iGluRs），是这种快速信号传导的组成部分。NMDARs的一个关键功能特征是门控——配体结合/解结合导致孔打开/关闭的过程。门控涉及配体诱导的配体结合域的构象变化，这些构象变化被转移到形成孔的跨膜域，增加通道打开的可能性。这种门控过程是药理干预的一个有希望的目标。我将讨论一个新的假设，即连接配体结合域和跨膜域（特别是M3-S2和S2-M4）的连接体中的静电相互作用强烈影响NMDARs中的门接能量学。在GluN2A亚基中，M3-S2和S2-M4连接体有许多带电残基，我有初步的数据表明这些连接体是近端的，如果电荷发生突变，门控动力学会发生显著改变。Aim 1将重点关注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，免疫印迹和取代半胱氨酸来确定这些带电残基如何相互作用以调节门控能量学。在目标2中，我将探讨连接中的静电相互作用如何有助于亚基特异性门控机制。GluN2和GluN3亚基赋予NMDARs不同的门控特性，并定义了NMDARs如何在天然突触中起作用。我假设亚基内连接剂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可能是亚基特异性的。总的来说，从这些研究中获得的知识将为NMDAR门控提供重要的见解，并为亚型和亚基特异性的潜在药物干预位点开辟途径。

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.