Electrostatic Interactions in the Transduction Pathway Alter NMDAR Gating

传导通路中的静电相互作用改变 NMDAR 门控

• 批准号: 8255045
• 负责人: Rashek Kazi
• 金额: $ 4.67万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8255045
• 关键词: Acute; Address; Adverse effects; Affect; Alzheimer' s Disease; Brain; Cells; Charge; Chronic; Complex; Cysteine; Data; Development; Disease; Electrostatics; Gated Ion Channel; Glutamate Receptor; Glutamates; Goals; Immunoblot Analysis; Intervention; Ion Channel; Kinetics; Knowledge; Lead; Learning; Ligand Binding; Ligand Binding Domain; Ligands; Mediating; Memory; Mental disorders; Mutate; N-Methyl-D-Aspartate Receptors; Nerve Degeneration; Neuraxis; Neurotransmitters; Parkinson Disease; Pathology; Pathway interactions; Perception; Process; Property; Relative (related person); Role; Schizophrenia; Seizures; Signal Transduction; Site; Stroke; Synapses; Synaptic Transmission; System; Testing; Transmembrane Domain; Variant; base; defined contribution; expectation; insight; mutant; nervous system disorder; novel; receptor; receptor function; research study; transmission process

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.

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。谷氨酸信号对于细胞间的快速传输、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不可或缺的一部分。 NMDAR 的一个关键功能特征是门控——配体结合/解除结合导致孔打开/关闭的过程。门控涉及配体诱导的配体结合域构象变化，这些构象变化被转移到孔形成跨膜域，增加通道打开的可能性。该门控过程是药物干预的一个有希望的目标。我将提出一个新的假设，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 NMDAR 中的门控能量学。在 GluN2A 亚基中，M3-S2 和 S2-M4 连接子具有许多带电残基，我的初步数据表明这些连接子是近端的，并且如果电荷突变，门控动力学会显着改变。目标 1 将重点关注 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、免疫印迹和取代的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，我将探讨连接子中的静电相互作用如何促进亚基特异性门控机制。 GluN2 和 GluN3 亚基赋予 NMDAR 不同的门控特性，并定义 NMDAR 在天然突触中的功能。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 和单通道记录中获得的见解来测试连接子中的静电相互作用如何是亚基特异性的。总体而言，从这些研究中获得的知识将为 NMDAR 门控提供重要的见解，并为亚型和亚基特异性的潜在药理干预位点开辟途径。