Mechanism of action of novel subunit-selective NMDA receptor modulators

描述（由申请人提供）：N-甲基-D-天冬氨酸选择性谷氨酸受体（NMDA受体）是介导脑和脊髓中兴奋性突触传递的配体门控离子通道。NMDA受体是由两个结合甘氨酸的NR 1亚基和两个结合谷氨酸的NR 2亚基组成的异多聚体，其中有四个家族成员（NR 2A、B、C、D）。NMDA受体参与许多正常脑功能，如神经元发育、学习、记忆，以及它们在神经病理学病症如中风、癫痫和神经精神障碍中的作用。自首次描述以来，NR 2B选择性NMDA受体拮抗剂艾芬地尔及其许多类似物已用于大量研究，探索该亚基在脑功能的几乎每个方面的作用，包括行为，认知，突触可塑性，神经元发育和神经病理学。然而，对于含NR 2A-、NR 2C-或NR 2D-受体，还没有描述高选择性拮抗剂。因此，我们开发了一种测定法来鉴定含有NR 2C和NR 2D的NMDA受体的非竞争性变构调节剂，随后筛选了约60，000种化合物用于新的亚基选择性拮抗剂和增效剂。结果鉴定出两类结构独特的化合物，与含NR 2A或NR 2B的受体或AMPA/红藻氨酸受体相比，它们是NR 1/NR 2C和NR 1/NR 2D的100-500倍选择性抑制剂。我们还确定了两种结构上不同的增效剂，它们对含NR 2C/D的受体具有选择性。这些新的非竞争性变构调节剂代表了研究NR 2C/D亚基在正常脑功能和神经系统疾病中的作用的突破性机会。该建议使用这些亚基选择性调节剂解决3个问题。1. NR 2C/D选择性抑制剂和增效剂的结构决定因素是什么？我们将利用定点诱变，利用NR 2A/B和NR 2C/D之间的序列差异，以确定介导三类NR 2C/D选择性增效剂和抑制剂作用的关键残基。2. NR 2C/D选择性抑制剂和增效剂的作用机制是什么？我们将分析电压钳下记录的宏观和单通道电流，以确定非竞争性NR 2C/NR 2D抑制剂以及NR 2C/NR 2D增效剂的作用机制。3. NR 2C/D调节剂如何改变突触信号和神经元兴奋性？我们将在脑片中评估皮质-底丘脑神经元突触处含NR 2D的NMDA受体和传入兴奋性突触处含NR 2C/D的NMDA受体对海马中间神经元的抑制和增强作用。实验将测试在NR 2C/D调节剂存在下对刺激串的突触反应。我们还将使用这些新的药理学工具来研究是否可以通过抑制或增强含NR 2C/D的受体来改变中间神经元和底丘脑神经元的兴奋性和尖峰频率。 公共卫生相关性：NMDA受体介导中枢神经系统中神经元之间的通信，因此在几乎所有的脑功能以及许多神经系统疾病中发挥重要作用。NMDA受体是由两个结合甘氨酸的NR 1亚基和两个结合谷氨酸的NR 2亚基组成的四聚体，其中有四种亚型（NR 2A-D）。由于不同的NR 2亚基差异表达，并具有不同的功能特性，它们在正常脑功能和疾病中发挥着不同的作用。虽然亚基选择性调节剂已被提出是有用的治疗剂在许多神经系统疾病，只有一个真正的选择性NR 2亚基选择性拮抗剂已被发现。艾芬地尔及其类似物对含NR 2B受体的抑制作用比含NR 2A、C、D的NMDA受体强500倍以上。自1993年首次描述艾芬地尔的NR 2B-选择性以来，没有描述新的高选择性亚基特异性化合物。药理学试剂的缺乏导致我们寻找作用于含NR 2C和NR 2D受体的新配体。我们已经发现了至少三类NMDA受体功能的拮抗剂，当与含NR 2A或NR 2B的受体相比时，它们对含NR 2C和NR 2D的受体的效力高100-500倍。目前的资助提出了实验，以确定这些新化合物在NR 2D亚基上的作用，它们如何控制NMDA受体功能，以及它们对突触传递和神经元兴奋性的影响。这些信息将提供深入了解受体功能，并首次定义NR 2C和NR 2D亚基在突触传递中的作用。

DESCRIPTION (provided by applicant): N-methyl-D-aspartate-selective glutamate receptors (NMDA receptors) are ligand-gated ion channels that mediate excitatory synaptic transmission in the brain and spinal cord. NMDA receptors are heteromultimers comprised of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four family members (NR2A,B,C,D). NMDA receptors are involved in many normal brain functions such as neuronal development, learning, memory, in addition to their roles in neuropathological conditions such as stroke, epilepsy, and neuropsychiatric disorders. Since its first description, the NR2B-selective NMDA receptor antagonist ifenprodil and its many analogues have been used in a multitude of studies exploring the role of this subunit in virtually every aspect of brain function including behavior, cognition, synaptic plasticity, neuronal development, and neuropathology. However, no highly selective antagonists have yet been described for NR2A-, NR2C-, or NR2D-containing receptors. We therefore developed an assay to identify non-competitive allosteric modulators of NR2C- and NR2D-containing NMDA receptors, and subsequently screened ~60,000 compounds for new subunit-selective antagonists and potentiators. The result was the identification of two structurally unique classes of compounds that are 100-500 fold selective inhibitors of NR1/NR2C and NR1/NR2D compared to NR2A- or NR2B-containing receptors or AMPA/kainate receptors. We also identified two structurally distinct classes of potentiators that are selective for NR2C/D-containing receptors. These new non-competitive allosteric modulators represent a breakthrough opportunity to study the role of the NR2C/D subunits in normal brain function and in neurological diseases. This proposal addresses 3 questions using these subunit-selective modulators. 1. What are the structural determinants of NR2C/D-selective inhibitors and potentiators? We will utilize site-directed mutagenesis, exploiting sequence differences between NR2A/B and NR2C/D, to identify key residues that mediate the actions of three classes of NR2C/D-selective potentiators and inhibitors. 2. What is the mechanism of action of NR2C/D selective inhibitors and potentiators? We will analyze macroscopic and single channel currents recorded under voltage clamp to define the mechanism of action of the non-competitive NR2C/NR2D inhibitors as well as NR2C/NR2D potentiators. 3. How do NR2C/D modulators alter synaptic signaling and neuronal excitability? We will evaluate the effect of inhibition and potentiation of NR2D-containing NMDA receptors at the cortical-subthalamic neuron synapse and NR2C/D-containing NMDA receptors at afferent excitatory synapses onto hippocampal interneurons in brain slices. Experiments will test the synaptic response to stimulus trains in the presence of NR2C/D modulators. We will also use these new pharmacological tools to investigate whether interneuron and subthalamic neuron excitability and spiking frequency can be altered through inhibition or potentiation of NR2C/D-containing receptors. PUBLIC HEALTH RELEVANCE: NMDA receptors mediate communication between neurons in the central nervous system, and thus play an important role in virtually all brain functions as well as numerous neurological diseases. The NMDA receptors are tetrameric assemblies comprised of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four subtypes (NR2A-D). Because different NR2 subunits are differentially expressed and have different functional properties, they serve a host of divergent roles in both normal brain function and disease. Although subunit-selective modulators have been proposed to be useful therapeutic agents in a number of neurological diseases, only one truly selective NR2 subunit-selective antagonist has been discovered. Ifenprodil and its analogues inhibit NR2B-containing receptors over 500-fold more potently than NR2A,C,D-containing NMDA receptors. Since the first description of the NR2B-selectivity of ifenprodil in 1993, no new highly selective subunit- specific compounds have been described. This lack of pharmacological reagents has led us to search for new ligands that act at NR2C- and NR2D-containing receptors. We have found at least three classes of antagonists of NMDA receptor function that are 100-500 fold more potent at NR2C- and NR2D-containing receptors when compared to NR2A- or NR2B-containing receptors. The current grant proposes experiments that define where on the NR2D subunit these novel compounds act, how they control NMDA receptor function, and what effect they have on synaptic transmission and neuronal excitability. This information will provide insight into receptor function, and define for the first time the role of the NR2C and NR2D subunits in synaptic transmission.