GABA activation of the M-current

M 电流的 GABA 激活

• 批准号: 10084328
• 负责人: Geoffrey W Abbott
• 金额: $ 33.8万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10084328
• 关键词: Acids; Age; Anions; Anticonvulsants; Binding; Binding Sites; Brain; CRISPR/Cas technology; Cardiac; Charge; Chemistry; Chlorides; Cnidaria; Data; Dependence; Development; Disease; Electrophysiology (science); Epilepsy; Exhibits; Family; Fasting; GABA Receptor; Germ-Line Mutation; Glutamates; Goals; Ion Channel; Ion Channel Gating; Ions; Ketone Bodies; Ketosis; Knowledge; Ligands; Mediating; Membrane; Membrane Potentials; Modality; Molecular; Movement; Mus; Muscarinic Acetylcholine Receptor; Mutagenesis; Mutant Strains Mice; Mutation; Nervous system structure; Neurons; Neurotransmitters; Organism; Pharmaceutical Preparations; Phase; Phylogenetic Analysis; Physiological; Physiology; Positioning Attribute; Postsynaptic Membrane; Potassium; Predisposition; Preparation; Process; Protein Isoforms; Receptor Activation; Recording of previous events; Regulation; Role; Seizures; Signal Transduction; Site; Slice; Structure; Structure-Activity Relationship; Synapses; Synaptic Transmission; Testing; Therapeutic; Therapeutic Effect; Time; analog; base; beta-Hydroxybutyrate; design; extracellular; gabapentin; gamma-Aminobutyric Acid; in vivo; ketogenic diet; molecular targeted therapies; nervous system disorder; neuronal excitability; neurotransmission; novel; novel therapeutic intervention; preference; presynaptic; receptor; response; sensor; synergism; voltage

Pentameric ligand-gated ion channels (LGICs) are activated by neurotransmitter binding to highly specialized, inter-subunit, extracellular binding pockets. In contrast, voltage-gated potassium (Kv) channels are activated by membrane depolarization, electromechanically communicated by the voltage sensor to the pore module. Kv channels composed of KCNQ2/3 heteromers generate the neuronal M-current, a ubiquitous and essential hyperpolarizing K+ current controlling excitability in mammalian CNS. This proposal is based on our recent discovery that KCNQ2/3 channels are directly activated by γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in vertebrate CNS, with sensitivity comparable to that of the most sensitive α/β/γ GABAA receptor LGICs. In contrast, the excitatory neurotransmitter glutamate, which is structurally related to GABA, has no effect on KCNQ2/3 activity. We have identified the KCNQ2/3 GABA binding site as KCNQ3-W265, and find the position is highly conserved in deuterostome clades, present in some Cnidarians, but absent in protostomes; it is also absent from cardiac-expressed KCNQ1. In addition, we have found that GABA analogs and metabolites exhibit similar structure-activity relationships (SARs) for KCNQ2/3 channel activation and anticonvulsant activity. The metabolites include β-hydroxybutyrate (BHB), the primary ketone body produced in response to fasting or ketogenic diets, which protect against seizures. We find that BHB is a potent KCNQ2/3 activator and anticonvulsant, uncovering a molecular target for the therapeutic effects of ketosis. Our findings show that despite their wide structural disparity, GABA activates both principal classes of inhibitory ion channels in vertebrate neurons, creating a new paradigm for regulation of Kv channel gating and inhibitory neurotransmission. We propose three Specific Aims directed towards a fuller understanding of the mechanisms, breadth and scope underlying this novel signaling modality. In Aim 1 we will elucidate the molecular requirements for GABA and BHB regulation of KCNQ channels and when this capability evolved. In Aim 2 we will define the KCNQ binding sites of key GABA analogs and metabolites we recently discovered to also activate KCNQs, and leverage synergy between these compounds to develop optimized, potent anticonvulsants. In Aim 3, we will utilize newly CRISPR-Cas9 generated mice bearing germline mutations in the KCNQ3 & 5 GABA/BHB binding sites to determine the importance and KCNQ isoform-dependence of the anticonvulsant actions of BHB and the ketogenic diet. We will then use cellular electrophysiological analysis to quantify the age- and KCNQ-isoform dependence of GABA and BHB modulation of native M-current and neuronal excitability. The project will thoroughly define the fundamental aspects of a novel, unexpected form of inhibitory neuronal signaling, with the dual goals of understanding its role in brain physiology and harnessing the knowledge to help develop advanced, safer therapeutics for epilepsy and other neurological disorders.

五聚合配体门控离子通道（LGICS）被神经递质与高度专业化， 亚基间，细胞外结合口袋。相反，电压门控钾（KV）通道被激活 膜去极化，电压传感器通过机电通信到孔模块。 KV 由KCNQ2/3异构体组成的通道产生神经元M-电流，无处不在且必不可少的 哺乳动物中枢神经系统中的k+电流控制兴奋性过高。该提议基于我们的最新 发现KCNQ2/3通道被主要抑制性γ-氨基丁酸（GABA）直接激活 脊椎动物中枢神经系统中的神经递质，其灵敏度与最敏感的α/β/γGABAA相当 受体LGIC。相反，与GABA结构相关的兴奋性神经递质谷氨酸 对KCNQ2/3活性没有影响。我们已经将KCNQ2/3 GABA绑定位点确定为KCNQ3-W265，并且 在某些cnidarians中找到该位置在氘代表中高度保守，但在 原生物；心脏表达的KCNQ1也不存在。此外，我们发现GABA类似物 对于KCNQ2/3通道激活和 抗惊厥活性。这些代谢物包括β-羟基丁酸（BHB），这是在 对禁食或生酮饮食的反应，可防止癫痫发作。我们发现BHB是潜在的KCNQ2/3 激活剂和抗惊厥药，揭示了酮症治疗作用的分子靶标。我们的发现 表明尽管结构差异很大，但GABA激活了两个主要类别的抑制离子 脊椎动物神经元中的通道，为调节KV通道门控和抑制性的新范式 神经传递。我们提出了三个针对对人的特定目标 这种新型信号传导方式的机理，广度和范围。在AIM 1中，我们将阐明 KCNQ通道的GABA和BHB调节的分子要求以及该能力演变时。在 AIM 2我们将定义我们最近发现的关键GABA类似物和代谢物的KCNQ结合位点 还激活KCNQ，并利用这些化合物之间的协同作用来开发优化，有效的 抗惊厥药。在AIM 3中，我们将利用新生成的CRISPR-CAS9产生的小鼠，并带有种系突变 KCNQ3和5 GABA/BHB结合位点，以确定该重要性和KCNQ同工型的重要性 BHB和生酮饮食的抗惊厥作用。然后，我们将使用细胞电生理分析 量化天然M-电流的GABA和BHB调制的年龄和KCNQ-异型依赖性 神经元令人兴奋。该项目将彻底定义小说，意外形式的基本方面 抑制性神经元信号传导，其双重目标是理解其在脑生理和利用的作用 有助于开发癫痫和其他神经系统疾病的先进，更安全的疗法的知识。