GABA activation of the M-current

M 电流的 GABA 激活

• 批准号: 10330997
• 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/10330997
• 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-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.

五聚体配体门控离子通道 (LGIC) 通过与高度专业化的神经递质结合而激活， 亚基间、细胞外结合袋。相反，电压门控钾 (Kv) 通道被激活 膜去极化，通过电压传感器以机电方式传送至孔模块。千伏 由 KCNQ2/3 异聚体组成的通道产生神经元 M 电流，这是一种普遍存在且必不可少的 超极化 K+ 电流控制哺乳动物中枢神经系统的兴奋性。这个建议是基于我们最近的 发现 KCNQ2/3 通道直接被主要抑制因子 γ-氨基丁酸 (GABA) 激活 脊椎动物 CNS 中的神经递质，其敏感性与最敏感的 α/β/γ GABAA 相当 受体 LGIC。相比之下，兴奋性神经递质谷氨酸，其结构与 GABA 相关， 对 KCNQ2/3 活性没有影响。我们已将 KCNQ2/3 GABA 结合位点鉴定为 KCNQ3-W265，并且 发现该位置在后口动物分支中高度保守，存在于一些刺胞动物中，但在 原口动物；心脏表达的 KCNQ1 中也不存在它。此外，我们还发现 GABA 类似物 和代谢物对于 KCNQ2/3 通道激活表现出相似的结构活性关系 (SAR) 抗惊厥活性。代谢物包括 β-羟基丁酸 (BHB)，它是在 对禁食或生酮饮食的反应，可以预防癫痫发作。我们发现 BHB 是一种有效的 KCNQ2/3 激活剂和抗惊厥剂，揭示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，GABA 仍能激活两类主要的抑制离子 脊椎动物神经元通道，创建 Kv 通道门控和抑制调节的新范例 神经传递。我们提出了三个具体目标，旨在更全面地了解 这种新颖的信号传导方式的机制、广度和范围。在目标 1 中，我们将阐明 GABA 和 BHB 调节 KCNQ 通道的分子要求以及这种能力何时进化。在 目标 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的、有效的 抗惊厥药。在目标 3 中，我们将利用新的 CRISPR-Cas9 生成的携带种系突变的小鼠 KCNQ3 和 5 GABA/BHB 结合位点，以确定其重要性和 KCNQ 同工型依赖性 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 GABA 和 BHB 对天然 M 电流调节的年龄和 KCNQ 亚型依赖性 神经元的兴奋性。该项目将彻底定义一种新颖的、意想不到的形式的基本方面 抑制性神经元信号传导的研究，具有了解其在大脑生理学中的作用和利用其双重目标 帮助开发先进、更安全的癫痫和其他神经系统疾病疗法的知识。