GABA activation of the M-current

五聚体配体门控离子通道（LGIC）被神经递质结合到高度特化的， 亚基间，细胞外结合口袋。相比之下，电压门控钾（Kv）通道被激活， 膜去极化，通过电压传感器机电地传送到孔模块。kV 由KCNQ 2/3异聚体组成的通道产生神经元M电流，这是一种普遍存在的和必需的 哺乳动物中枢神经系统兴奋性超极化钾电流控制。这项建议是根据我们最近的 发现KCNQ 2/3通道直接被γ-氨基丁酸（GABA）激活， 脊椎动物CNS中的神经递质，灵敏度与最敏感的α/β/γ GABAA相当 受体LGIC。相反，兴奋性神经递质谷氨酸，结构上与GABA相关， 对KCNQ 2/3活性无影响。我们已经确定KCNQ 2/3 GABA结合位点为KCNQ 3-W265， 我发现这个位置在后口动物分支中是高度保守的，存在于一些刺胞动物中，但在 原口类;它也不存在心脏表达的KCNQ 1。此外，我们发现GABA类似物 和代谢物对KCNQ 2/3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-羟基丁酸酯（BHB），即在体内产生的主要酮体。 反应禁食或生酮饮食，防止癫痫发作。我们发现BHB是一个有效的KCNQ 2/3 激活剂和抗惊厥剂，揭示了酮症治疗作用的分子靶点。我们的研究结果 表明尽管它们在结构上有很大差异，但GABA激活两种主要的抑制离子 在脊椎动物神经元中的Kv通道，为Kv通道门控和抑制的调节创造了新的范例 神经传递我们提出了三个具体目标，旨在更全面地了解 机制，广度和范围的基础上，这种新的信号形式。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 目的2我们将确定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， 抗惊厥药在目标3中，我们将利用新产生的携带种系突变的CRISPR-Cas9小鼠， KCNQ 3和5 GABA/BHB结合位点，以确定KCNQ亚型的重要性和依赖性， 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， 神经元兴奋性该项目将彻底定义一个新颖的，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，其双重目标是了解其在大脑生理学中的作用， 知识，以帮助开发先进的，更安全的治疗癫痫和其他神经系统疾病。

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.