GABA activation of the M-current

五聚体配体门控离子通道(LGIC)由神经递质结合到高度专门化的， 亚基间、胞外结合袋。相反，电压门控钾(Kv)通道由 膜去极化，由电压传感器以机电方式与孔道模块通信。KV 由KCNQ2/3异构体组成的通道产生神经元M-电流，这是一种普遍而必要的 哺乳动物中枢神经系统中控制兴奋性的超极化K+电流。这项建议是基于我们最近 发现KCNQ2/3通道可被主要抑制物γ-氨基丁酸直接激活 脊椎动物中枢神经递质，其敏感性与最敏感的α/β/γGABA相当 受体LGICs。相比之下，与GABA结构相关的兴奋性神经递质谷氨酸， 对KCNQ2/3活性无影响。我们已经确定KCNQ2/3 GABA结合位点为KCNQ3-W265，并且 发现该位置在后口支系中高度保守，存在于一些蛇形动物中，但在 它也不存在于心脏表达的KCNQ1中。此外，我们还发现GABA类似物 和代谢产物表现出相似的结构-活性关系(SARS)的KCNQ2/3通道激活和 抗惊厥活性。代谢产物包括β-羟基丁酸酯(BHb)，主要的酮体产生于 对禁食或生酮饮食的反应，以防止癫痫发作。我们发现BHB是一种有效的KCNQ2/3 激活剂和抗惊厥剂，揭示了酮病治疗效果的分子靶点。我们的发现 表明尽管它们的结构差异很大，但GABA激活了两种主要的抑制离子 脊椎动物神经元中的通道，创造了调节Kv通道门控和抑制的新范式 神经传递。我们提出了三个具体目标，旨在更全面地理解 这种新的信号方式背后的机制、广度和范围。在目标1中，我们将阐明 GABA和BHB调节KCNQ通道的分子需求以及这种能力何时演变。在……里面 目的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 也激活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，我们将利用新产生的CRISPR-Cas9小鼠携带胚系突变 KCNQ3和5 GABA/BHB结合位点决定KCNQ的重要性和KCNQ异构体依赖性 BHB的抗惊厥作用与生酮饮食。然后我们将使用细胞电生理分析来 量化天然M-电流和BHB对GABA和BHB调制的年龄和KCNQ-亚型依赖性 神经元兴奋性。该项目将彻底定义一种新颖的、意想不到的形式的基本方面 抑制性神经元信号转导的研究，其双重目标是了解其在大脑生理学中的作用和利用 帮助开发先进、更安全的癫痫和其他神经疾病治疗方法的知识。

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.