Physiological roles of Kv1.1 RNA editing

描述（由申请人提供）：电压门控钾通道在大脑中形成电信号方面发挥重要作用。电压门控钾通道的Kv1.1亚型与调节动作电位传播有关，Kv1.1基因内的突变与发作性共济失调1型（EA1）相关，EA1是一种显性人类神经系统疾病，临床表现多种多样，包括应激诱导的共济失调、肌震颤、神经性肌强直和癫痫。编码Kv1.1通道亚基的RNA转录物经历腺苷至肌苷RNA编辑事件，其中基因组编码的异亮氨酸密码子（AUU）在成熟mRNA中转化为缬氨酸密码子（IUU）以改变编码的通道蛋白的位置400处的氨基酸同一性。这种氨基酸残基位于高度 已知通道的保守离子传导孔和RNA编辑改变异源表达系统中钾通道失活的速率。为了确定这种非同义氨基酸改变的生理重要性，我们开发了仅表达非编辑的（I）或编辑的（V）通道同种型的遗传修饰的小鼠系。在初步分析中，未编辑的Kv1.1（I）表达小鼠显示出与EA1一致的表型特征，表明通过编辑调节Kv1.1活性是运动控制和癫痫易感性的重要调节因子。拟议的研究将侧重于Kv1.1突变动物的分子、行为和神经生理学表征，特别关注在未编辑的Kv1.1（I）小鼠的初步研究中观察到的EA1样表型。这些研究还将确定Kv1.1编辑与已建立的EA1小鼠模型之间的关系，该模型被设计为携带与这种疾病相关的人类突变。初步表征将量化参与Kv1.1信号传导的蛋白质的基因表达的潜在补偿性变化，以及确定纯合Kv1.1（I）动物死于不完全渗透致死的时间范围，从而深入了解Kv1.1编辑的发育重要性。由于EA1中观察到的运动功能障碍会因应激而加重，因此将在控制和应激条件下对运动协调的各个方面进行行为分析。为了评估突变Kv1.1的脑电活动的改变， 在小鼠中，脑电图（EEG）研究将监测自发性癫痫发作，而化学惊厥剂将用于确定诱发性癫痫发作奥尔兹。为了评估先前表征的EA1突变是否通过错义氨基酸掺入或编辑破坏来改变Kv1.1功能，我们将使用体外和体内模型系统来量化EA1突变体的编辑特征。最后，我们将采用电生理技术在小脑切片浦肯野神经元，以检查潜在的Kv1.1通道动力学的改变。预计拟议的研究不仅将为RNA编辑对Kv1.1功能调节的重要性提供重要见解，而且还将为Kv1.1转录物的编辑改变如何导致运动和神经功能障碍提供重要见解。

DESCRIPTION (provided by applicant): Voltage-gated potassium channels play an important role in shaping electrical signals in the brain. The Kv1.1- subtype of voltage-gated potassium channel has been implicated in modulating action potential propagation and mutations within the Kv1.1 gene are associated with episodic ataxia type-1 (EA1), a dominant human neurological disorder with widely variable clinical manifestations, including stress-induced ataxia, myokymia, neuromyotonia, and epilepsy. RNA transcripts encoding the Kv1.1 channel subunit are subject to an adenosine-to- inosine RNA editing event in which a genomically-encoded isoleucine codon (AUU) is converted to a valine codon (IUU) in the mature mRNA to alter the amino acid identity at position 400 of the encoded channel protein. This amino acid residue lies in the highly conserved ion-conducting pore of the channel and RNA editing is known to alter the rate of potassium channel inactivation in heterologous expression systems. To determine the physiological importance of this non-synonymous amino acid alteration, we have developed genetically- modified mouse lines that solely express either the non-edited (I) or edited (V) channel isoforms. In preliminary analyses, non-edited Kv1.1 (I)-expressing mice displayed phenotypic characteristics consistent with EA1, suggesting that modulation of Kv1.1 activity through editing is an important regulator of motor control and seizure susceptibility. The proposed studies will focus upon molecular, behavioral and neurophysiological characterization of Kv1.1 mutant animals with a particular focus on EA1-like phenotypes that have been observed in preliminary studies for non-edited Kv1.1 (I) mice. These studies will also determine the relationship between Kv1.1 editing compared to an established mouse model of EA1, engineered to bear a human mutation associated with this disorder. The initial characterization will quantify potential compensatory changes in gene expression for proteins involved in Kv1.1 signaling, as well as determine the time-frame in which homozygous Kv1.1 (I) animals die of an incompletely penetrant lethality, providing insights into the developmental importance of Kv1.1 editing. Behavioral analyses of various aspects of locomotor coordination will be performed under both control and stressed conditions since the motor dysfunction observed in EA1 is exacerbated by stress. In order to assess alterations in brain electrical activity for mutant Kv1.1 mice, electroencephalography (EEG) studies will monitor spontaneous seizures and chemical convulsants will be used to determine induced-seizure thresh- olds. To assess whether previously characterized EA1 mutations alter Kv1.1 function by missense amino acid incorporation or disruption of editing, we will use both in vitro and in vivo model systems to quantify editing profiles for EA1 mutants. Finally, we will employ electrophysiological techniques on Purkinje neurons in cerebellar slices to examine potential alterations in Kv1.1 channel kinetics. It is anticipated that the proposed studies will not only provide critical insights into he importance of RNA editing for the regulation of Kv1.1 function, but also into how altered editing for Kv1.1 transcripts may result in locomotor and neurological dysfunction.