Physiological roles of Kv1.1 RNA editing

Kv1.1 RNA 编辑的生理作用

基本信息

批准号：
8718280
负责人：
Elizabeth Anne Ferrick Kiddie
金额：
$ 2.73万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8718280
关键词：
Action Potentials Adenosine Affect Amino Acid Sequence Amino Acids Animals Ataxia Behavior Behavioral Biological Models Brain Cell physiology Cells Characteristics Chemicals Clinical Codon Nucleotides Communication Complex Convulsants Development Diagnosis Disease Electroencephalography Embryo Engineering Epilepsy Event Frequencies Functional disorder Gene Expression Genes Human In Vitro Inosine Ions Isaacs syndrome Isoleucine Kinetics Knowledge Lead Link Messenger RNA Molecular Monitor Motor Motor Seizures Mus Mutant Strains Mice Mutation Myokymia Nervous System Physiology Neurologic Dysfunctions Neurons Patients Pattern Phenocopy Phenotype Physiological Physiology Play Positioning Attribute Potassium Channel Predisposition Process Property Protein Isoforms Proteins RNA RNA Editing RNA Processing Regulation Research Personnel Role Seizures Shapes Signal Transduction Slice Stress Structure System Techniques Time Transcript Ursidae Family Valine Voltage-Gated Potassium Channel base brain electrical activity electrical property in vivo in vivo Model insight motor control mouse model mutant nervous system disorder neurophysiology novel patch clamp postnatal postsynaptic public health relevance therapeutic development voltage clamp

项目摘要

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.

描述（由申请人提供）：电压门控钾通道在塑造大脑的电信号方面起着重要作用。 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.编码KV1.1通道亚基的RNA转录本受到腺苷对肌苷RNA编辑事件的约束，其中基因组编码的异亮氨酸密码子（AUU）被转换为成熟mRNA中的Valine Codon（iuU），以改变编码通道的400位位置400的氨基酸鉴定。该氨基酸残基在高度已知通道的保守离子传导孔和RNA编辑可以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，我们开发了遗传修饰的小鼠系，这些小鼠系仅表达非编辑（i）或编辑（v）通道同工型。在初步分析中，未编辑的KV1.1（I）表达小鼠表现出与EA1一致的表型特征，这表明通过编辑对KV1.1活性的调节是运动控制和癫痫敏感性的重要调节剂。拟议的研究将重点关注KV1.1突变动物的分子，行为和神经生理学表征，特别关注于EA1样表型，这些表型在非编辑的KV1.1（I）小鼠的初步研究中已经观察到。与已建立的EA1小鼠模型相比，这些研究还将确定KV1.1编辑之间的关系，该模型具有与该疾病相关的人类突变。最初的表征将量化参与KV1.1信号传导的蛋白质的基因表达的潜在补偿性变化，并确定纯合KV1.1（I）动物死于不完全渗透杀伤力的时间框架，从而洞悉了KV1编辑的发展重要性。在对照和应力条件下，将对运动协调的各个方面进行行为分析，因为在EA1中观察到的运动功能障碍会受到压力的加剧。为了评估突变体KV1.1的脑电活动的变化小鼠，脑电图（EEG）的研究将监测自发癫痫发作，将化学抽搐剂用于确定诱导的塞氏阈值。为了评估先前表征的EA1突变是否通过识别氨基酸掺入或编辑中断改变KV1.1功能，我们将使用体外和体内模型系统来量化EA1突变体的编辑曲线。最后，我们将在小脑切片中的Purkinje神经元上采用电生理技术，以检查KV1.1通道动力学中的潜在变化。预计拟议的研究不仅将提供有关RNA编辑在调节KV1.1功能的重要性的关键见解，而且还可以使KV1.1转录本的编辑变化可能导致运动和神经功能障碍。