Cellular Regulation of Sodium-activated Potassium Channels

钠激活钾通道的细胞调节

基本信息

批准号：
10584753
负责人：
LEONARD K KACZMAREK
金额：
$ 43.07万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-01 至 2027-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10584753
关键词：
3&apos Untranslated Regions Action Potentials Animal Model Animals Antisense Oligonucleotides Binding Biological Assay C-terminal Cell Line Cell membrane Cerebral cortex Characteristics Coupled Cytoplasm Development Electrophysiology (science)Ensure Epilepsy FMRP Focal Seizure Genes Glutamates Goals Immunoprecipitation In Situ Hybridization Interneurons Ion Channel Ions Ligation Location Messenger RNA Mus Mutant Strains Mice Mutation Neurons Pattern Peptides Physiological Potassium Channel Process Property Protein Biosynthesis Protein Isoforms Proteins RNA RNA Splicing Reporter SCN1A protein SCN2A protein SCN8A gene Seizures Signaling Protein Sodium Syndrome Testing Therapeutic Time Translations Up-Regulation autism spectrum disorder beta Actin cell cortex cell growth regulation cell type childhood epilepsy differential expression disease-causing mutation early onset excitatory neuron experimental study gain of function gain of function mutation hippocampal pyramidal neuron infancy inhibitor inhibitory neuron mRNA Translation migration mouse model mutant neuronal excitability novel therapeutic intervention response severe intellectual disability voltage

项目摘要

ABSTRACT The KCNT1 gene encodes a Na+-activated K+ channel (KNa channel) termed Slack. These channels are bound directly to regulators of mRNA translation in neurons. Mutations in KCNT1 give rise to several childhood epilepsies as well as autism. These gain-of-function mutations produce a 3-20-fold increase in K+ current and all result in very severe intellectual disability. In a mouse model of epilepsy expressing the Slack-R455H mutation, KNa currents are increased in both excitatory and inhibitory cortical neurons. The characteristics of the increased KNa current are, however, different in the two types of cells, and the firing of excitatory neurons is increased while that of GABAergic interneurons is suppressed. This proposal will test the hypothesis that excitatory and inhibitory cortical neurons preferentially express either Slack-A or Slack-B splice isoforms, which differ substantially in their activation properties. In situ hybridization and immunolocalization experiments, coupled with the use of antisense oligonucleotides, will localize and suppress the expression of each isoform. The second part of the proposal will investigate the finding that, in addition to increased KNa currents, cortical neurons in Slack mutant mice have greatly increased Na+ currents and levels of NaV subunits. Because both physiological and pathophysiological (Slack mutant) activation of Slack channels triggers increased translation of mRNAs for β-actin and for mRNA translation reporter constructs, we will test whether this mechanism contributes to increased synthesis of NaV channel subunits. The ability of Slack channels to stimulate translation will be tested using reporter constructs containing 5’ and 3’ UTR domains of NaV genes, as well as assays of the rate of synthesis of nascent NaV peptides in cortical neurons. Finally, experiments will determine if specific domains in the cytoplasmic C-terminal domain of Slack channels, or K+ flux through the channels, are required for stimulation of mRNA reporter constructs or increased Na+ channel synthesis. The findings will provide essential information on the major unsolved question of how neurons coordinate expression of Na+ and K+ channels to regulate intrinsic excitability, and on how disruption of this process leads to abnormal firing and severe intellectual disability.

抽象的 KCNT1 基因编码 Na+ 激活的 K+ 通道（KNa 通道），称为 Slack。这些通道是绑定的。 KCNT1 的突变直接影响神经元中 mRNA 翻译的调节因子，从而导致多个童年。这些功能获得性突变导致 K+ 电流和自闭症增加 3-20 倍。在表达 Slack-R455H 的癫痫小鼠模型中，所有这些都会导致非常严重的智力障碍。突变时，兴奋性和抑制性皮层神经元中 KNa 电流均增加的特点。然而，两种类型的细胞中增加的 KNa 电流是不同的，兴奋性神经元的放电也不同。增加，而 GABA 能中间神经元则受到抑制。该提议将检验以下假设：兴奋性和抑制性皮层神经元优先表达 Slack-A 或 Slack-B 剪接亚型，原位杂交和免疫定位实验有很大不同，与反义寡核苷酸的使用相结合，将定位并抑制每种亚型的表达。该提案的第二部分将调查以下发现：除了 KNa 电流增加之外，皮质 Slack 突变小鼠的神经元大大增加了 Na+ 电流和 NaV 亚基水平。 Slack 通道的生理和病理生理（Slack 突变体）激活会触发翻译增加 β-肌动蛋白的 mRNA 和 mRNA 翻译报告基因构建体，我们将测试这种机制是否有助于增加 NaV 通道亚基的合成 Slack 通道的刺激能力。将使用包含 NaV 基因的 5' 和 3' UTR 结构域的报告构建体以及最后，实验将确定皮质神经元中新生 NaV 肽的合成率。如果 Slack 通道的细胞质 C 端结构域中存在特定结构域，或通过通道的 K+ 通量，刺激 mRNA 报告基因构建体或增加 Na+ 通道合成所需。提供有关神经元如何协调 Na+ 和 Na+ 表达的主要未解决问题的重要信息 K+ 通道调节内在兴奋性，以及该过程的破坏如何导致异常放电和严重智力障碍。