Cellular Regulation of Sodium-activated Potassium Channels

钠激活钾通道的细胞调节

• 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
• 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' 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

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.

摘要 KCNT1基因编码Na+激活的K+通道（KNa通道），称为Slack。这些渠道是绑定的 直接作用于神经元中mRNA翻译的调节因子。KCNT1的突变引起了几种儿童 癫痫和自闭症。这些功能获得性突变使K+电流增加3 - 20倍， 都会导致严重的智力残疾在表达Slack-R455 H的癫痫小鼠模型中 突变后，兴奋性和抑制性皮质神经元中的KNa电流均增加。的特点 然而，增加的KNa电流在两种类型的细胞中是不同的，兴奋性神经元的放电是 GABA能中间神经元的活性受到抑制。这一提议将检验以下假设： 兴奋性和抑制性皮质神经元优先表达Slack-A或Slack-B剪接异构体， 在它们的活化性质上有很大的不同。原位杂交和免疫定位实验， 结合反义寡核苷酸的使用，将定位并抑制每种同种型的表达。 该提案的第二部分将调查这一发现，除了增加KNa电流，皮质 在Slack突变小鼠的神经元中，Na+电流和NaV亚单位的水平大大增加。因为两 Slack通道的生理和病理生理（Slack突变体）激活触发翻译增加 β-肌动蛋白和mRNA翻译报告基因构建的mRNA，我们将测试这种机制是否 有助于增加NaV通道亚基的合成。松弛通道的能力，以刺激 将使用含有NaV基因的5 '和3' UTR结构域的报告构建体以及 皮质神经元中新生NaV肽合成速率的测定。最后，实验将确定 如果Slack通道的细胞质C末端结构域中的特定结构域，或K+通过通道流动， 是刺激mRNA报告构建体或增加Na+通道合成所必需的。其成果将 提供关于神经元如何协调Na+表达的主要未解决问题的基本信息， K+通道调节内在兴奋性，以及如何破坏这一过程导致异常放电， 严重智力残疾。