Novel approach to reveal the PKA sensitive potassium channels mediating the sAHP

项目摘要 慢后超极化(SAHP)是钙激活的钾电导，它伴随着 神经元的活动，控制动作电位发射的数量，从而控制神经元的兴奋性。 重要的是，SAHP是几乎所有皮质神经调节剂和 激活第二信使cAMP及其主要下游效应蛋白激酶的神经递质 A(PKA)；包括去甲肾上腺素(NE)、5-羟色胺、多巴胺和血管活性肠肽(VIP)。作为一名 结果，SAHP的变化与几个认知功能相关，包括获得或提取 回忆。尽管在过去的十年里，在鉴定分子组成方面取得了进展 SAHP，在SAHP期间开放的钾通道的身份，以及这些通道是如何 目前对PKA的调控机制尚不清楚。这项提议寻求使用一种新的替代传统克隆的方法 将钾通道连接到SAHP的方法。我们的战略是高度创新的，因为它结合了 计算和实验方法。首先，我们利用最近出版的单细胞 从小鼠皮质中提取外显子组，以确定含有SAHP的皮质区域中存在的候选钾通道。 然后，为了进一步筛选这些候选者，我们开发了一种新的策略来预测钾上的PKA底物 渠道，这是松散地基于我们以前开发的扫描-x程序。新的平台拥有一个 各种独特的改进，包括基于概率的评分和对多个PKA主题的处理 独立于预测器生成。实验上，我们将使用CRISPR/Cas9基因组编辑系统来 测试哪些钾通道对SAHP有贡献。这种最先进的方法使我们能够比较 并将多个基因同时在短时间内用于子宫内的效果进行对比 电穿孔(IUE)，这在标准的转基因小鼠方法中是不可能的。

Project Summary The slow afterhyperpolarization (sAHP) is a calcium-activated potassium conductance that follows a surge of neuronal activity, controlling the number of action potentials fired, and consequently neuronal excitability. Importantly, the sAHP is the major potassium conductance targeted by almost all cortical neuromodulators and neurotransmitters that activate the second messenger cAMP and its main downstream effector protein kinase A (PKA); this includes norepinephrine (NE), serotonin, dopamine, and vasoactive intestinal peptide (VIP). As a result, changes to the sAHP correlate with several cognitive functions, including acquisition or retrieval of memories. Although progress over the last ten years has been made to identify the molecular components of the sAHP, the identity of the potassium channels that open during the sAHP, and how these channels are modulated by PKA are still not clear. This proposal seeks to use a new alternative to traditional cloning approaches to link potassium channels to the sAHP. Our strategy is highly innovative as it combines both a computational and experimental approach. First, we make use of the recent publication of the single cell exome from mouse cortex to identify candidate potassium channels present in sAHP-containing cortical areas. Then, to further filter these candidates, we developed a new strategy to predict PKA substrates on potassium channels, which is loosely based on our previously developed scan-x program. The new platform boasts a variety of unique improvements including probability-based scoring and the treatment of multiple PKA motifs independently in predictor generation. Experimentally, we will use the CRISPR/Cas9 genome editing system to test which potassium channels contribute to the sAHP. This state-of-the-art methodology allows us to compare and contrast the effects of multiple genes simultaneously and in a short time frame using in utero electroporation (IUE), which would not be possible with a standard transgenic mouse approach.