Cellular mechanisms controlling the expression and activity of sodium channels

负责大脑活动和肌肉收缩的电信号是通过打开称为钠通道的小蛋白来触发的，因为钠通道能够控制钠离子进入细胞内的流动。目前已知九种钠通道亚型--因其电压依赖性开放而被标记为NAV。它们中的每一个都具有特定的生化和药理特性，一旦打开，就会产生具有独特性质的电流(INA)。细胞通过表达NAF来利用这种多样性，这些NAV赋予INA所需的属性，以确保神经元和肌肉对发育过程中激素和神经递质的变化做出足够的反应，或者适应它们的环境。细胞如何选择表达一种NAV而不是另一种NAV尚不清楚。此外，对环境或代谢条件的某些形式的适应会触发NAV亚型的表达，这些亚型在某些类型的细胞中通常是不存在的。然而，在环境或生理应激过程中，NAV基因表达调控的细胞和遗传机制是完全未知的。我们未来五年的研究计划分为两个部分。我们首次提出使用心脏和神经细胞模型来确定Nav1.1(神经元通道)和NaV1.5(心脏通道)基因启动子的元件，它们分别限制和促进每个通道的表达。为此，我们构建了DNA结构(质粒)，使每个通道的启动子驱动一种绿色荧光蛋白(GFP)的表达，该蛋白可以通过共聚焦显微镜观察到。我们将逐渐缩短每个启动子的长度，直到我们可以将它们缩小到几个核苷酸的活性区域。接下来，我们将构建由Nav1.1启动子驱动NaV1.5表达的杂交DNA载体，反之亦然，以鉴定抑制元件。然后，我们将使用我们正在开发的一种新方法和质谱学来提取和鉴定每个启动子上附着的转录因子。在第二部分中，我们将确定有选择地调节NAV贩运和活动的胞内级联。我们将测量它们的mRNA和蛋白的表达，并用INA的电学测量来补充数据。将使用特定的激活剂和抑制剂来剖析每个贩运组成部分。一旦确定，我们将把这些机制与压力刺激联系起来，以调节它们的活动。我们的研究计划还包括开发一种识别转录因子的新方法，并为了解可兴奋细胞如何调节其电反应和基因组靶标以调节钠通道的表达奠定了基础。这些知识应该被证明有助于开发新的化合物，可以用来增强或降低哺乳动物的表达兴奋性，而且由于果蝇中介导电兴奋性的分子通常与人类的分子相似，这也将增强我们控制昆虫种群的能力。

The electrical signal responsible for brain activity and muscle contraction is triggered by opening of small proteins called sodium channels, for their ability to control the flow of Na+ getting inside the cells. Nine sodium channel isotypes -labeled NaV because of their voltage-dependent opening- are currently known. Each of them possesses specific biochemical and pharmacological characteristics which, upon opening, generate an electrical current (INa) with unique properties. Cells exploit this diversity by expressing NaVs that confer to INa the attributes needed to ensure adequate response of neurons and muscles to variations in hormone and neurotransmitter during development or, to adapt to their environment. How cells select to express one NaV over the other is unknown. Moreover, some forms of adaptation to environmental or metabolic conditions trigger expression of NaV isoforms normally absent in some cell types. However, the cellular and genetic mechanisms regulating expression of NaVs during environmental or physiological stress is utterly unknown. Our research program of the next 5 years is divided in two parts. We first propose to use cardiac and neuronal cell models to determine the elements of the gene promoter of NaV1.1 (a neuronal channel) and NaV1.5 (a cardiac channel) that respectively restrict and promote the expression of each channel. To this end, we made DNA constructions (plasmids) such that the promoter of each channel is driving expression of a green fluorescent protein (GFP) that can be visualized by confocal microscopy. We will gradually shorten the length of each promoter until we can narrow them down an active region of a few nucleotides. We will next construct hybrid DNA plasmids with the promoter of NaV1.1 driving expression of NaV1.5 and vice--versa to identify repressor elements. We will thereafter use a novel method we are developing and mass spectrometry to pull down and identify transcription factors attached to each promoter. In the second part we will identify the intracellular cascades that selectively modulate the trafficking and the activity of NaVs. We will measure expression of their mRNA and protein and complement the data with electrical measurement of INa. Specific activators and inhibitors will be used to dissect each trafficking component. Once identified, we will link these mechanisms to stress stimulus known to modulate their activity. Our research program also includes the development of  a novel method to identify transcription factors and a basis to understand how excitable cells regulate their electrical response and genomic targets to modulate expression of sodium channels. Such knowledge should prove useful to develop new compounds that can be used to enhance or reduce expression excitability in mammals and since molecules mediating electrical excitability in the fruit fly are generally similar to those in humans it will also enhance our arsenal to control insect populations.