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由称为钠通道的小蛋白质的打开触发的，因为它们能够控制Na+进入细胞内的流动。目前已知有九种钠通道同种型，由于其电压依赖性开放而被标记为NaV。它们中的每一种都具有特定的生物化学和药理学特征，这些特征在打开时产生具有独特性质的电流（INa）。细胞通过表达NaV来利用这种多样性，NaV赋予INa所需的属性，以确保神经元和肌肉在发育过程中对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而不是另一种尚不清楚。此外，对环境或代谢条件的某些形式的适应触发在某些细胞类型中通常不存在的NaV同种型的表达。然而，在环境或生理应激期间调节NaVs表达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部分。我们首先建议使用心脏和神经元细胞模型来确定分别限制和促进每个通道的表达的NaV1.1（神经元通道）和NaV1.5（心脏通道）的基因启动子的元素。为此，我们制作了DNA构建体（质粒），使得每个通道的启动子驱动可通过共聚焦显微镜观察到的绿色荧光蛋白（GFP）的表达。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。我们接下来将构建具有驱动NaV1.5表达的NaV1.1启动子的杂交DNA质粒，反之亦然，以鉴定阻遏物元件。此后，我们将使用我们正在开发的一种新方法和质谱法来拉下并鉴定附着在每个启动子上的转录因子。 在第二部分中，我们将确定选择性地调节NaVs的运输和活性的细胞内级联。我们将测量它们的mRNA和蛋白质的表达，并通过INa的电测量来补充数据。将使用特定的激活剂和抑制剂来剖析每一种贩运成分。一旦确定，我们将把这些机制与已知的调节其活动的应激刺激联系起来。 我们的研究计划还包括开发一种新的方法来识别转录因子，并为了解易兴奋细胞如何调节其电反应和基因组靶点来调节钠通道的表达奠定基础。这样的知识应该证明是有用的，以开发新的化合物，可用于增强或降低表达兴奋性哺乳动物和由于介导的分子电兴奋性在果蝇中通常是类似的那些在人类中，它也将增强我们的武器库，以控制昆虫种群。

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.