Cellular mechanisms controlling the expression and activity of sodium channels

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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.