Regulation of Na+ channel gene expression modulates the electrical excitability of muscle cells. A decrease in muscle Na+ channel expression results in at least one identified syndrome, acute quadriplegic myopathy, and may also play a role in other disease or age-related dysfunction. The mechanisms that control expression of the skeletal muscle type 1 Na+ channel (SkM1) will be studied to determine how the transcription factors (TFs) that regulate it work together to modulate specific temporal and spatial aspects of SkMl expression. Previous work indicates that the myogenic basic helix-loop-helix (bHLH) proteins myogenin and MRF4 recruit other novel TFs, including both a transrepressor and a transactivator, in regulating SkM 1 expression. Likely these novel TFs regulate expression of other genes as well and therefore they will be cloned. Certain aspects of SkM1 expression, such as a preferential expression at neuromuscular junctions and the relatively late postnatal increase in mRNA levels, cannot be replicated in cultured muscle cells. Therefore several strategies will be used to study the interaction between the bHLH factors and novel TFs in vivo. The SkM1 cis-regulatory sequences driving expression of an epitope-tagged Na+ channel will be introduce into transgenic mice, and the level, temporal, and spatial distribution of the transgene assessed by immunocytochemistry, Western blotting or RNAse protection assays relative to the endogenous SkM1. Mutations in the binding sites of the novel TFs or the bFILH factors will be tested to determine what aspect of SkM I expression these sites control. In addition, the role of the bHLH factors will be evaluated by determining what changes in the temporal or spatial distribution of SkM1 expression occurs in myogenin and MRF4 knockout animals. Future work will utilize recombinant adenovirus to introduce the bHLH factors, novel TFs, or dominant/negatives of these factors in vivo. The effect of these IFs on the spatial and temporal expression of not only SkMI but also other muscle genes such as the acetyicholine receptor will be studied. These experiments will validate in vitro findings, and, in addition, will provide the first temporally integrated analysis of ion channel gene expression, and will yield important insights into how ion channel genes are regulated in developmental, adulthood, and perhaps under pathological conditions.
