The long term goal of this research is to test the hypothesis that sodium channel beta subunits communicate between extracellular matrix or cell adhesion molecules and the neuronal cytoskeleton. Beta subunits are members of the immunoglobulin superfamily and contain cell adhesion molecule domains. An extracellular matrix protein, tenascin-R (TN-R) is a functional modulator of sodium channel beta and alpha subunits. Transfected cells expressing sodium channel beta1, beta2 or alpha subunits are initially attracted to and then are repelled from TN-R plated on a nitrocellulose substrate. Sodium channel beta subunits interact homophilically in drosophila S2 cells and recruit ankyrin to points of cell- cell contact. It is proposed that sodium channel beta subunits communicate extracellular signals to the neuronal cytoskeleton. This interaction may be important to sodium channel placement and function during normal formation in the CNS. Alternatively, this interaction may be important in axonal fasciculation or de-fasciculation or establishment of neuronal polarity. The goal of this proposal is to determine the molecular basis of the interactions of beta subunits with each other and with TN-R.
The specific aims are proposed: 1. To determine the structural domains in beta1, beta1A, and beta2 that mediate the functional effects of TN-R on cell body migration. 2. To determine the structural domains present in beta1 and beta2 that mediate extracellular homophilic binding and intracellular recruitment of ankyrin. 3. To determine whether beta1 and beta2 exhibit heterophilic binding to each other or to neurofascin and, if so, whether the interaction is cis or trans. Truncation mutants which eliminate the intracellular COOH-terminal domains of beta1 and beta2, introduction of a COOH-terminal signal peptide which eliminates the transmembrane domain and adds a glycophosphatidyinositol lipid anchor to beta 1 or beta2, and point mutations of amino acids in beta1 and beta2 that are predicted to be located in the extracellular Ig fold will be constructed and expressed in mammalian cells and tested in cell migration assays. The effect of TN-R n beta1A, and beta1 isoform with a novel COOH terminal domain, will also be tested. These mutant constructs will be tested for homophilic binding and ankyrin recruitment in S2 cells. The experiments described in this proposal will lead to a greater understanding of the events involved in sodium channel placement during normal brain development, and may contribute to the understanding of nodal formation and axonal fasciculation. This basic knowledge may provide a framework for the development of therapeutic agents to treat demyelinating disease such as multiple sclerosis.
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