The expression of voltage-gated sodium (Na) channels is a crucial aspect of neuronal differentiation. As the molecular basis of electrical excitability in most neurons, Na channels play a central role in the production and propagation of action potentials and ultimately in the coding and transfer of information in the nervous system. Despite the importance of Na channel expression, the mechanisms regulating the Na channel gene family are unknown. Therefore, as part of a long term goal to understand the regulation of the Na channel gene family, the specific aims of this proposal are designed to elucidate cellular and molecular mechanisms controlling the expression of the type II Na channel gene. First, regulatory elements in the 5' and 3' flanking regions of the type II gene and the proteins that interact with them will be identified and characterized. Genomic fragments of the 5' and 3' flanking regions that have already been isolated will be dissected by deletional analysis and fused to a reliably assayed reporter gene. The biological activity of the fusion genes in neuronal and non-neuronal cell lines will be determined in transient expression assays. Upon identification of the precise sequences necessary for the neuronal specific expression of the gene, the interaction of specific DNA-binding proteins with these sequences will be analyzed using mobility shift assays and nuclear extracts from neuronal and non- neuronal cells. Results of this analysis will then be used in initial attempts to clone the DNA-binding proteins by screening lambdagt11 expression libraries with DNA recognition-site probes. Second, the mechanism(s) underlying the induction of the type II gene by nerve growth factor (NGF) will be clarified using molecular biological approaches. The influence of NGF on the stability of type II Na channel mRNA will be determined using in vivo pulse chase labeling and filter binding assays, as well as RNA synthesis inhibitors and Northern Blot analysis. Biochemical and molecular biological reagents will be used to alter the activity of protein kinase A and determine its role in the NGF induction reagents will be used to alter the activity of protein kinase A and determine its role in the NGF induction of type II Na channel mRNA. The ability of biochemical treatments that activate protein kinase A to mimic the induction of type II Na channel mRNA by NGF, as well as the ability of NGF to induce type II Na channel mRNA in mutant cells deficient in A kinase activity and in cells stably transfected with an A kinase inhibitor, will be determined using Northern Blot analysis. Third, other growth factors influencing the expression of the type II Na channel will be identified using both electrophysiological and molecular biological approaches. In particular, the effects of fibroblast growth factor and epidermal growth factor will be determined using whole-cell patch clamp recordings and previously developed cell lines stably transfected with Na channel fusion genes. The results will provide a basis for understanding the changes in Na channel expression that occur in response to injury and disease, and will clarify the processes underlying neuronal differentiation and development of the nervous system.
