NA+ CHANNELS IN NORMAL & ABNORMAL NERVE MEMBRANE

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The central theme of the proposed studies is to characterize the cellular and molecular bases underlying the evolution of ion channel expression in the nervous system. This is pursued in a unique model system of NB2a neuroblastoma cell, which have retained two distinct pathways of the differentiation program of normal development. These cells, when induced to differentiate with db cAMP or retinoic acid, extend either axonal or dendritic processes respectively. This Research Plan focuses upon one phenotypic characteristic of neuronal differentiation, the expression of isoforms of the Na+ channel, which we have previously shown to differ between "axonal" and "dendritic" NB2a cells. The application's broad long-term objectives are: 1) To determine the cellular and molecular mechanisms controlling expression of Na+ channel isoforms in nerve cells; 20 To determine whither Na+ channel isoforms with specialized functional properties arise as products of distinct genes; and to determine the differences in their molecular structure which account for these specialized properties, and the roles they serve; 3) To apply these studies to further our understanding of nerve excitability mechanisms in normal and disease states. The specific aims and experimental design are: 1) To characterize the events of protein synthesis which account for the different time courses of expression of the two Na+ channel subtypes during differentiation of axonal and dendritic NB2a cells. This will include a comparison of the lifetime and turnover rates of the Na+ channel subtypes; 2) To characterize transcripts of mRNA species involved in expression of the two Na+ channel subtypes in differentiated NB2a cells, to determine whether the two Na+ channel subtypes are products of distinct gene species, and to determine the role of mRNA transcription in Na+ channel expression; 3) To establish a model system for studying the reciprocal changes in expression of the two Na+ channel subtypes which occur with innervation and denervation. The major experimental methods used include: 1) characterizing expression of Na+ channel subtypes during NB2a cell differentiation by measurement of high- and low-affinity [3H]- STX receptors; 2) characterizing Na+ channel mRNA species by hybridization with a rat brain Na+ channel II cRNA probe. The health relatedness of the project is that the distribution and function of the various Na+ channel isoforms in normal and abnormal excitable membranes determines the normal physiology and pathophysiology of impulse conduction in these tissues. The NB2a cells provide a model system to begin studying function and distribution of Na+ channel isoforms in excitable membranes. These properties are relevant in : 1) normal function of the nervous system; 2) nervous system diseases such as multiple sclerosis, Alzheimer's disease, and muscular dystrophy; and 3) cardiovascular impulse conduction, arrhythmia generation, and action of anti-arrhythmic agents.