The voltage-sensitive sodium channel is an integral component of impulse conduction by neuronal cells, transmitting the initial inward current during an action potential. The channel has been purified from a number of different tissues, and consists of one large subunit term alpha which is associated in some tissues with one or two small subunits termed beta. Full-length cDNA clones have been isolated which encoded the alpha subunits from rat brain, muscle and heart, and expression of these in Xenopus oocytes has shown that the alpha subunit is sufficient for functional sodium channels. The goals of this proposal are to determine the roles of phosphorylation. We have previously shown that the cytoplasmic liner between domains III and IV (LIII-IV) is critical for sodium channel inactivation. The first major goal of the studies proposed here is to investigate the mechanisms by which LIII-IV functions during fast inactivation. Specifically, we will investigate the structural properties of LIII-IV that are essential for inactivation by constructing insertion, deletion and substitution mutations, followed by electrophysiological analysis in Xenopus oocytes. We will also determine whether LIII-IV functions as an inactivating particle in trans by using synthetic peptides on excised patches. We will attempt to identify the region on the channel with which LIII-IV interacts (the docking site), and we will examine the specificity of this interaction. Finally, the role of LIII-IV in gating charge immobilization will be investigated. The second major goal of the studies proposed here is to investigate the functional significance of phosphorylation of the sodium channel alpha subunit by protein kinase A (PKA). There are five consensus PKA phosphorylation sites in the cytoplasmic linker between domains I and II (LI-II) of the rat brain sodium channel. The effects of phosphorylation at each of those sites will be determined by site specific mutagenesis followed by electrophysiological analysis. We will investigate whether phosphorylation at those sites is essential for insertion of the channels into the cell membrane, and we will test whether LI-II functions as an inhibitory loop in the channel. Rat skeletal muscle sodium channels do not contain any consensus PKA sites in LI-II, and the functional effects of this difference between the brain and muscle channels will be examined by construction chimeric channels. Finally, the role of electrostatic interactions in the functional effects of phosphorylation will be tested by substituting negatively charged amino acids for the serine residues that are normally phosphorylated. These studies should enhance our knowledge about the normal function of the voltage-sensitive sodium channel, ultimately helping to understand the pathological processes that affect channel function in disease and to design pharmaceutical that interact with channels of specific tissues.
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