The long-term goal of this research are to analyze structure-function relationships in the sodium channel, and to correlate these to diseases of excitability. The specific experimental aims of this proposal are to produce physical models of channel conformation during slow inactivation, and include: 1) testing the hypothesis that (a) the S4 4 membrane-spanning segments are in their depolarized-favored position during slow inactivation, (b) that slow inactivation is produced by electrostatic interactions between charged residues in two or more S4 membrane-spanning segments; (2) testing the hypothesis that (a) slow inactivation is limited by electrostatic interactions between the III-IV intracellular linker and S4 membrane-spanning, (b) segments the domain III-IV intracellular linker is in the bound position during slow inactivation; (3) testing the hypothesis that specific channel interactions associated with slow inactivation can be localized from skeletal muscle/cardiac muscle channel chimeras. The methodology that will be used to achieve these aims includes a combination of molecular biological manipulation of sodium channel structure and electrophysiological (patch clamp) assessment of sodium channel function. The health-relatedness of this proposal is that sodium channels form the primary basis for action potentials in nerves, muscles, and secretory cells, and that slow inactivation is a critical determinant of the number of channels available for opening and, therefore, cell excitability. Modification of cell excitability due to sodium channel mutations leads to a variety of disease states including non-dystrophic myotonia, cardiac arrhythmia, and epilepsy. Differences in excitability amongst sodium channel subtypes may also form the basis of firing patterns and post-synaptic integration in the central nervous system, proven, or suspected, to be the basis of many of these differences in cell excitability. However, the structural underpinnings of slow inactivation, and its interactions with other sodium channel properties, as yet remain unknown. The physical models of sodium channel conformation during slow inactivation that this proposal will produce contribute crucial information regarding the structural substrates of slow inactivation. This information is a critically-necessary first step to developing treatments for diseases of excitability and to a basic understanding of sodium channel function.
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