Two forms of voltage-sensitive sodium channels (NaChs) exist in rat and human striated muscles, SkM1 (skeletal muscle subtype 1) and H1 (heart subtype 1). These NaChs have a common structural motif, four internal highly homologous repeat domains (D) separated by one homologous and two quite divergent interdomain segments. The isotypes differ in their electrophysiological and pharmacological characteristics, notably, in activation and inactivation kinetics, single channel conductance and sensitivity to block by various divalent metal ions or toxins. The expression of the subtypes also differs in their tissue distribution, time of occurrence during development and response to denervation. Our goal is to help understand which of the distinct electrophysiological or structural features of a channel subtype are responsible for a cell choosing to express a given NaCh subtype. First, we will study chimeric channels comprised of domains and interdomain segments obtained from hSkM1, hH1, rSkM1, rH1 (hereafter called rSkM2/rH1). Three of these channels have been shown to be electrophysiologically and pharmacologically distinguishable by our collaborators (mainly Dr. R. Horn) and the one most recently discovered, hSkM1, is currently being characterized. Based on these results, we will extend the studies to higher resolution with clustered (block) and site-specific point mutations. The goal is to define the parts of the channel protein which form the extracellular mouth of the ion permeation pathway by determining toxin and divalent ion binding sites. We will then proceed to analyze by similar mutational strategies: (1) inactivation, (2) the effects of compounds that block open channels from the inside of the cell, and (3) interactions of these blockers with the inactivation gate. Third, we will create and study autologous and heterologous complementary hemichannels (D1-D2 & D3-D4) and partial channels (e.g. D1-D2-D3 & D4). Successful combinations, those which support voltage-dependent Na ion conductance, will facilitate the mutagenesis process and help to delineate interdomain interactions by the characterization of these NaChs electrophysiologically and pharmacologically. Fourth, we will attempt to determine the functions of individual segments of channels by deletional, insertional and hybrid mutagenesis experiments. The creation and analysis of hybrid channels formed from NaCh and KCh elements electrophysiologically and pharmacologically is designed to define interdomain interaction surfaces, the inactivation gate and the ion selectivity region. Such experimental data may help to define the three-dimensional structure of the channel.
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