The long-term objective of this application is to elucidate the molecular correlates of the biophysical properties of voltage-dependent sodium channels, using a combination of patch recording and molecular biology. Three clones of channels, derived from mammalian striated muscle, will be expressed, both transiently and stably, in frog oocytes and mammalian cell lines. The clones include both cardiac and skeletal isoforms from rat and human sources. The projects are divided into four categories. First, the extracellular mouth of the channels will be studied with an emphasis on the permeation pathway. The three isoforms differ with respect to single channel conductance, block by extracellular toxins (tetrodotoxin and mu- conotoxin), and extracellular divalent cations. Regions of the channels suspected to be located near the outer mouth will be swapped among isoforms, and selected point mutations will be constructed and tested. Second, the process of inactivation will be studied in order to test in some detail the 'ball-and-chain' hypothesis. Rates and voltage dependence of inactivation from the open state will be estimated from single channel data. Inactivation will be removed, either enzymatically or by mutation, and open channel block at the intracellular mouth will be studied, using either fast blockers (e.g., tetramethylammonium) or slow blockers (e.g., lidocaine derivatives or 'ball peptides'). Voltage dependence of blockers will be compared with that of the natural inactivation process, and competition between blockers and the inherent gate will be measured. Mutants of the postulated 'ball' and 'ball receptor' will be examined. Third, hemichannels will be coexpressed using mixtures of the first two, and last two, homologous domains of the alpha subunit. Homogenous and heterogeneous channels will be examined for their biophysical properties and their tendencies to segregate. Fourth, the functional modulation of cardiac sodium channels by kinases will be examined.
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