Sodium channels underlie the rapid depolarization of action potentials in most excitable cells and are involved in nervous conduction, voluntary muscle contraction, cardiac excitation-contraction coupling, as well as ion channel linked muscle diseases. They serve as receptors for cardiac antiarrhythmic agents, agents used to intervene in episodes of skeletal muscle derived periodic paralysis, as well as anticonvulsant agents. Thus, these important excitability proteins are essential to normal physiological behavior and are important targets for therapeutic intervention. There is considerable evidence for an inter-dependence between Na+ channel gating and channel block by local anesthetic-like agents. However at present little is known about the molecular determinants of channel gating and pharmacology. The long term aims of this proposal are to determine the role of the two principal protein subunits of human Na+ channel (alpha and beta1 subunits) and how their interactions modulate channel gating and pharmacology. Specific regions of the Na= channel (amino acid domains) will be manipulated through protein engineering and recombinant DNA methods with the goal of identifying their role in channel gating (opening and inactivation), interactions with the beta1 subunit, and in drug binding. The methods include patch clamp and high speed cut-open oocyte voltage clamp of channels expressed in Xenopus oocytes or in mammalian cells. A major strategy will be to capitalize on the natural functional and structural diversity of distinct Na+ channels to guide experiments. Three distinct human Na+ channels will be studied; the human cardiac Na+ channel, hH1; the human skeletal muscle Na+ channel, hSkm1; and a newly identified channel cloned from human ventricle, hNav2.1. One rationale for using these channels is that although highly conserved, functional differences combined with sequence differences provide clues for identifying and manipulating important domains in the protein. The need to understand state dependent drug block and channel gating mode changes is fundamental. The Na+ channel is the simplest system to begin these investigations at the molecular level. It offers significant advantages for successful protein structure-function studies. Ion channels have a functional signature (the single channel current) that can be measured with excellent, and functionally relevant temporal resolution at the level of a single protein molecule. The results will improve understanding of the function of these newly identified human proteins and will help identify protein domains involved with channel gating and binding of therapeutically relevant pharmacological agents.
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