The Na+ channel is responsible for the conduction of electrical impulses throughout excitable tissues including the heart. They are the primary targets for local anesthetics as well as related antiarrhythmic drugs. Several hereditary cardiac and muscular diseases have now been linked to mutations in Na+ channels, e.g., one form of congenital long QT syndrome (LQT3). The long-term goals of this proposal are to understand the molecular basis for the function of Na+ channels. We have evidence to suggest that there are significant overlaps in the region which are important for the two fundamental properties of the channels; namely gating and permeation. Specifically, two consecutive residues (W402 and E403) in the 55-56 region (P loop) in Domain I of the mu1 skeletal muscle Na+ channel, both of which have been shown to line the pore of the channel, are important in the gating of the channel. We hypothesize that the negatively charged residues in the pore region of the channel (e.g.,E403 residue) interact with nearby positively charged residues to stabilize the activation gate of the channel. This hypothesis can be directly tested using site-directed mutagenesis combined with heterologous expression in oocytes and a mammalian expression system. We will determine the mechanisms by which the negatively charged residues affect the gating of the channel using cysteine mutagenesis combined with sulfhydryl modifications. Using data obtained from the mu1 skeletal muscle Na+ channel as groundwork, we will study the homologous mutations (e.g., E375C) in the hH1 cardiac Na+ channel isoform. The goals are two folds: to determine whether the change in gating seen with e.g., E403C is a generalized phenomenon and to establish whether there are isoform-specific differences in the activation gating machinery between the two channels. We will study the nearby positively charged residues in the S5-S6 linker and S4 transmembrane segment in Domain I, which may interact electrostatically with the negatively charged residues. The combined techniques of molecular cloning and site-directed mutagenesis together with electrophysiologic recording promise to provide new insights into the structure-function relationship of ion channels. Definition of the regions determining ion transport, selectivity and gating hold promises for new and more mechanistic approaches for our diagnosis and therapy of cardiovascular ion channel diseases.
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