verbatim): The long-term purpose of this project is to understand cardiac Na+ channel function at the molecular level, and to use the understanding to develop strategies for control of lethal arrhythmias. This project has three interdependent goals for the next five years: 1) resolving the molecular configuration of the Na+ channel permeation path/selectivity region, including the sites for local anesthetic drug binding, 2) examining the roles of charged vestibule residues in permeation and selectivity, and 3) determining the functional abnormalities resulting from naturally occurring channel mutants, especially alpha-subunit interactions. The Na+ channel is a major participant in most serious or lethal arrhythmias, and it is the target of some of our most powerful antiarrhythmic drugs. Considerable progress has been made in identifying the molecular structure of the outer vestibule (outer third of the permeation path) by effects of point mutations on permeation and selectivity and by determining the complimentary surface for binding of the pore-blocking toxins. Our unifying structural hypothesis is that the Na+ channel is related through evolution to the structurally determined KcsA bacterial channel, and that it spore motif of a helix teepee and a selectivity motif of helix-turn-strand is applicable to the Na+ channel. We propose to extend the complimentary interaction surface for outer vestibule toxins to identify the structure of the """"""""turrets"""""""" in the outer path, perhaps also attempting to resolve the isoform selectivity of u-conotoxin in the process. These important carboxyls in the outer vestibule will be examined by mutation and pH titration for their contribution to dehydration of the permeating ions. The residues facing the inner pore will be identified by sequential mutation of the residues to cysteine, with access determined by methanesulfonate derivative interaction. Their roles in local anesthetic drug binding will be determined, in order to find all of the molecular parts of the drug binding site. State-dependent access of these residues will be examined to determine the changes in the site with channel gating. Each experimental step is developed based on the best molecular model we can develop, and in turn the model is improved as new experimental information is obtained in these experiments or published by others.
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