The overall goal of the research proposed in this application is to understand the molecular and structural basis of cardiac arrhythmias caused, at least in part, by inherited mutations of the SCN5A gene, and to determine novel mutation-targeted therapeutic strategies to treat them. The central hypothesis is that one step in the genesis of these arrhythmias is the perturbation of membrane electrical activity caused by alteration in the biophysical properties of the SCN5A gene product, Nav1.5. Most disease-associated Nav.15 mutations that alter channel gating affect channel inactivation. The overall goal of the research proposed in this application is to understand the molecular and structural basis of cardiac arrhythmias caused, at least in part, by inherited mutations of SCN5A, the gene coding for Nav1.5, the alpha subunit of the primary heart Voltage-gated Na+ channel, and to determine novel mutation-targeted therapeutic strategies to treat them. The research proposed builds upon work done during the previous funding period and continues to focus upon the molecular basis of cardiac arrhythmias caused by inherited Nav1.5 mutations, but emphasizes the role of the Nav1.5 carboxy terminal (C-T) domain in these arrhythmias. In the past we have relied on homology models to gain insight into structural mechanisms that might contribute to mutation-altered channel function. Analysis of the physiological and pharmacological properties of mutant Nav1.5 channels in HEK 293 cells will be complemented by expression in cardiac myocytes to provide a framework to test the predictions of studies in heterologous expression systems with consequences in a physiologically relevant environment. There are two specific aims of this project. [[The first aim is to test the hypothesis that the inactivation gate forms a complex with the C-T domain by directly determining the three dimensional structure of the Nav1.5 C-T in the absence and presence of the channel inactivation gate using x-ray crystallography. We will further test the hypothesis that predicted structural motifs of the Nav1.5 C-T domain are necessary to maintain this complex and that disruption of C-T structure as well as identified sites of interaction with the inactivation gate disrupt Nav1.5 channel inactivation.]] The second aim is to investigate mutation-specific pharmacology and biophysical consequences of LQT-3 and BrS Syndrome Nav1.5 mutations and to determine the physiological consequences and pharmacological modulation of these mutant channels in HEK 293 cells and in cardiac myocytes. We hypothesize that information gained from these cellular and molecular experiments can be translated directly to improved therapeutic intervention in humans based on specific properties of mutant gene products, and also shed light on the possible interrelationship of these and other inherited disorders in the heart due to defects in Na+ channel inactivation.
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