UNIVERSITY OF CHICAGO, THE

The overall aim of this application is to understand the fundamental basis of the interaction of local anesthetic/ antiarrhythmic drugs (LA) with the cardiac voltage-gated sodium (Na) channel (NaV1.5). Our multiple-PI format brings together the strengths of two research laboratories and four senior scientists to this proposal. Both laboratories have demonstrated expertise in this area of investigation but have used different approaches; the laboratory of Dr. Sheets at the University of Utah (in ongoing and close collaboration with Dr. Hanck since 1985) has investigated drug and toxin interactions with the voltage-sensor domain of Na channels while the laboratory of Drs. Hanck, Fozzard and Lipkind at the University of Chicago have studied the pore domain of the Na channel, focusing recently on the inner pore, the site of LA binding. This application gives us the opportunity to combine the expertises of both laboratories to investigate the interactions between the voltage-sensor domain and the pore domain in determining the action of LA's and, in special circumstances, anticonvulsant drugs. The experiments will use wild-type (WT) and site-directed mutant Na channels heterologously expressed in mammalian cells and oocytes combined with molecular modeling of the Na channel pore. Aim 1. The LA binding site in the pore. A. Construct a new LA binding site in pore domains other than DIII and DIV. B. Determine the role of DIII-Leu1461 in the coupling of DIV to DIII. C. Determine the role of Ile397 in DI in LA action. D. Measure the symmetry of the pore at the selectivity filter (DEKA ring), and determine the contribution of selectivity filter residues to electrostatic interactions of charged LA's within the channel. Aim 2. The link between fast inactivation and LA affinity. A. Determine the contribution to LA affinity by interaction of the inactivation lid with the intracellular ends of the S6's and their S4-S5 linkers. B. Correlate the size and flexibility of LA and anticonvulsant drugs to their EC50's in mutant channels with fast inactivation removed by mutagenesis of the inactivation lid. C. Investigate the basis of the recently reported lidociane-induced Brugada Syndrome NaV1.5 channelopathy, V232I+L1308F. Aim 3. LA interactions with the closed channel and with early steps in activation. A. Investigate the mechanism of lipophilic block by the neutral LA, benzocaine, on the S6 segments of closed Na channels. B. Determine the residues at the bundle crossing by crosslinking Cys substitutions between S6 segments. C. Use crosslinked Na channels to better define the LA binding site formed by the S6 segments. PUBLIC HEALTH RELEVANCE: Conduction disorders and arrhythmias of the heart and seizure disorders of the CNS are major contributors to human morbidity and mortality, and the Na channel is profoundly involved in most of these disease processes. The results from the proposed studies investigating local anesthetic/antiarrhythmic drugs and anticonvulsant drugs with Na channels will advance our overall understanding of the molecular mechanisms of drug interactions with voltage-gated ion channels and with the cardiac Na channel in particular. Together, the experimental findings should contribute to the formation of a better molecular model of these drug interactions with the cardiac Na channel, and may contribute to the design of future medications that will better treat diseases involving the Na channel in electrical signaling of excitable tissues.

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