ABSTRACT: This project seeks to characterize a novel mechanism of cardiac action potential (AP) conduction and develop new approaches to treat arrhythmias based on the insights gained. We have reported that the Nav1.5 subunit of the Na+ channel is concentrated at the Cx43 (Gja1) gap junction (GJ) edge in an intercalated disk nanodomain called the perinexus. The high concentration of Na+ channels and narrow inter- membrane width at the perinexus are consistent with theoretical predictions of a structural unit that may support ephaptic conduction. The hypothesis tested is that the ? subunit of the Na+ channel complex is the central organizing element of this unit: The ephapse. Our premise is that the adhesion function of ?1 (Scn1b) promotes the formation of specialized regions of inter-membrane contact within intercalated disks. This arrangement is envisaged as enabling trans-interacting Na+ channels to form between myocytes, facilitating cell-to-cell conduction of AP. Furthermore, we propose that agonists that maintain ephapse adhesion could constitute new therapies to mitigate arrhythmogenic conduction defects following myocardial infarction. In support of the hypothesis, data is provided from super-resolution, electron, and immuno-electron microscopy and smart patch clamp (SPC) studies that Nav1.5 and ?1 subunits, as well as active Na+ channels, are concentrated in the perinexus. Moreover, we have developed a novel ?1 inhibitor - a 19 amino acid mimetic of the adhesion domain of ?1 (?adp1). In electric cell-substrate impedance sensing assays, ?adp1 caused loss of adhesion between cells heterologously over-expressing ?1. Infusion of ?adp1 into hearts resulted in dose-dependent: (1) Widening of the perinexal cleft consistent with ?1 de-adhesion; (2) Conduction slowing; and (3) Increased ventricular arrhythmia incidence. Importantly, while inhibition of ?1 adhesion appeared to have no effect on whole-cell Na+ currents, SPC revealed a selective decrease in GJ-associated Na+ channel activity. Finally, preliminary data is shown for a rationally designed agonist of ?1-mediated trans-interaction (dbl-?adp) that promotes adhesion between ?1 over-expressing cells. To test the hypotheses that: (1) ?1 is required for stabilizing trans-interacting Na+ channels at the ephapse; and (2) ?1-mediated adhesion is an anti-arrhythmic target: Aim 1 will determine the requirement of ?1 and Cx43 GJs for the hypothesized ephaptic mechanism. Mice encoding Scn1b and Gja1 null alleles, together with the ?1 trans-adhesion inhibitor ?adp1, will be used in studies of the structure and autonomous function of the hypothesized ephapse. Aim 2 will determine the role of ?1 adhesion on Na+ channel activity and remodeling and AP conduction in myocyte monolayers. A GJ-plaque-forming, but functionally incompetent Cx43 mutant (L90V) will be used in a strategy to study the contribution of the ephaptic mechanism to conduction independent of GJ coupling. Aim 3 will develop efficient molecules that stabilize ?1 adhesion based on our first-generation agonist dbl-?adp and test the anti-arrhythmic efficacy of these compounds in the pro-arrhythmic setting of acute myocardial infarction.