Pore-forming (Nav1.5) and auxiliary (β1; SCN1b) subunits of cardiac sodium channels are enriched at the cardiomyocyte intercalated disk (ID). Mathematical models suggest that this may facilitate conduction via ephaptic mechanisms. We previously demonstrated anisotropic conduction slowing during acute interstitial edema (AIE), possibly due to weakened ephaptic coupling. Here we assessed Nav1.5 and β1 localization to ID microdomains using electron microscopy (EM) and super-resolution microscopy (gSTED, STORM) and used optical mapping and computer modeling to investigate the implications for ephaptic conduction in the heart. gSTED and STORM revealed Nav1.5 and β1 enrichment within ID regions not containing dense clusters of Cx43 and N-Cadherin. Notably, both were identified within the perinexus, a microdomain surrounding Cx43 gap junctions. Overall, 22% of Nav1.5 was located within perinexal regions while only 2% was within Cx43 clusters. EM revealed closer membrane apposition at perinexal ( 10nm) under control conditions. AIE increased intermembrane distance at perinexal, but not at non-perinexal sites. Functionally, this correlated with decreased transverse conduction velocity (CV-T; 15.2±0.3 vs. 19.6±0.1cm/s) and increased anisotropic ratio (AR; 3.0±0.2 vs. 2.8±0.1) relative to control, in perfused guinea pig ventricles. Next, we investigated AIE effects on Nav1.5 function in conduction. Nav1.5 blockade (0.5 µM flecainide) by itself decreased CV (18%) without changing AR. However, Nav1.5 inhibition during AIE preferentially decreased CV-T (13.0±0.6cm/s), increased AR (3.3±0.2) and increased spontaneous arrhythmias (7/9 vs. 4/11) compared to AIE alone. Notably, only a computer model including ephaptic coupling and the ID localization of Nav1.5 could recapitulate these results. In summary, sodium channel complexes localized to ID microdomains such as the perinexus may enable ephaptic conduction in the heart. Further, Nav1.5 functional availability and perinexal membrane spacing emerge as novel determinants of anisotropic conduction.