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Regulation of cardiac ion channels is essential for functional expression of ion channels and excitability of cardiac cells. Previous results with dystrophin-deficient (mdx) mice showed that disruption of the dystrophin-syntrophin macromolecular complex impairs excitability and impulse propagation by interfering with the interaction between the PDZ domain-binding motif of the cardiac sodium ion channel Nav1.5 and syntrophin at the lateral membrane of cardiomyocytes. In addition, interfering with the association between Nav1.5 and another PDZ domain-containing protein, SAP97 that is located at the intercalated discs, led to reduction of sodium current (INa). To study the in vivo significance of this PDZ domain-binding motif on Nav1.5 function, we created and characterized a homozygous knock-in mouse strain which removed this motif by truncating the last three amino acids, Ser-Ile-Val (ΔSIV), of the Nav1.5 C-terminus. Western blots detected a 25% decrease in Nav1.5 protein level in ΔSIV hearts, while levels of associating proteins were unaffected. Immunostainings revealed loss of Nav1.5 expression at the lateral cardiomyocyte membrane but not at the intercalated discs. This reduction corresponded to a 36% decrease of whole-cell INa in ΔSIV cardiomyocytes and a 62% reduction of INa, specifically at the lateral membrane, in macropatch recordings. A significant slowing of 35% of maximal upstroke velocity of the cardiac action potential was detected. On surface ECGs, ΔSIV mice displayed a pronounced QRS-interval prolongation, indicating impeded ventricular conduction. This was confirmed with optical mapping of ΔSIV hearts showing prolongation of activation time and increased anisotropy due to a preferential decrease in transversal conduction velocity. We also investigated the effects of a specific mutation within the PDZ domain-binding motif of Nav1.5 (p.V2016M) that was found in a patient with Brugada syndrome. This mutation changes the last amino acid of the PDZ domain-binding motif from valine to methionine (SIV to SIM) at the Nav1.5 C-terminus. HEK293 cell transfections of mutant V2016M channels revealed a 24% decrease in cell surface expression compared to WT in biotinylation experiments. Patch-clamp recordings of V2016M mutant channels revealed a 49% decrease in peak INa. Pull-down experiments with Nav1.5 WT or mutated C-terminus GST fusion proteins showed a significant decrease in the interaction of Nav1.5 and SAP97. Taken together, these results emphasize the importance of the Nav1.5 PDZ domain-binding motif in correct ion channel expression, protein interaction, and cardiac function. [ABSTRACT FROM AUTHOR]