Background:14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial.Aims:In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5.Methods and Results:Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded byYWHAH) co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The amplitude of Nav1.5-mediated sodium peak current density significantly increased upon co-expression with difopein.Conclusions:Our findings illustrate that the direct implication of 14-3-3 in regulating Nav1.5 is not evident in the heterologous expression system used here. Potentiation of Nav1.5-mediated current by difopein could be explained by the inhibition of 14-3-3/ligand interactions with other partner proteins of the channel complex (e.g., PKA, PKC, CaMKII) and should be further investigated.