The Ca(2+)-independent transient outward potassium current (I(to)) encoded by the Kv4 family of potassium channels, is central to normal repolarization of cardiac myocytes. KChIPs are a group of Ca(2+)-binding accessory subunits that modulate Kv4-encoded currents. However, the biophysical effects of KChIP2 on Kv4 currents raise questions about the role that KChIP2 plays in forming the native I(to). Previous heterologous expression studies demonstrated that the Na channel beta1 subunit modulates the gating properties of Kv4.3 to closely recapitulate native I(to) suggesting that Na(v)beta1 may modulate the function of Kv4-encoded channels in native cardiomyocytes. Therefore we hypothesized the existence of a structural or functional complex between subunits of I(to) and I(Na). In co-immunoprecipitation of proteins from neonatal rat ventricular myocardium (NRVM), Na(v)beta1 was pulled-down by Kv4.x antibodies suggesting a structural association between subunits that comprise I(to) and I(Na). Remarkably, post-transcriptional gene silencing of KChIP2 in NRVM, using small interfering RNAs specific to KChIP2, suppressed both cardiac I(to) and I(Na) consistent with a functional coupling of these channels. KChIP2 silencing suppressed Na channel alpha and beta1 subunit mRNA levels, leaving Kv4.x mRNAs unaltered, but reducing levels of immunoreactive proteins. Post-transcriptional gene silencing of Na(v)beta1 reduced its protein expression. Silencing of Na(v)beta1 also reduced mRNA and protein levels of its alpha-subunit, Na(v)1.5. Surprisingly, silencing of Na(v)beta1 also produced a reduction in KChIP2 mRNA and protein as well as Kv4.x proteins resulting in remarkably decreased I(Na) and I(to). These data are consistent with a novel structural and functional association of I(Na) and I(to) in NRVMs.