Post-transcriptional gene silencing of KChIP2 and Navβ1 in neonatal rat cardiac myocytes reveals a functional association between Na and Ito currents

The Ca2+-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 Ca2+-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 beta 1 subunit modulates the gating properties of Kv4.3 to closely recapitulate native Ito suggesting that Na-v beta 1 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 beta 1 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 beta 1 subunit mRNA levels, leaving Kv4.x mRNAs unaltered, but reducing levels of immunoreactive proteins. Post-transcriptional gene silencing of Na-v beta 1 reduced its protein expression. Silencing of Na-v beta 1 also reduced mRNA and protein levels of its alpha-subunit, Na(v)1.5. Surprisingly, silencing of Na-v beta 1 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. (C) 2008 Elsevier Inc. All rights reserved.