PKC Activation Decreases Kv1.5 Protein Expression through Accelerating Endocytic Channel Degradation

Abstract

The voltage-gated potassium channel Kv1.5 is primarily expressed in the atria and conducts the ultra-rapidly delayed rectifier K+ current (IKur), which is important for atrial repolarization and closely related to atrial fibrillation. Elevated stress is associated with several cardiovascular outcomes, including atrial fibrillation in which Kv1.5 is thought to play a role. Since protein kinase C (PKC) activation is associated with stress, we examined the effects of PKC activation on Kv1.5 channels. Previous studies have shown that PKC regulates Kv1.5 channel when co-expressed with Kvβ1.2 or Kvβ1.3. In the present study, using Western blot analysis and whole-cell patch clamp recording in human embryonic kidney cells stably expressing Kv1.5 alone, we found that PKC activation by phorbol 12-myristate 13-acetate (PMA) at 10 nM nearly abolished Kv1.5 channel expression and current within 3 hours. Inhibition of PKC by Bisindolylmaleimide I completely prevented PMA-induced Kv1.5 channel reduction. Co-immunoprecipitation experiments showed that PMA treatment enhanced ubiquitination of Kv1.5 channels; proteasome inhibitors MG-132 or lactacystin partially prevented the PMA-induced Kv1.5 channel reduction. We also found that transfection of dominant-negative Rab5 partly impeded PMA-induced Kv1.5 channel reduction. Furthermore, truncation of the N-terminus of Kv1.5 up to residue 209 (ΔN209) abolished PMA-mediated reduction in expression and current of the channel. Intriguingly, deletion of a distinct motif in the N-terminus for non-receptor tyrosine kinase Src also abolished PMA-mediated reduction in Kv1.5 expression and current. We propose that interplay between PKC and Src mediates degradation of mature Kv1.5 channels partially through ubiquitination and Rab5-mediated endocytosis. These findings extend our understanding of the regulation of Kv1.5 channel by PKC activation. (Supported by the Canadian Institutes of Health Research).