Hypoxia Inhibits Kv1.5 Currents Through Reactive Oxygen Species-Mediated Post Translational Cysteine Modification
Kv1.5 , ROS , Reactive Oxygen Species , AF , Atrial Fibrillation , Ultra-Rapid Delayed Rectifier Potassium Current , HEK
The voltage-gated potassium channel Kv1.5 plays an important role in atrial repolarization and vascular tone of pulmonary arteries. Dysfunction of Kv1.5 channels can lead to atrial fibrillation and pulmonary hypertension, both of which are associated with hypoxia. In the present study, we investigated the effects of hypoxia on Kv1.5 channels. We induced hypoxia by culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in hypoxic conditions (0.5% O2) and then recorded Kv1.5 current using whole-cell voltage clamp. We observed that 3 hours of hypoxic culture reduced current after 3 hours. Using immunofluorescent staining and western blot analysis, we found that hypoxic culture-induced decrease in current was not caused by the internalization of mature Kv1.5 channels from the membrane. However, we found that hypoxia-induced current reduction is mediated through reactive oxygen species (ROS). First, hydrogen peroxide (H2O2) treatment decreased current in a manner similar to hypoxic culture. Second, the addition of dithiothreitol (DTT), a reducing agent, abolished the effects of hypoxia-induced current reduction. Therefore, our results demonstrate that disrupting ROS-mediated posttranslational modification abolishes the hypoxia-induced reduction in Kv1.5 current. Furthermore, hypoxia-induced reduction in Kv1.5 current required specific cysteine residues within the Kv1.5 channel; using point mutations, our results showed that mutation of Cys581 abolished hypoxia-induced current reduction. Therefore, we have demonstrated that hypoxia decreases IKv1.5 through the production of ROS and that the subsequent posttranslational modifications involve Cys581.