Functional Modulation of Cardiac ATP-Sensitive Potassium Channels by Nitric Oxide via Intracellular Signaling

Abstract

ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. Nitric oxide (NO) has been shown to stimulate cardiac KATP channels; however, the mechanistic details remain poorly understood. Here we sought to delineate the intracellular mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in combination with pharmacological, genetic and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the cardiac-type KATP) channels in transfected HEK293 cells, which was abolished by selective suppression of cGMP-dependent protein kinase (PKG), extracellular signal-regulated protein kinase (ERK)1/2, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and reactive oxygen species (ROS) (hydrogen peroxide H2O2 in particular), respectively. Importantly, NO donors potentiated function of sarcKATP channels preactivated by the channel opener pinacidil in adult rabbit ventricular myocytes, through destabilizing the longest closed state and facilitating opening transitions, and the potentiation was nullified when PKG, calmodulin, CaMKII or ERK1/2 was inhibited. Exogenous H2O2 also stimulated ventricular sarcKATP channels in intact cells in an ERK1/2- and CaMKII-dependent manner. Genetic ablation of CaMKIIδ, the predominant cardiac CaMKII isoform, diminished PKG stimulation of mouse ventricular sarcKATP channels (compared with wild-type controls). Kinase activity and Western blot assays further supported that NO-PKG activation augmented CaMKII activity in ventricular myocytes, which was mediated by ERK1/2. Collectively, we demonstrate that NO stimulates ventricular sarcKATP channels via a cGMP/PKG/ROS(H2O2)/ERK/calmodulin/CaMKII signaling cascade that alters channel gating. This novel signaling pathway may control cardiac excitability and mediate, in part, cytoprotection against ischemia-reperfusion injury, by opening myocardial KATP channels.