If H2O2is generated endogenously after, and hence positioned downstream of, activation of ERK1/2 and CaMKII, the effectiveness of exogenous H2O2to stimulate sarcKATPchannels should not be compromised by suppression of either kinase. opener pinacidil in rabbit ventricular myocytes, through reducing the event and the dwelling time of the long closed claims whilst increasing the rate of recurrence of channel opening; in contrast, all these changes were SLC7A7 reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2potentiated pinacidil-preactivated sarcKATPchannel activity in undamaged cardiomyocytes, but the H2O2-induced KATPchannel activation was obliterated when ERK1/2 or CaMKII activity was suppressed, implying that H2O2is definitely situated upstream of ERK1/2 and CaMKII for KATPchannel modulation. Furthermore, genetic ablation (i.e. knockout) of CaMKII, the predominant cardiac CaMKII isoform, diminished ventricular sarcKATPchannel activation elicited by activation of PKG, unveiling CaMKII as a crucial player. Additionally, evidence from kinase activity and Western blot analyses exposed that activation of NOPKG signalling augmented CaMKII activity in rabbit ventricular KPT276 myocytes and, importantly, CaMKII activation KPT276 by PKG occurred in KPT276 an ERK1/2-dependent manner, placing ERK1/2 upstream of CaMKII. Taken together, these findings suggest that NO modulates ventricular sarcKATPchannels via a novel sGCcGMPPKGROS(H2O2)ERK1/2calmodulinCaMKII ( isoform in particular) signalling cascade, which heightens KATPchannel activity by destabilizing the long closed says while facilitating closed-to-open state transitions. This pathway may contribute to regulation of cardiac excitability and cytoprotection against ischaemiareperfusion injury, in part, by opening myocardial sarcKATPchannels. == Introduction == Vital in the adaptive response to (patho)physiological stress, the ATP-sensitive potassium (KATP) channel functions as a high-fidelity metabolic sensor, which couples intracellular metabolic state to membrane excitability (Ashcroft,1988; Miki & Seino,2005; Nichols,2006) and serves a homeostatic role ranging from blood glucose regulation to cardioprotection (Olson & Terzic,2010). The KATPchannel is usually a hetero-octameric protein composed of four inwardly rectifying potassium channel subunits (Kir6.x) and four sulphonylurea receptors (SURx; Shyng & Nichols,1997; Babenkoet al.1998), whose molecular (subunit) composition exhibits tissue specificity. For example, in cardiac (ventricular) and skeletal muscles the KATPchannels are composed of Kir6.2 and SUR2A subunits (Inagakiet al.1996; Okuyamaet al.1998), whereas in central neurons and pancreatic -cells they consist of Kir6.2 and SUR1 subunits KPT276 (Aguilar-Bryanet al.1998). Whilst it is appreciated that KATPchannels are directly regulated by intracellular ATP, MgADP (Nichols,2006) and phosphatidylinositol-4,5-bisphophate (PIP2; Fan & Makielski,1997; Baukrowitzet al.1998; Shyng & Nichols,1998), how these important channels are modulated by more complicated intracellular signalling processes is far less comprehended. The gaseous messenger nitric oxide has a fundamental biological role in protecting the heart against ischaemiareperfusion injury (Bolli,2001). It has been suggested that NO shortens action potential duration (APD)90and increases maximal diastolic potential in the heart, by activating sarcolemmal KATP(sarcKATP) channels via a cGMP-dependent mechanism (Bakeret al.2001). Nitric oxide also potentiates the action KPT276 of potassium channel openers (KCOs) around the KATPchannel in single ventricular cells, yet with conflicting findings on whether cGMP is usually involved (Shinbo & Iijima,1997; Hanet al.2002). The intracellular mechanism by which NO modulates cardiac KATPchannels has remained largely unknown. In the present study, we combined single-channel patch-clamp recordings with pharmacological and biochemical approaches to delineate the intracellular signalling mechanism responsible for NO modulation of cardiac sarcKATPchannels. Human embryonic kidney (HEK) 293 cells expressing recombinant cardiac-type KATP(i.e. Kir6.2/SUR2A) channels and ventricular cardiomyocytes freshly isolated from adult rabbits as well as from CaMKII gene-null and wild-type mouse models expressing endogenous KATPchannels were used. Specifically, we investigated the involvement in NO signal transduction of soluble guanylyl cyclase (sGC), cGMP-dependent protein kinase (PKG), reactive oxygen species (ROS), hydrogen peroxide (H2O2), calmodulin, calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated protein kinase (ERK)1/2 of the mitogen-activated protein kinase (MAPK) family. Here we show that functional modulation of ventricular sarcKATPchannels by NO induction is usually mediated by intracellular signalling via a novel sGCcGMPPKGROS(H2O2)ERK1/2calmodulinCaMKII (CaMKII isoform in particular) signalling pathway that alters the open and closed properties of the channel, enhancing channel activity. == Methods == == Ethical approval == All protocols involving animals were approved by.