Stimulation of neuronal K_ATP channels by cGMP-dependent protein kinase: involvement of ROS and 5-hydroxydecanoate-sensitive factors in signal transduction

Abstract

Recently, we demonstrated that neuronal KATP channels are functionally enhanced by activation of a nitric oxide (NO)/cGMP/ cGMP-dependent protein kinase (PKG) signaling cascade. In this study, we further investigated the intracellular mechanism underlying PKG stimulation of neuronal K ATP channels. By performing singlechannel recordings in transfected HEK293 and neuroblastoma SH-SY5Y cells, we found that the increase of Kir6.2/SUR1 (i.e., the neuronal-type KATP) channel currents by PKG activation in cellattached patches was diminished by 5-hydroxydecanoate (5-HD), an inhibitor of the putative mitochondrial K ATP channel; N-(2-mercaptopropionyl)glycine, a reactive oxygen species (ROS) scavenger, and catalase, a hydrogen peroxide (H 2O2)-decomposing enzyme. These reagents also ablated NO-induced K ATP channel stimulation and prevented the shifts in the single-channel open-and closed-time distributions resulting from PKG activation and NO induction. Bath application of H 2O2 reproduced PKG stimulation of Kir6.2/SUR1 but did not activate tetrameric Kir6.2LRKR368/369/370/371AAAA channels. Moreover, neither the PKG activator nor exogenous H 2O2 was able to enhance the function of KATP channels in the presence of Ca 2ϩ chelators and calmodulin antagonists, whereas the stimulatory effect of H 2O2 was unaffected by 5-HD. Altogether, in this report we provide novel evidence that activation of PKG stimulates neuronal K ATP channels by modulating intrinsic channel gating via a 5-HDsensitive factor(s)/ROS/Ca 2ϩ /calmodulin signaling pathway that requires the presence of the SUR1 subunit. This signaling pathway may contribute to neuroprotection against ischemic injury and regulation of neuronal excitability and neurotransmitter release by modulating the function of neuronal K ATP channels. nitric oxide; hydrogen peroxide; single channel; patch clamp; adenosine 5=-triphosphate-sensitive potassium channel THE ATP-SENSITIVE POTASSIUM (K ATP ) channel serves as an important metabolic sensor by coupling intracellular metabolic status to changes in transmembrane potassium fluxes and cell excitability (4, 51, 54). Cellular functions regulated by these channels include neuronal excitability, neurotransmitter and hormone release, vascular tone, heart rate, and protection of neurons and cardiomyocytes under metabolic stress (66). The K ATP channel is an octameric protein (9, 68) composed of four pore-forming subunits (Kir6.2 or Kir6.1) (34, 62), which are members of the inwardly rectifying potassium channel family, and four regulatory subunits (the sulfonylurea receptors SUR1, SUR2A, or SUR2B) (1, 35, 37), which are members of the ATP-binding cassette protein superfamily. K ATP channels are widely distributed, and their molecular compositions exhibit tissue specificity. For instance, K ATP channels in central neurons are composed of Kir6.2 and SUR1 subunits, whereas in cardiac and skeletal muscles they are composed of Kir6.2 and SUR2A subunits (35, 56). The Kir6.2 subunit possesses ATP binding sites responsible for channel inhibition (...

Activation of cGMP-Dependent Protein Kinase Stimulates Cardiac ATP-Sensitive Potassium Channels via a ROS/Calmodulin/CaMKII Signaling Cascade

“…The single-channel conductance remained the same. The specificity of KT5823 at 1 µM to selectively inhibit activation of PKG but not that of PKA has been verified in our recent study [25]. Our new data thus indicate that zaprinast stimulated the cardiac-type K ATP channel Kir6.2/SUR2A via activation of a cGMP/PKG mechanism in intact cells.…”

“…PKG activation has been demonstrated to account for NO-induced ROS generation in rat cardiomyocytes as well as the anti-infarct effect of NO in intact, isolated heart [36], which suggests that ROS generation may be induced by PKG activation in cardiac tissues. Interestingly, our recent findings also suggest that ROS are required for the acute, stimulatory action of PKG on neuronal-type K ATP channels [25]. Would ROS serve as intermediate signals inducible upon activation of PKG to mediate an acute effect of the enzyme on cardiac-type K ATP channels?…”

“…Our recent study suggests that calmodulin mediates PKG stimulation of neuronal K ATP channels in intact cells [25]. Whether calmodulin is required for the functional effect of PKG activation on cardiac-type K ATP channels is not known.…”

A cGMP-dependent protein kinase (PKG) controls synaptic transmission tolerance to acute oxidative stress at the Drosophila larval neuromuscular junction

“…In addition to being a classic oxidative stress paradigm, H 2 O 2 has been shown to modulate neuromuscular activity in vertebrates (Giniatullin and Giniatullin 2003) and manipulate neuronal signal transduction in mammalian cell culture (Chai and Lin 2010). This study exposed Drosophila larval NMJ preparations to 2.25 mM H 2 O 2 and demonstrates that PKG manipulation alters synaptic transmission tolerance to acute oxidative toxicity.…”

“…The distinct mechanisms underlying the PKG pathway's potential for modulating neuronal function and ultimately neuroprotection during acute trauma remain only partially understood. Previous published data indicate a link between K ϩ conductance and PKG activity (Chai and Lin 2010;Dawson-Scully et al 2010;Renger et al 1999;White et al 1993), which leads to speculation that these ion channels are potential downstream targets of this intracellular signaling mechanism. However, which specific K ϩ channel(s) mediate PKG pathway effects is still a matter of debate.…”