| ATP-sensitive potassium (KATP) channels are gated by intracellular ATP and ADP, and serve as a metabolic sensor to couple cellular metabolic status and energetic demands with cell membrane electrical activity. The activity of KATP channels has been implicated in hormone release, cardiovascular adaptive response to stress, maintenance of neuronal electrical stability and protection of neurons, among others. The function of the KATP channel is also regulated by cellular modulators in addition to adenine nucleotides. In this dissertation project, I aim to understand how the cGMP/cGMP-dependent protein kinase (PKG) signaling mechanism modulates the function of K ATP channels.;In the first part of project, by performing single-channel recordings in human embryonic kidney (HEK) 293 cells and human neuroblastoma SH-SY5Y cells transiently transfected with Kir6.2/SUR1 (i.e., pancreatic/neuronal KATP) channels, I uncovered that the elevation of intracellular cGMP levels by applying either a cGMP analogue 8-Br-cGMP or zaprinast, a cGMP-specific phosphodiesterase (PDE) inhibitor, increased the activity of Kir6.2/SUR1 channels in intact cells. The obligatory role of the PKG kinase activity was demonstrated by using selective PKG kinase inhibitors. Interestingly, the application of purified PKG proteins directly to the intracellular surface of Kir6.2/SUR1 channels in excised membrane patches resulted in current suppression rather than stimulation, suggesting that in addition to an indirect, stimulatory action, PKG also suppresses KATP channels, possibly by direct phosphorylation of the channel or its associated regulatory proteins. Moreover, neither the direct inhibition nor the indirect stimulation by PKG was observed on the Kir6.2LRKR368/369/370/371AAAA channel expressed without the SUR subunit, implying that the SUR subunit of the channel is responsible for the dual effects of PKG. Furthermore, exogenously generated nitric oxide (NO) renders the Kir6.2/SUR1 channel activation in both cell types. This study thus suggests that PKG exerts dual regulation of neuronal KATP channels, and the NO/soluble guanylyl cyclase (sGC)/cGMP/PKG signaling pathway positively modulates the function of neuronal/pancreatic-type KATP channels.;In the second part of the project, I further explored the signaling mechanism downstream of PKG that leads to the stimulation of the Kir6.2/SUR1 channel. Using electrophysiological and pharmacological tools to study KATP channels expressed in both transfected HEK and SH-SY5Y cells, I found that the PKG stimulation of Kir6.2/SUR1 channels was disrupted by mitochondrial KATP (mitoKATP) channel blockers, reactive oxygen species (ROS) scavengers and catalase, a decomposing enzyme of hydrogen peroxide (H 2O2). Moreover, H2O2 was capable of evoking KATP channel activation, and the effect was not prevented by mitoKATP channel blockers. Furthermore, NO stimulation of the Kir6.2/SUR1 channels also depends on the activities of ROS and 5-HD sensitive factor (possibly the mitoKATP channel). Notably, H2O 2 stimulates Kir6.2/SUR1 channels in an indirect manner in intact cells, which, reminiscent of the indirect action of PKG, requires the SUR subunit. This study thus suggested a novel NO/cGMP/PKG/5-HD-sensitive factor/ROS (H2O2) signaling pathway responsible for the NO/PKG-induced stimulation of pancreatic/neuronal-type KATP channels. |
