Regulation of Neuronal KATP Channels by Signaling Elicited by cGMP-Dependent Protein Kinase Activation

Abstract

The ATP-sensitive potassium (KATP) channel couples intracellular metabolic state to cell excitability. Recently, we have demonstrated that activation of the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling cascade results in stimulation of Kir6.2/SUR1 (i.e. the neuronal-type KATP) channels. To understand how PKG activation induces plasma-membrane KATP channel stimulation, in the present study we investigated the potential involvement of the mitochondrial KATP (mitoKATP) channel and reactive oxygen species (ROS) in signal transduction. By performing single-channel recordings in transfected human embryonic kidney (HEK) 293 cells and neuroblastoma SH-SY5Y cells, we found that the enhancement of Kir6.2/SUR1 channel currents by PKG activation observed in cell-attached patches was diminished by the selective mitoKATP channel inhibitor 5-hydroxydecanoic acid (5-HD), ROS scavengers, and catalase, an enzyme that decomposes hydrogen peroxide (H2O2). 5-HD, ROS scavengers and catalase also significantly attenuated Kir6.2/SUR1 channel stimulation induced by NO donors. Moreover, bath application of H2O2 increased the activity of Kir6.2/SUR1 channels in cell-attached but not inside-out patches, and the stimulatory effect was not affected by 5-HD, excluding ROS as a signal upstream of the mitoKATP channel to mediate Kir6.2/SUR1 channel stimulation. In addition, H2O2 failed to stimulate tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit in intact cells. Altogether, these novel findings suggest that PKG stimulates neuronal KATP channels via opening of mitoKATP channels and ROS generation in a SUR1 subunit-dependent manner, implicating functional coupling between mitoKATP and plasma-membrane KATP channels upon PKG activation. The NO/cGMP/PKG/mitoKATP/ROS signaling cascade may contribute to neuroprotection under ischemic conditions by enhancing the function of plasma-membrane KATP channels whose activation reduces cell excitability.