Identification of subdomains in NADPH oxidase-4 critical for the oxygen-dependent regulation of TASK-1 K+ channels.
Hypoxic inhibition of K+ current is a critical O2-sensing mechanism. Previously, it was demonstrated that the cooperative action of TASK-1 and NADPH oxidase-4 (NOX4) mediated the O2-sensitive K+ current response. Here we addressed the O2-sensing mechanism of NOX4 in terms of TASK-1 regulation. In TASK-1 and NOX4-coexpressing human embryonic kidney 293 cells, hypoxia (5% O2) decreased the amplitude of TASK-1 current (hypoxia-DeltaI(TASK-1)). To examine whether reactive oxygen species (ROS) mediate the hypoxia-DeltaI(TASK-1), we treated the cells with carbon monoxide (CO) which is known to reduce ROS generation from the heme-containing NOX4. Unexpectedly, CO failed to mimic hypoxia in TASK-1 regulation, rather blocked the hypoxia-DeltaI(TASK-1). Moreover, the hypoxia-DeltaI(TASK-1) was neither recovered by H2O2 treatment nor prevented by antioxidant such as ascorbic acid. However, the hypoxia-DeltaI(TASK-1) was noticeably attenuated by succinyl acetone, a heme synthase inhibitor. To further evaluate the role of heme, we constructed and expressed various NOX4 mutants, such as HBD(-) lacking the heme binding domain, NBD(-) lacking the NADPH binding domain, FBD(-) lacking the FAD binding domain, and HFBD(-) lacking both heme and FAD domains. The hypoxia-DeltaI(TASK-1) was significantly reduced in HBD(-)-, FBD(-)-, or HFBD(-)-expressing cells, versus wild-type NOX4-expressing cells. However, NBD(-) did not affect the TASK-1 response to hypoxia. We also found that p22 is required for the NOX4-dependent TASK-1 regulation. These results suggest that O2 binding with NOX4 per se controls TASK-1 activity. In this process, the heme moiety and FBD seem to be responsible for the NOX4 regulation of TASK-1, and p22 might support the NOX4-TASK-1 interaction.
Park
Su Jung
SJ
Department of Physiology, Seoul National University College of Medicine, Seoul National University, Seoul, Republic of Korea.
Chun
Yang-Sook
YS
Park
Kyung Sun
KS
Kim
Sung Joon
SJ
Choi
Si-On
SO
Kim
Hye-Lim
HL
Park
Jong-Wan
JW
D002248:Carbon Monoxide / Q000378:metabolism / Q000494:pharmacology; D015687:Cell Hypoxia; D002460:Cell Line; D004789:Enzyme Activation; D005294:Ferrochelatase / Q000378:metabolism; D006801:Humans; D009154:Mutation; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D010100:Oxygen / Q000502:physiology*; D024683:Potassium Channels, Tandem Pore Domain / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species / Q000378:metabolism
D009419:Nerve Tissue Proteins; D024683:Potassium Channels, Tandem Pore Domain; D017382:Reactive Oxygen Species; C420132:potassium channel subfamily K member 3; D002248:Carbon Monoxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D005294:Ferrochelatase; D010100:Oxygen