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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Identification of subdomains in NADPH oxidase-4 critical for the oxygen-dependent regulation of TASK-1 K⁺ channels

Department of ¹Physiology, Seoul National University College of Medicine, Department of ²Pharmacology, Seoul National University College of Medicine, ³Ischemia/Hypoxia Disease Institute, and ⁴Kidney Research Institute, Medical Research Center, Seoul National University, Seoul, Republic of Korea

Submitted 8 September 2008 ; accepted in final form 21 July 2009

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-I_TASK-1). To examine whether reactive oxygen species (ROS) mediate the hypoxia-I_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-I_TASK-1. Moreover, the hypoxia-I_TASK-1 was neither recovered by H₂O₂ treatment nor prevented by antioxidant such as ascorbic acid. However, the hypoxia-I_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-I_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.

hypoxia; p22; background K⁺ current; carbon monoxide