Kim, Eun Young, Anderson, Marc, Wilson, Cory ORCID: 0000-0003-3948-3831, Hagmann, Henning, Benzing, Thomas and Dryer, Stuart E. (2013). NOX2 interacts with podocyte TRPC6 channels and contributes to their activation by diacylglycerol: essential role of podocin in formation of this complex. Am. J. Physiol.-Cell Physiol., 305 (9). S. C960 - 12. BETHESDA: AMER PHYSIOLOGICAL SOC. ISSN 1522-1563

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Abstract

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathophysiology of glomerular diseases. TRPC6 channels are typically activated by diacylglycerol (DAG) during PLC-dependent transduction cascades. TRPC6 channels can also be activated by reactive oxygen species (ROS). We previously showed that podocin is required for DAG analogs to produce robust activation of TRPC6 channels in podocytes. Here we show that endogenous TRPC6 channels in immortalized podocytes reciprocally coimmunoprecipitate with the catalytic subunit of the NADPH oxidase NOX2 (gp91(phox)). The NOX2-TRPC6 interaction was not detected in cells stably expressing a short hairpin RNA targeting podocin, although NOX2 and TRPC6 were present at normal levels. Application of a membrane-permeable DAG analog [1-oleoyl-2-acetylsn- glycerol (OAG)] increased generation of ROS in podocytes, but this effect was not detected in podocin knockdown cells. OAG also increased steady-state surface expression of the NOX2 regulatory subunit p47(phox). In whole cell recordings, TRPC6 activation by OAG was reduced in podocytes pretreated with the NOX2 inhibitor apocynin, by the pan-NOX inhibitor diphenylene iodonium, and by tempol, a ROS quencher. Cholesterol depletion and disruption of lipid rafts by methyl-beta-cyclodextrin reduced activation of podocyte TRPC6 channels by OAG and also eliminated the NOX2-TRPC6 interaction as assessed by coimmunoprecipitation. These data suggest that active NOX2 assembles with TRPC6 at podocin-organized sterol-rich raft domains and becomes catalytically active in response to DAG. The localized production of ROS contributes to TRPC6 activation by chemical stimuli such as DAG. Podocin appears to be necessary for assembly of the NOX2-TRPC6 complex in lipid rafts.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Kim, Eun YoungUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Anderson, MarcUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wilson, CoryUNSPECIFIEDorcid.org/0000-0003-3948-3831UNSPECIFIED
Hagmann, HenningUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Benzing, ThomasUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Dryer, Stuart E.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-472706
DOI: 10.1152/ajpcell.00191.2013
Journal or Publication Title: Am. J. Physiol.-Cell Physiol.
Volume: 305
Number: 9
Page Range: S. C960 - 12
Date: 2013
Publisher: AMER PHYSIOLOGICAL SOC
Place of Publication: BETHESDA
ISSN: 1522-1563
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
GLOMERULAR SLIT DIAPHRAGM; GENERATING NADPH OXIDASES; CELL-FREE SYSTEM; PLASMA-MEMBRANE; MEDIATED ACTIVATION; SURFACE EXPRESSION; OXIDATIVE STRESS; LIPID RAFTS; CHOLESTEROL; PROTEINMultiple languages
Cell Biology; PhysiologyMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/47270

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