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Please use this identifier to cite or link to this item: http://hdl.handle.net/10373/221

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Title: PKC-{delta} sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells
Authors: Brown, Sean G.
Thomas, Alison
Dekker, Lodewijk V.
Tinker, Andrew
Leaney, Joanne L.
Affiliation: University of Abertay Dundee. School of Social and Health Sciences
Keywords: Phosphatidylinositol 4,5-bisphosphate
Phorbol 12-myristate 13-acetate
Receptor for activated C kinase
A kinase anchoring protein
Issue Date: 27-Apr-2005
Publisher: American Physiological Society
Type: Journal Article
Refereed: peer-reviewed
Rights: Published version (c)American Physiological Society, available from DOI: 10.1152/ajpcell.00025.2005
Citation: Brown, S. G., et al. 2005. PKC-{delta} sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells. American Journal of Physiology: Cell Physiology. 289: pp.543-556. [Online] Available from: DOI: 10.1152/ajpcell.00025.2005
Abstract: G protein-gated inward rectifier (Kir3) channels are inhibited by activation of Gq/11-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP2). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M3 receptor activation. In perforated patch mode, staurosporine prevented the Gq/11-mediated, M3 receptor, inhibition of channel activity. Recovery from M3-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP2, were still irreversibly inhibited by M3 receptor stimulation. When adenosine A1 receptors were co-expressed, inclusion of PIP2 rescued the A1-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-{delta} mediates channel inhibition because recombinant PKC-{delta} inhibited channel activity, M3-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-{delta} constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-{delta} to the plasma membrane on M3 receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-{delta} after M3 receptor activation in HEK-293 cells.
URI: http://hdl.handle.net/10373/221
ISSN: 0363-6143
Appears in Collections:Social & Health Sciences Collection

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