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sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels following M3 receptor activation in HEK293 cells
1 Medicine, University College London, London, United Kingdom
* To whom correspondence should be addressed. E-mail: rmhasgb{at}ucl.ac.uk.
G protein-gated inward rectifier (Kir3) channels are inhibited by activation of Gq/11-coupled receptors and this has been postulated to involve the signalling 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 HEK293 cells following 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, labelled phosphate is incorporated into the channel. We suggest that PKC- 
mediates channel inhibition since recombinant PKC- inhibited channel activity, M3-mediated inhibition of the channel was counteracted by overexpression of 2 types of dominant negative PKC- constructs and, using confocal microscopy, we have demonstrated translocation of GFP-tagged PKC- to the plasma membrane upon M3 receptor stimulation. Thus, Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent upon the activity of PKC- following M3-receptor activation in HEK293 cells.
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