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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

PKC- sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M₃ receptor activation in HEK-293 cells

Department of Medicine, University College of London, London, United Kingdom

Submitted 20 January 2005 ; accepted in final form 12 April 2005

G protein-gated inward rectifier (Kir3) channels are inhibited by activation of G_q/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 G_q/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- mediates channel inhibition because recombinant PKC- inhibited channel activity, M₃-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC- constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC- 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- after M₃ receptor activation in HEK-293 cells.

phosphatidylinositol 4,5-bisphosphate; phorbol 12-myristate 13-acetate; receptor for activated C kinase; A kinase anchoring protein; carbachol; 5'-N-ethylcarboxyamidoadenosine

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