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

Sean G. Brown, Alison Thomas, Lodewijk V. Dekker, Andrew Tinker, Joanne L. Leaney

Research output: Contribution to journalArticlepeer-review

30 Citations (Scopus)


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-δ mediates channel inhibition because recombinant PKC-δ inhibited channel activity, M3-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-δ constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-δ 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-δ after M3 receptor activation in HEK-293 cells.
Original languageEnglish
Pages (from-to)C543-C556
Number of pages14
JournalAmerican Journal of Physiology - Cell Physiology
Issue number3
Publication statusPublished - 27 Apr 2005
Externally publishedYes


  • A kinase anchoring protein
  • Phosphatidylinositol 4,5-bisphosphate
  • Phorbol 12-myristate 13-acetate
  • Receptor for activated C kinase
  • Carbachol
  • 5'-N-ethylcarboxyamidoadenosine


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