title: Quantitative properties and receptor reserve of the DAG and PKC branch of G(q)-coupled receptor signaling.
abstract: Gq protein-coupled receptors (GqPCRs) of the plasma membrane activate the phospholipase C (PLC) signaling cascade. PLC cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers diacylgycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), leading to calcium release, protein kinase C (PKC) activation, and in some cases, PIP2 depletion. We determine the kinetics of each of these downstream endpoints and also ask which is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels in single living tsA-201 cells. We measure DAG production and PKC activity by Förster resonance energy transfer-based sensors, and PIP2 by KCNQ2/3 channels. Fully activating endogenous purinergic receptors by uridine 5'triphosphate (UTP) leads to calcium release, DAG production, and PKC activation, but no net PIP2 depletion. Fully activating high-density transfected muscarinic receptors (M1Rs) by oxotremorine-M (Oxo-M) leads to similar calcium, DAG, and PKC signals, but PIP2 is depleted. KCNQ2/3 channels are inhibited by the Oxo-M treatment (85%) and not by UTP (<1%), indicating that depletion of PIP2 is required to inhibit KCNQ2/3 in response to receptor activation. Overexpression of A kinase-anchoring protein (AKAP)79 or calmodulin (CaM) does not increase KCNQ2/3 inhibition by UTP. From these results and measurements of IP3 and calcium presented in our companion paper (Dickson et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210886), we extend our kinetic model for signaling from M1Rs to DAG/PKC and IP3/calcium signaling. We conclude that calcium/CaM and PKC-mediated phosphorylation do not underlie dynamic KCNQ2/3 channel inhibition during GqPCR activation in tsA-201 cells. Finally, our experimental data provide indirect evidence for cleavage of PI(4)P by PLC in living cells, and our modeling revisits/explains the concept of receptor reserve with measurements from all steps of GqPCR signaling.
authors: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.
authors: Björn H
authors: BH
authors: Falkenburger
authors: Eamonn J
authors: EJ
authors: Dickson
authors: Bertil
authors: B
authors: Hille
mesh_terms: D054758:A Kinase Anchor Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019852:Diacylglycerol Kinase / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D051658:KCNQ2 Potassium Channel / Q000378:metabolism; D051659:KCNQ3 Potassium Channel / Q000378:metabolism; D007700:Kinetics; D015778:Minor Histocompatibility Antigens; D054801:Phosphoinositide Phospholipase C / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D043643:Receptor, Muscarinic M1 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015290:Second Messenger Systems; D015398:Signal Transduction
chemical_list: D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D002147:Calmodulin; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; D051659:KCNQ3 Potassium Channel; C496916:KCNQ3 protein, human; D015778:Minor Histocompatibility Antigens; D043643:Receptor, Muscarinic M1; D011956:Receptors, Cell Surface; D015544:Inositol 1,4,5-Trisphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; C402182:phosphatidylinositol phosphate 4-kinase; D011493:Protein Kinase C; D054801:Phosphoinositide Phospholipase C; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium
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