PubMed 23630338

Referenced in: none

Automatically associated channels: Kv7.2

Title: Quantitative properties and receptor reserve of the DAG and PKC branch of G(q)-coupled receptor signaling.

Authors: Björn H Falkenburger, Eamonn J Dickson, Bertil Hille

Journal, date & volume: J. Gen. Physiol., 2013 May , 141, 537-55

PubMed link:

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., 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.