Abstract
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC50 concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M1 mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABAA receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-Gq/11α-binding, catalytically-inactive D110A,K220RG protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCε, but not Ca2+/diacylglycerol-sensitive eGFP-PKCβII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCβII, but not PKCε, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.
Highlights
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture
Effects of synaptic activity on M1 muscarinic acetylcholine (mACh) receptor desensitization Our previous work has extensively characterized the desensitization of M1 mACh receptors in immature (< 10 days in vitro, hereafter DIV), non-synaptically active hippocampal neurons (Willets et al 2004, 2005)
While agonist addition caused minimal translocation of eGFP-PKCbII, picrotoxin treatment (100 lmol/L) induced rapid, transient eGFP-PKCbII translocations to the plasma membrane, which mirrored the changes in intracellular [Ca2+] (Fig. 8c). Recent studies from this laboratory have provided strong evidence that endogenous GRK2 and GRK6 can regulate the responsiveness of M1 mACh receptor signalling in cultured rat hippocampal neurons (Willets et al 2004, 2005)
Summary
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Picrotoxin-driven translocation of myristoylated, alaninerich C kinase substrate was accompanied by translocation of PKCbII, but not PKCe, and was dependent on PKC and Ca2+/ calmodulin Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons. In the current study we have investigated mechanisms of regulation in mature hippocampal neurons that form synaptic interconnexions in culture (Bacci et al 1999) and in which glutamate-mediated synaptic activity can be dramatically increased by suppressing inhibitory GABAergic inputs with picrotoxin, or suppressed with tetrodotoxin (TTx) (Bacci et al 1999; Nash et al 2004; Young et al 2005). Our current data suggest that increased synaptic activity enhances hippocampal M1 mACh receptor desensitization through mechanisms involving endogenous GRK2 and Ca2+-sensitive protein kinase C (PKC) isoenzyme(s)
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