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The regulation of M 1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons 1
Author(s) -
Willets Jonathon M.,
Nelson Carl P.,
Nahorski Stefan R.,
Challiss R. A. John
Publication year - 2007
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2007.04931.x
Subject(s) - picrotoxin , muscarinic acetylcholine receptor m1 , muscarinic acetylcholine receptor m2 , oxotremorine , muscarinic acetylcholine receptor , chemistry , muscarinic acetylcholine receptor m5 , medicine , protein kinase c , endocrinology , agonist , biology , muscarinic acetylcholine receptor m3 , microbiology and biotechnology , receptor , gabaa receptor , biochemistry , signal transduction
To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M 1 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 EC 50 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 M 1 mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA A receptors with picrotoxin. The picrotoxin‐mediated effect on M 1 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‐G q/11 α‐binding, catalytically‐inactive D110A,K220R G protein‐coupled receptor kinase 2 mutant, decreased the extent of M 1 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 Ca 2+ /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 Ca 2+ /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 Ca 2+ /calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M 1 mACh receptor desensitization in neurons.