regulation of M₁ muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

• Published in: Journal of neurochemistry 2007-12, Vol.103 (6), p.2268-2280
• Main Authors: Willets, Jonathon M, Nelson, Carl P, Nahorski, Stefan R, Challiss, R.A. John
• Format: Article
• Language: English
• Subjects: Animals; Animals, Newborn; Biochemistry; Biological and medical sciences; Calcium Signaling - drug effects; Calcium Signaling - physiology; Calmodulin - drug effects; Calmodulin - metabolism; Cell culture; Cell receptors; Cell structures and functions; Cells, Cultured; Central nervous system; Central neurotransmission. Neuromudulation. Pathways and receptors; Enzyme Activation - drug effects; Enzyme Activation - physiology; Fundamental and applied biological sciences. Psychology; G protein-coupled receptor kinase; G-Protein-Coupled Receptor Kinase 2 - genetics; G-Protein-Coupled Receptor Kinase 2 - metabolism; GABA Antagonists - pharmacology; hippocampal neuron; Hippocampus - drug effects; Hippocampus - metabolism; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); muscarinic acetylcholine receptor; Muscarinic Agonists - pharmacology; Neurology; Neurons - drug effects; Neurons - metabolism; Neurotransmitters; Phosphorylation - drug effects; protein kinase C; Protein Kinase C - drug effects; Protein Kinase C - metabolism; Protein Kinases - drug effects; Protein Kinases - metabolism; Protein Transport - drug effects; Protein Transport - physiology; Rats; receptor desensitization; Receptor, Muscarinic M1 - drug effects; Receptor, Muscarinic M1 - metabolism; Signal transduction; Synapses - drug effects; Synapses - metabolism; synaptic activity; Synaptic Transmission - drug effects; Synaptic Transmission - physiology; Vertebrates: nervous system and sense organs
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/j.1471-4159.2007.04931.x

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.