Intracellular distribution of functional M1-muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Authors

  • Junsuke Uwada,

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    • These authors contributed equally to this work.

  • Abu Syed Md Anisuzzaman,

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    • These authors contributed equally to this work.

  • Atsushi Nishimune,

    1. Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, and Organization for Life Science Advancement Programs, School of Medicine, University of Fukui, Eiheiji, Fukui, Japan
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  • Hatsumi Yoshiki,

    1. Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, and Organization for Life Science Advancement Programs, School of Medicine, University of Fukui, Eiheiji, Fukui, Japan
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  • Ikunobu Muramatsu

    1. Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, and Organization for Life Science Advancement Programs, School of Medicine, University of Fukui, Eiheiji, Fukui, Japan
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Address correspondence and reprint requests to Ikunobu Muramatsu, Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, University of Fukui School of Medicine, 23-3 Matsuoka-Shimoaizuki, Eiheiji, Fukui 910-1193, Japan. E-mail: muramatu@u-fukui.ac.jp

Abstract

J. Neurochem. (2011) 118, 958–967.

Abstract

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M1-mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M1-mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-3H]-benzilate ([3H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-3H] scopolamine ([3H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M1-mAChR antibody also detected both cell-surface and intracellular M1-mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca2+ mobilization were completely inhibited by a cell-impermeable M1 antagonist, muscarinic toxin -7 and the Gq/11 inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of Gq/11 (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M1-mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.

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