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

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 M-1-mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M-1-mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl[phenyl-4-H-3]-benzilate ([H-3]-QNB) whereas the hydrophilic probe, 1-[N-methyl-H-3] scopolamine ([H-3]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M-1-mAChR antibody also detected both cell-surface and intracellular M-1-mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca2+ mobilization were completely inhibited by a cell-impermeable M-1 antagonist, muscarinic toxin -7 and the G(q/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 G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M-1-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.