Sequestration of Muscarinic Cholinergic Receptors in Permeabilized Neuroblastoma Cells

: The feasibility of using a permeabilized preparation of human SH‐SY‐5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin‐O, or the α‐toxin from Staphylococcus aureus to oxotremorine‐M (Oxo‐M) for 30 min resulted in a 25–30% reduction in the number of cell surface mAChRs, as monitored by the loss of N[3H]methyl‐ scopolamine ([3H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 μM digitonin, the Oxo‐M‐mediated reduction in [3H]NMS binding was time (t1/2∼ 5 min) and concentration (EC50∼ 10 μM) dependent and was agonist specific (Oxo M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [3H]quinuclidinyl benzilate, occurred following Oxo‐M treatment. The loss of [3H]NMS sites observed in the presence of Oxo‐M was unaffected by omission of either ATP or Ca2+, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5′‐O‐(2‐thiodiphosphate). mAChRs sequestered in response to Oxo‐M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 μM). The results indicate (a) that permeabilized SH‐SY‐5Y cells support an agonist‐induced sequestration of mAChRs, the magnitude of which is ∼ 65–70% of that observed for intact cells, (b) that when internalized, mAChRs are located in a cellular compartment to which [3H]NMS has only a limited access despite the removal of the plasma membrane barrier, and (c) that the production of phosphoinositide‐derived second messengers is not a prerequisite for mAChR sequestration.