Routes of delivery of muscarinic acetylcholine receptors to the plasma membrane in NG108-15 cells

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Abstract
1 We have examined the routes of delivery of muscarinic acetylcholine receptors to the plasma membrane in unstimulated and agonist-stimulated NG108-15 cells. Delivery of receptors to the plasma membrane was measured by irreversibly alkylating receptors already at the cell surface with propyl-<***>benzilylcholine mustard (PrBCM) and then following the recovery of binding of the polar radioligand [3H]-N-methylscopolamine ([3H]-NMS) in intact cells. 2 In unstimulated cells, recovery of [3H]-NMS binding after 2 h of incubation at 37°C was 30% of binding in control cells. Binding affinity of [3H]-NMS was unchanged. In cells that had been pre-exposed to carbachol (0.5 mm) for 30 min, initial [3H]-NMS binding was reduced by 38% but recovery of binding was increased from 30% to 43% of control binding. 3 When the cells were pre-incubated for 1 h with the protein synthesis inhibitor cycloheximide (20 μg ml−1), recovery of [3H]-NMS binding was reduced by similar extents in unstimulated (30% to 9%) and carbachol-stimulated (43% to 19%) cells. Incubation of the cells at 20°C instead of 37°C reduced recovery of [3H]-NMS binding from 30% to 9% in unstimulated cells and from 43% to 23% in carbachol-stimulated cells. 4 Depletion of cellular ATP by addition of antimycin (50 nm) and deoxyglucose (50 mm), reduced recovery of binding to 12% in unstimulated cells and to 6% in carbachol-stimulated cells. 5 These results indicate that in unstimulated NG108-15 cells, delivery of muscarinic receptors to the plasma membrane is almost exclusively through the synthetic pathway. In agonist-stimulated cells, receptor sequestration into an intracellular compartment (probably endosomes) occurs. Under these circumstances, delivery of receptors to the plasma membrane along the synthetic route is unaffected but an additional route of delivery (recycling) now operates.