Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization

Desensitization of human muscarinic acetylcholine receptor m2 subtypes (hm2 receptors) stably expressed in chinese hamster ovary cells was measured as decreases in the carbamylcholine-stimulated [S-35]GTP gamma S binding activity in membrane preparations after pretreatment of cells with carbamylcholine. The extent of carbamylcholine-stimulated [S-35]- GTP gamma S binding activity was found to decrease to 64% following pretreatment of cells with 10 mu M carbamylcholine for 30 min, and under the same conditions 51-59% of hm2 receptors were sequestered/internalized as assessed by decreases in the [H-3] N-methylscopolamine binding activity on the cell surface, A similar reduction in the carbamylcholine-stimulated [S-35] GTP gamma S binding activity was observed by pretreatment of cells with 5 nM propylbenzylylcholine mustard, which irreversibly bound to and inactivated 58% of the hm2 receptors, When the cells were pretreated with 10 mu M carbamylcholine in the presence of 0.32 M sucrose, which is known to inhibit clathrin-mediated endocytosis, no sequestration/ internalization of hm2 receptors was observed, and the extent of carbamylcholine-stimulated [S-35]GTP gamma S binding activity did not change. These results indicate that desensitization of hm2 receptors may be caused by reduction of receptor number on the cell surface through sequestration/internalization rather than by loss of the function of receptors.