Muscarinic receptor stimulation induces translocation of an α-synuclein oligomer from plasma membrane to a light vesicle fraction in cytoplasm

The close correspondence between the distribution of brain alpha -synuclein and that of muscarinic M-1 and M-3 receptors suggests a role for this protein in cholinergic transmission. We thus examined the effect of muscarinic stimulation on alpha -synuclein in SH-SY5Y, a human dopaminergic cell line that expresses this protein. Under basal conditions, alpha -synuclein was detected in all subcellular compartments isolated as follows: plasma membrane, cytoplasm, nucleus, and two vesicle fractions. The lipid fractions contained only a 45-kDa alpha -synuclein oligomer, whereas the cytoplasmic and nuclear fractions contained both the oligomer and the monomer. This finding suggests alpha -synuclein exists physiologically as a lipid-bound oligomer and a soluble monomer. Muscarinic stimulation by carbachol reduced the alpha -synuclein oligomer in plasma membrane over a 30-min period, with a concomitant increase of both the oligomer and the monomer in the cytoplasmic fraction. The oligomer was associated with a light vesicle fraction in cytoplasm that contains uncoated endocytotic vesicles. The carbachol-induced alteration of alpha -synuclein was blocked by atropine. Translocation of the alpha -synuclein oligomer in response to carbachol, stimulation corresponds closely with the time course of ligand-stimulated muscarinic receptor endocytosis. The data suggest that the muscarine receptor stimulated release of the alpha -synuclein oligomer from plasma membrane, and its subsequent association with the endocytotic vesicle fraction may have a role in muscarine receptor endocytosis. We propose that its function may be a transient release of membrane-bound phospholipase D-2 from alpha -synuclein inhibition, thus allowing this lipase to participate in muscarinic receptor endocytosis.