Cholesterol Modulates the Rate and Mechanism of Acetylcholine Receptor Internalization

Virginia Borroni,Francisco J Barrantes

doi:10.1074/jbc.m110.211870

Virginia Borroni, Francisco J Barrantes

Open Access

https://doi.org/10.1074/jbc.m110.211870

Copy DOI

Abstract

Stability of the nicotinic acetylcholine receptor (AChR) at the cell surface is key to the correct functioning of the cholinergic synapse. Cholesterol (Chol) is necessary for homeostasis of AChR levels at the plasmalemma and for ion translocation. Here we characterize the endocytic pathway followed by muscle-type AChR in Chol-depleted cells (Chol(-)). Under such conditions, the AChR is internalized by a ligand-, clathrin-, and dynamin-independent mechanism. Expression of a dominant negative form of the small GTPase Rac1, Rac1N17, abolishes receptor endocytosis. Unlike the endocytic pathway in control CHO cells (1), accelerated AChR internalization proceeds even upon disruption of the actin cytoskeleton. Under Chol(-) conditions, AChR internalization is furthermore found to require the activity of Arf6 and its effectors Rac1 and phospholipase D. The Arf6-dependent mechanism may constitute the default endocytic pathway followed by the AChR in the absence of external ligands, membrane Chol levels acting as a key homeostatic regulator of cell surface receptor levels.

Highlights

acetylcholine receptor (AChR) internalization in the CHO-K1/A5 clonal cell line and C2C12 muscle cells was recently reported to occur via a novel endocytic mechanism [1] that does not involve the canonical dynamin, clathrin, or caveolin pathways [6]
We have recently found that cholesterol (Chol), an endogenous lipid present at the postsynaptic membrane, contributes to the homeostasis of receptor levels at the plasmalemma [9, 10]
In order to verify whether CDx or its complex with Chol did modify cell membrane Chol content, we monitored changes in the fluorescence of the fluorescent Chol analog fPEG-Chol after treatment with CDx or Chol1⁄7CDx. fPEG-Chol is a soluble derivative of Chol that preferentially partitions into Chol-rich membrane domains, acting as a Chol sensor in cells [19]

Summary

EXPERIMENTAL PROCEDURES

Materials—Methyl-␤-cyclodextrin (CDx), nystatin, latrunculin A, and cytochalasin D were purchased from Sigma. Alexa Fluor-labeled ␣BTX and Alexa Fluor-labeled antibodies were purchased from Molecular Probes, Inc. MAb210 antibody against the main immunogenic region of the ␣ subunit was a gift from Dr J. Lindstrom (University of Pennsylvania Medical Center, Philadelphia, PA). The plasmid coding for dynK44A-HA was provided by S.

Cholesterol Modulation of AChR Endocytosis

RESULTS

DISCUSSION