Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

Abstract

The distribution of nicotinic acetylcholine receptor (AChR) clusters at the cell membrane was studied in CHO-K1/A5 cells using fluorescence microscopy. Di-4-ANEPPDHQ, a fluorescent probe that differentiates between liquid-ordered (Lo) and liquid-disordered (Ld) phases in model membranes, was used in combination with monoclonal anti-AChR antibody labeling of live cells, which induces AChR clustering. The so-called generalized polarization (GP) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with or devoid of antibody-induced AChR clusters, respectively. AChR clusters were almost equally distributed between Lo and Ld domains, independently of receptor surface levels and agonist (carbamoylcholine and nicotine) or antagonist (α-bungarotoxin) binding. Cholesterol depletion diminished the cell membrane mean di-4-ANEPPDHQ GP and the number of AChR clusters associated with Ld membrane domains increased concomitantly. Depolymerization of the filamentous actin cytoskeleton by Latrunculin A had the opposite effect, with more AChR clusters associated with Lo domains. AChR internalized via small vesicles having lower GP and lower cholesterol content than the surface membrane. Upon cholesterol depletion, only 12% of the AChR-containing vesicles costained with the fluorescent cholesterol analog fPEG-cholesterol, i.e., AChR endocytosis was essentially dissociated from that of cholesterol. In conclusion, the distribution of AChR submicron-sized clusters at the cell membrane appears to be regulated by cholesterol content and cytoskeleton integrity.

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu

“…Together with Toshihide Kobayashi we employed fPEGcholesterol to follow the dynamics of cholesterol internalization and to test whether nAChR endocytosis followed the same route. Most of the internalized receptor followed a pathway different from that of fPEG-cholesterol (Kamerbeek et al, 2013), in agreement with our early studies with Satyajit Mayor showing that the receptor utilized a RAC-dependent, clathrin-and dynamin-independent endocytic route (Kumari et al, 2008).…”

“…In parallel, we used a dual-channel recording setup to measure di-4-ANEPPDHQ GP in different regions of the plasma membrane ( Figure 1 ). The nAChR showed a punctiform distribution in both Ld and Lo domains ( Kamerbeek et al, 2013 ). We observed a decrease in di-4-ANEPDHQ GP values upon cholesterol depletion, a change that was correlated with the diminution of nAChR aggregates associated with Lo domains ( Figure 1 ).…”

Alzheimer’s Disease as a Membrane Disorder: Spatial Cross-Talk Among Beta-Amyloid Peptides, Nicotinic Acetylcholine Receptors and Lipid Rafts

“…Furthermore, Chol depletion affects the maintenance of the nAChR in the plasma membrane by several mechanisms. Treatments of cells with methyl-β-cyclodextrin, which extracts Chol from the membrane, enhanced nAChR internalization by endocytosis with a marked decrease of the number of nAChR domains, concomitantly with a gain-of-function of the remaining nAChR (Borroni et al, 2007; Borroni and Barrantes, 2011; Kamerbeek et al, 2013). Furthermore, chronic treatments with mevinolin, an inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase and hence of Chol synthesis, inhibited the trafficking of the receptor toward the membrane surface, which caused low nAChR cell-surface expression, and increased the intracellular nAChR pools (Pediconi et al, 2004).…”

Lovastatin Differentially Regulates α7 and α4 Neuronal Nicotinic Acetylcholine Receptor Levels in Rat Hippocampal Neurons

“…An increase in α4 nAChR protein stability may explain the increase observed here. nAChRs are very sensitive to membrane cholesterol levels; ion translocation properties, membrane domain localization, rate and mechanism of internalization, and exocytic trafficking are strongly dependent on membrane cholesterol in muscle nAChR [ 13 , 14 , 15 , 16 , 19 ]. The results presented in this paper indicate that the cholesterol dependence may be a hallmark not only of the muscle-type nAChR but of the entire nAChR family.…”

“…Cholesterol in particular is necessary for the maintenance of nAChRs at the plasmalemma and for ion translocation [ 4 ]. Previous studies have shown that cholesterol levels modulate the trafficking, membrane domain localization, and function of muscle-type nAChR heterologously expressed in CHO-K1/A5 cells and in endogenously expressed nAChR in C2C12 myotubes [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Little is known, however, on the effects of cholesterol modulation on neuronal nAChR.…”