Cell-surface and internalized nicotinic acetylcholine receptor platforms distribute between liquid-ordered and liquid-disordered cholesterol sensitive domains

Abstract

The correlation between the physical state of the membrane andnicotinic acetylcholine receptor (AChR) clusters was studied inCHO-K1/A5 cells using fluorescence microscopy. For this purposedi-4-ANEPPDHQ, a fluorescent probe that differentiates liquid-ordered (Lo) from liquid-disordered (Ld) phases in model mem-branes (Jin et al., 2005), was used in combination with AChRlabeling of live cells with anti-AChR antibodies. The latter results inthe formation of AChR spots visible with conventional wide-fieldmicroscopy, consisting of nanometer-sized clusters which can beresolved by superresolution microscopy (Kellner et al., 2007). Theso-called generalized polarization (‘‘GP’’) of di-4-ANEPPDHQ wasmeasured in regions of the cell-surface membrane associated with ordevoid of AChR spots, respectively. Under control conditionsAChR spots were almost equally distributed between Lo and Lddomains. This distribution was independent of AChR surface levelsand antagonist binding. Cholesterol depletion mediated by methyl-b-cyclodextrin treatment produced a diminution in the mean GP ofdi-4-ANEPPDHQ in the plasmalemma and a concomitant increasein the number of AChR spots associated with Ld membranedomains. Disruption of the actin cytoskeleton with Latrunculin Ahad the opposite effect. The fate of AChR-lipid domain associationwas further followed upon internalization of the fluorescent spots.Upon endocytosis, the AChR was found in large, aggregatedvesicles and in small, individual vesicles. AChR-containing largevesicles had a higher GP value and a higher cholesterol content, asevidenced by the fluorescence of the probe fPEG-cholesterol. Uponcholesterol depletion, AChR internalization occurred via smallvesicles, and no colocalization with fPEG-cholesterol was observed.The association of AChR aggregates with lipid domains of differentcholesterol content may thus bear on the organization of the AChRat the cell surface and on the ensuing receptor endocytic routes(Borroni et al., 2007; Kumari et al., 2008; Borroni and Barrantes,2011)