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

KAMEERBECK C.B; BORRONI M.V; PEDICONI MF; BARRANTES F.J.

Buenos Aires

Congreso; Synapses and dendritic spines in health and disease; 2012

International Society for Neurochemistry

Cell-surface and internalized nicotinic acetylcholine receptor platforms distribute between liquid-ordered and liquid-disordered cholesterol sensitive domains C. B. Kamerbeek1, M. V. Borroni1, M. F. Pediconi1, F. J. Barrantes2 1Instituto de Investigaciones Bioquı´micas de Bahı´a Blanca, Consejo de Investigaciones Cientı´ficas y Te´cnicas (CONICET) / Universidad Nacional del Sur, Bahı´a Blanca 2Laboratory of Molecular Neurobiology, Institute of Biomedical Research, Catholic University of Argentina, and CONICET, 1107 Buenos Aires, Argentina The correlation between the physical state of the membrane and nicotinic acetylcholine receptor (AChR) clusters was studied in CHO-K1/A5 cells using fluorescence microscopy. For this purpose di-4-ANEPPDHQ, a fluorescent probe that differentiates liquidordered (Lo) from liquid-disordered (Ld) phases in model membranes (Jin et al., 2005), was used in combination with AChR labeling of live cells with anti-AChR antibodies. The latter results in the formation of AChR spots visible with conventional wide-field microscopy, consisting of nanometer-sized clusters which can be resolved by superresolution microscopy (Kellner et al., 2007). The so-called generalized polarization (??GP??) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with or devoid of AChR spots, respectively. Under control conditions AChR spots were almost equally distributed between Lo and Ld domains. This distribution was independent of AChR surface levels and antagonist binding. Cholesterol depletion mediated by methylb- cyclodextrin treatment produced a diminution in the mean GP of di-4-ANEPPDHQ in the plasmalemma and a concomitant increase in the number of AChR spots associated with Ld membrane domains. Disruption of the actin cytoskeleton with Latrunculin A had the opposite effect. The fate of AChR-lipid domain association was further followed upon internalization of the fluorescent spots. Upon endocytosis, the AChR was found in large, aggregated vesicles and in small, individual vesicles. AChR-containing large vesicles had a higher GP value and a higher cholesterol content, as evidenced by the fluorescence of the probe fPEG-cholesterol. Upon cholesterol depletion, AChR internalization occurred via small vesicles, and no colocalization with fPEG-cholesterol was observed. The association of AChR aggregates with lipid domains of different cholesterol content may thus bear on the organization of the AChR at the cell surface and on the ensuing receptor endocytic routes (Borroni et al., 2007; Kumari et al., 2008; Borroni and Barrantes, 2011).