No abstract

The structural and functional properties of the nicotinic acetylcholine receptor (AChR), the archetype molecule in the superfamily of Cys-looped ligand-gated ion channels, are strongly dependent on the lipids in the vicinal microenvironment. The influence on receptor properties is mainly exerted by the AChR-vicinal ("shell" or "annular") lipids, which occur in the liquid-ordered phase as opposed to the more disordered and "fluid" bulk membrane lipids. Fluorescence studies from our laboratory have identified discrete sites for fatty acids, phospholipids, and cholesterol on the AChR protein, and electron-spin resonance spectroscopy has enabled the establishment of the stoichiometry and selectivity of the shell lipid for the AChR and the disclosure of lipid sites in the AChR transmembrane region. Experimental evidence supports the notion that the interface between the protein moiety and the adjacent lipid shell is the locus of a variety of pharmacologically relevant processes, including the action of steroids and other lipids. I surmise that the outermost ring of M4 helices constitutes the boundary interface, most suitable to convey the signals from the lipid microenvironment to the rest of the transmembrane region, and to the channel inner ring in particular.

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.