Cholesterol influences ion-channel function, distribution and clustering in the membrane, endocytosis, and exocytic sorting of the nicotinic acetylcholine receptor (AChR). We report the occurrence of a cholesterol recognition motif, here coined “CARC”, in the transmembrane regions of AChR subunits that bear extensive contact with the surrounding lipid, and are thus optimally suited to convey cholesterol-mediated signaling from the latter. Three cholesterol molecules could be docked on the transmembrane segments of each AChR subunit, rendering a total of 15 cholesterol molecules per AChR molecule. The CARC motifs contribute each with an energy of interaction between 35 and 52 kJ.mol⁻¹, adding up to a total of about 200 kJ.mol⁻¹ per receptor molecule, i.e. ∼40% of the lipid solvation free energy/ AChR molecule. The CARC motif is remarkably conserved along the phylogenetic scale, from prokaryotes to human, suggesting that it could be responsible for some of the above structural/functional properties of the AChR.

Novel effects of cholesterol (Chol) on nicotinic acetylcholine receptor (AChR) cell-surface stability, internalization and function are reported. AChRs are shown to occur in the form of submicron-sized (240–280 nm) domains that remain stable at the cell-surface membrane of CHO-K1/A5 cells over a period of hours. Acute (30 min, 37°C) exposure to methyl-β-cyclodextrin (CDx), commonly used as a diagnostic tool of endocytic mechanisms, is shown here to enhance AChR internalization kinetics in the receptor-expressing clonal cell line. This treatment drastically reduced (∼50%) the number of receptor domains by accelerating the rate of endocytosis (t_1/2 decreased from 1.5–0.5 h). In addition, Chol depletion produced ion channel gain-of-function of the remaining cell-surface AChR, whereas Chol enrichment had the opposite effect. Fluorescence measurements under conditions of direct excitation of the probe Laurdan and of Förster-type resonance energy transfer (FRET) using the intrinsic protein fluorescence as donor both indicated an increase in membrane fluidity in the bulk membrane and in the immediate environment of the AChR protein upon Chol depletion. Homeostatic control of Chol content at the plasmalemma may thus modulate cell-surface organization and stability of receptor domains, and fine tune receptor channel function to temporarily compensate for acute AChR loss from the cell surface.

Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined "CARC"). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC-CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif-protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution.