Steroids and free fatty acids (FFA) are noncompetitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is purportedly located at the lipid-AChR interface, but their exact mechanism of action is still unknown. Here we studied the effect of structurally different FFA and steroids on the conformational equilibrium of the AChR in Torpedo californica receptor-rich membranes. We took advantage of the higher affinity of the fluorescent AChR open channel blocker, crystal violet, for the desensitized state than for the resting state. Increasing concentrations of steroids and FFA decreased the K D of crystal violet in the absence of agonist; however, only cisunsaturated FFA caused an increase in K D in the presence of agonist. This latter effect was also observed with treatments that caused the opposite effects on membrane polarity, such as phospholipase A 2 treatment or temperature increase (decreasing or increasing membrane polarity, respectively). Quenching by spin-labeled fatty acids of pyrene-labeled AChR reconstituted into model membranes, with the label located at the ␥M4 transmembrane segment, disclosed the occurrence of conformational changes induced by steroids and cis-unsaturated FFA. The present work is a step forward in understanding the mechanism of action of this type of molecules, suggesting that the direct contact between exogenous lipids and the AChR transmembrane segments removes the AChR from its resting state and that membrane polarity modulates the AChR activation equilibrium by an independent mechanism.

tains a relatively large extracellular domain and four hydrophobic membrane-spanning segments referred to as M1-M4, ending in a short extracellular carboxyl terminal domain. Three concentric rings can be distinguished in the AChR transmembrane (TM) region ( 1, 2 ): the M2 TM segments of all subunits outline the inner ring and form the walls of the ion channel proper; M1 and M3 constitute the middle ring; and the M4 segments form the outer ring, which is in closest contact with the AChR lipid microenvironment.One outstanding characteristic of the mature neuromuscular junction (NMJ) is the high density of AChR clusters at the postsynaptic membrane ( 3 ). Neural agrin, an extracellular proteoglycan, initiates the cascade of events in the AChR clustering process in the myotubes by fi rst activating MuSK (muscle-specifi c receptor tyrosine kinase), which subsequently induces the activation of several other intracellular enzymes. Finally, association of AChR with rapsyn, a cytoplasmic peripheral membrane protein colocalized with AChR in vivo, mediates binding to the cytoskeleton ( 3, 4 ), leading in turn to effi cient receptor clustering ( 5 ). Lipids have been postulated to be involved in AChR nanodomain organization and clustering, presumably at early, preinnervation stages of development ( 6 ). The plasma membrane organization of living cells is currently considered to be a mosaic of macroscopic and stable or transient and short-scale segregated domains (reviewed by Ref. 7 ). Lipid domains termed lipid "rafts" are highly enriched in both cholesterol (Chol) and sphingolipids.Abstract The nicotinic acetylcholine receptor (AChR) is in intimate contact with the lipids in its native membrane. Here we analyze the possibility that it is the intrinsic properties of the AChR that determine its partition into a given lipid domain. Torpedo AChR or a synthetic peptide corresponding to the AChR ␥ M4 segment (the one in closer contact with lipids) was reconstituted into "raft "-containing model membranes. The distribution of the AChR was assessed by Triton X-100 extraction in combination with fl uorescence studies, and lipid analyses were performed on each sample. The infl uence of rapsyn, a peripheral protein involved in AChR aggregation, was studied. Raft -like domain aggregation was also studied using membranes containing the ganglioside GM1 followed by GM1 crosslinking. The ␥ M4 peptide displays a marked preference for raft -like domains. In contrast, AChR alone or in the presence of rapsyn or ganglioside aggregation exhibits no such preference for raft-like domains, but it does cause a signifi cant reduction in the total amount of these domains. The results indicate that the distribution of the AChR in lipid domains cannot be due exclusively to the intrinsic physicochemical properties of the protein and that there must be an external signal in native cell membranes that directs the AChR to a specifi c membrane domain. The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane glycoprotein composed of four dif...

The mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ Förster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR.