The position of the double bond in monounsaturated free fatty acids is essential for the inhibition of the nicotinic acetylcholine receptor.

Free fatty acids (FFAs) are non-competitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is supposedly located at the lipid-AChR interface. To elucidate the mechanism involved in this antagonism, we studied the effect that FFAs with a single double-bond at different positions (ω6, ω9, ω11 and ω13 cis-18:1) have on different AChR properties. Electrophysiological studies showed that only two FFAs (ω6 and ω9) reduced the duration of the channel open-state. The briefest component of the closed-time distribution remained unaltered, suggesting that ω6 and ω9 behave as allosteric blockers. Fluorescence resonance energy transfer studies indicated that all FFAs locate at the lipid-AChR interface, ω6 being restricted to annular sites and all others occupying non-annular sites. The perturbation of the native membrane order by FFAs was evaluated by DPH (1,6-diphenyl-1,3,5-hexatriene) and Laurdan fluorescence polarization studies, with the greatest decrease observed for ω9 and ω11. AChR conformational changes produced by FFAs present at the lipid bilayer were evaluated by fluorescence quenching studies of pyrene-labeled AChR and also using the AChR conformational-sensitive probe crystal violet. All cis-FFAs produced AChR conformational changes at the transmembrane level, but only ω9, ω11 and ω13 perturbed the resting state. Thus, the position and isomerism of the torsion angle of unsaturated FFAs are probably a key factor in terms of AChR blockage, suggesting that FFAs with a unique cis double bond at a superficial position inside the membrane directly inhibit AChR function by perturbing a potential conserved core structure for AChR gating at that level.

Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution.

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 fluorescence studies, and lipid analyses were performed on each sample. The influence 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 gammaM4 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 significant 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 specific membrane domain.

Modulation of nicotinic acetylcholine receptor conformational state by free fatty acids and steroids.

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 cis-unsaturated 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 gammaM4 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.