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

Abstract

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...

Research update: Alpha7 nicotinic acetylcholine receptor mechanisms in Alzheimer's disease

“…Interestingly, cholesterol depletion with methylβ-cyclodextrin significantly attenuated these responses, suggesting that Aβ-sensitive α7 nAChRs reside within lipid rafts at presynaptic sites. These findings further reinforce that the lipid composition surrounding nAChR receptor transmembrane domains is an important variable contributing to nAChR functional profiles (74,75…”

Boundary lipids of the nicotinic acetylcholine receptor: Spontaneous partitioning via coarse-grained molecular dynamics simulation

“…Observed partitioning into the l do phase could be considered inconsistent with interpretations of some experiments, (74,95) which suggest minimal nAChR partitioning preference in symmetric model membranes or an actual preference for an l o phase in asymmetric model membranes. These experiments used only monounsaturated acyl chains, and may have had less well-defined domains.…”

“…To test for an intrinsic nAChR domain preference, Barrantes and co-workers checked for enrichment of nAChRs in the detergent resistant membrane (DRM). nAChRs were not enriched in the DRM of a model, domainforming mixture (1:1:1 Chol: palmitoyloleoylphosphatidylcholine(POPC): sphingomyelin) (74) but inducing compositional asymmetry across leaflets did yield nAChR enrichment in the DRM fraction (95) . While more precise and robust experimental methods for determining partitioning preference and specific boundary lipids such as mass spectrometry have been applied for other transmembrane proteins (96,97) , they have not been applied to complex heteromers like nAChR.…”

“…Even in the specific case of cholesterol-nAChR interactions, results from different approaches have suggested complex behavior and even contradictory interpretations. Results have indicated that both cholesterol enrichment (19)(20)(21) and cholesterol depletion (73) cause gain of function, that anionic phospholipids are unnecessary for native function (19)(20)(21) or must be (65,66) included in a reconstitution mixture, that cholesterol increases nAChR clustering (18,47,48) and directly interacts with nAChR (29,32) , but nAChR does not consistently partition into cholesterol-rich domains (74) . We suggest here that some of these apparent contradictions may be explained by competition between cholesterol and other lipids found in native membranes, primarily lipids with polyunsaturated fatty acyl chains (PUFAs).…”

Cholesterol and nicotinic acetylcholine receptor: An intimate nanometer-scale spatial relationship spanning the billion year time-scale

“…A pool of nAChRs has indeed been found in Lo domains in mammalian cells [27,29,80,127,146,154]. In contrast, when reconstituted in a sphingomyelin-cholesterol-POPC (1:1:1) model system, purified nAChR protein from Torpedo was found not to exhibit any preference for Lo domains in vitro [17]. However, inclusion of some sphingomyelin molecular species (brain sphingomyelins, 16:0, 18:0, or 24:1 sphingomyelins) that generate bilayer asymmetry by enriching the sphingolipid content of the outer leaflet of the lipid bilayer appear to favor the partitioning of the nAChR in Lo domains [100].…”