The nicotinic acetylcholine receptor and its surrounding lipids: a long-standing relationship.

ANTOLLINI, S.S.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades Biomédicas; 2017

The muscle nicotinic acetylcholine receptor (AChR) isone of the key players of the post-synaptic componentsin neuromuscular junction. It is an integral membraneprotein that belongs to the Cys-loop superfamily of ligand-gatedion channels and is composed of four subunitsin a pentameric arrangement (a2bgd and a2bed in embryonic and adult muscle of vertebrates, respectively).Each subunit has a large N-terminal extracellular domain,four transmembrane segments (M1-M4), a smallcytoplasmic domain between M3 and M4, and a shortC-terminal extracellular domain. Summing up, the AChRhas two well defined structural domains: the neurotransmitter-bindingsite extracellular domain and the transmembranedomain containing the ion pore. Whereas theextracellular domain is the location site of agonists ordifferent activators/inhibitors, the transmembrane regionexhibits extensive contact with the surrounding lipidsthrough structural motifs remarkably conserved alongphylogenic evolution. It is known that a correct allostericcoupling between both domains is crucial for AChR function,which is strongly dependent on lipid surrounding.We have previously demonstrated that exogenous hydrophobicmolecules, such as free fatty acids or steroids,disturb this coupling through the lipid-AChR interfase. Itis also known that the AChR is present in high-densityclusters at the top of folds in the muscle cell membrane,and that these clusters localize in heterogeneous membranedomains highly enriched in cholesterol (Chol) andsphingolipids. We studied the influence of different lipidhost compositions on the distribution of purified AChRreconstituted in membrane containing Lo domains by fluorescenceresonance energy transfer efficiency betweenthe AChR intrinsic fluorescence and Laurdan or dehydroergosterolfluorescence, and by analyzing the distributionof AChR in detergent-resistant and detergent-solublefractions (1% Triton X-100, 4°C). When the AChR wasreconstituted in a brain sphingomyelin (bSM), Chol andPOPC (1:1:1) model system it lacked preference for Lodomains. However, the change of bSM by 16:0-SM or18:0-SM resulted in the preferential partitioning of AChRin Lo domains, which was not the case with 24:1 SM.Although all these SM formed Lo domains, differences insize, amount and/or lipid order of each Lo domain wereobserved, showing a direct correlation with the tendencyof the AChR to localize in such domains. We further studiedanother membrane condition resulting from inducingtransbilayer asymmetry. Enrichment in bSM in the externalhemilayer resulted in an increase of the amount ofexternal domains with a higher lipid order and a markedincrease of AChR in these Lo domains. Other asymmet-ric conditions were also studied. Thus, a change in theproperties/size/location of Lo domains impacts on theAChR preference for this fraction, clearly indicating thatmembrane lipid surrounding influences both the couplingbetween agonist-binding and channel-gating domainsand the spatial localization of the AChR in the membrane.