Membrane lipid rafts are necessary for the maintenance of the alpha 7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons

Calcium-permeable neurotransmitter receptors are concentrated into structur ally and biochemically isolated cellular compartments to localize calcium-m ediated events during neurotransmission. The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopicall y visible clusters, and thus the association of a particular protein with l ipid rafts can result in its redistribution on the cell surface. The presen t study asks whether lipid rafts participate in the formation and maintenan ce of the calcium-permeable alpha7-subunit nicotinic acetylcholine receptor (alpha 7nAChR) clusters found in somatic spines of ciliary neurons. Lipid rafts and alpha 7nAChR become progressively colocalized within somatic spin es during synaptogenesis. To determine whether these rafts are required for the maintenance of alpha 7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-beta -cyclodextrin. T his treatment caused the dispersion of lipid rafts and the redistribution o f alpha 7nAChR into small clusters over the cell surface, suggesting that t he integrity of lipid rafts is required to maintain the receptor clustering . However, lipid raft dispersion also caused the depolymerization of the F- actin cytoskeleton, which can also tether the receptor at specific sites. T o assess whether interaction between rafts and alpha 7nAChR is independent of F-actin filaments, the lipid raft patches were stabilized with a combina tion of the cholera toxin B subunit (CTX), which specifically binds to the raft component ganglioside GM1, and an antibody against CTX. The stabilized rafts were then treated with latrunculin-A to depolymerize F-actin. Under these conditions, large patches of CTX persisted and were colocalized with alpha 7nAChR, indicating that the aggregates of receptors can be maintained independently of the underlying F-actin cytoskeleton. Moreover, it was fou nd that the alpha 7nAChR is resistant to detergent extraction at 4 degrees C and floats with the caveolin-containing lipid-rich fraction during densit y gradient centrifugation, properties that are consistent with a direct ass ociation between the receptor and the membrane microdomains.