Abstract
Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically isolated cellular compartments to localize calcium-mediated events during neurotransmission. The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopically visible clusters, and thus the association of a particular protein with lipid rafts can result in its redistribution on the cell surface. The present study asks whether lipid rafts participate in the formation and maintenance of the calcium-permeable alpha7-subunit nicotinic acetylcholine receptor (alpha7nAChR) clusters found in somatic spines of ciliary neurons. Lipid rafts and alpha7nAChR become progressively colocalized within somatic spines during synaptogenesis. To determine whether these rafts are required for the maintenance of alpha7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-beta-cyclodextrin. This treatment caused the dispersion of lipid rafts and the redistribution of alpha7nAChR into small clusters over the cell surface, suggesting that the 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. To assess whether interaction between rafts and alpha7nAChR is independent of F-actin filaments, the lipid raft patches were stabilized with a combination 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 alpha7nAChR, indicating that the aggregates of receptors can be maintained independently of the underlying F-actin cytoskeleton. Moreover, it was found that the alpha7nAChR is resistant to detergent extraction at 4 degrees C and floats with the caveolin-containing lipid-rich fraction during density gradient centrifugation, properties that are consistent with a direct association between the receptor and the membrane microdomains.