Cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism

A number of studies have demonstrated that cholera toxin (CT) is found in d etergent-insoluble, cholesterol-enriched domains (rafts) in various cells, including neurons. We now demonstrate that even though CT is associated wit h these domains at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism, at both early and late stage s of neuronal development. CT transport to the Golgi apparatus, and its sub sequent degradation, is inhibited by hypertonic medium (sucrose), and by ch lorpromazine; the former blocks clathrin recruitment, and the latter causes aberrant endosomal accumulation of clathrin. Moreover, both internalizatio n of the transferrin receptor (Tf-R), which occurs via a clathrin-dependent mechanism, and CT internalization, are inhibited to a similar extent by su crose. In contrast, the cholesterol-binding agents filipin and methyl-beta -cyclodextrin have no effect on the rate of CT or Tf-R internalization. Fin ally, once internalized, CT becomes more detergent-soluble, and chlorpromaz ine treatment renders internalized CT completely detergent-soluble. We prop ose two models to explain how, despite being detergent-insoluble at the cel l surface, CT is nevertheless internalized via a raft-independent mechanism in hippocampal neurons.