A membrane microdomain called raft has been under extensive study since the
assembly of various signal-transducing molecules into this region has been
envisaged. This domain is isolated as a low buoyant membrane fraction afte
r the extraction with a nonionic detergent such as Triton X-100. The charac
teristic low density of this fraction is ascribed to the enrichment of seve
ral lipids including cholesterol. To clear the molecular mechanism of raft
formation, several extraction methods were applied to solubilize raft compo
nents. Cholesterol extraction using methyl-beta-cyclodextrin was found to b
e effective to solubilize NAP-22, a neuron-enriched Ca2+-dependent calmodul
in-binding protein as well as one of the main protein components of brain r
aft. Purified NAP-22 bound to the liposomes that were made from phosphatidy
lcholine and cholesterol. This binding was dependent on the amount of chole
sterol in liposomes. Calmodulin inhibited this binding in a dose-dependent
manner. These results suggest that the presence of a calcium-dependent regu
latory mechanism works on the assembly of raft within the neuron.