title: Lipid raft localization of GABA A receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells.
abstract: The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na(+), K(+)-ATPase, was found in discrete rafts of lower density, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy "hot spots" at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-beta-cyclodextrin. In contrast, both the GABA(A) receptor and Na(+), K(+)-ATPase were largely soluble in ice cold Triton X-100. This indicates that Brij 98 extraction defines an unusual type of cholesterol-independent lipid rafts that harbour membrane proteins also associated with underlying scaffolding/cytoskeletal proteins such as gephyrin (GABA(A) receptor) and ankyrin G (Na(+), K(+)-ATPase). By providing an ordered membrane microenvironment, lipid rafts may contribute to the clustering of the GABA(A) receptor and the Na(+), K(+)-ATPase at distinct functional locations on the cell surface.
authors.affiliation: Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, 3 Blegdamsvej, DK-2200 Copenhagen N, Denmark.
authors.forename: Stine-Mathilde
authors.initials: SM
authors.lastname: Dalskov
authors.forename: Lissi
authors.initials: L
authors.lastname: Immerdal
authors.forename: Lise-Lotte
authors.initials: LL
authors.lastname: Niels-Christiansen
authors.forename: Gert H
authors.initials: GH
authors.lastname: Hansen
authors.forename: Arne
authors.initials: A
authors.lastname: Schousboe
authors.forename: E Michael
authors.initials: EM
authors.lastname: Danielsen
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017487:Ankyrins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000378:metabolism* / Q000648:ultrastructure; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D005677:G(M1) Ganglioside / Q000378:metabolism; D007150:Immunohistochemistry; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D009433:Neural Inhibition / Q000502:physiology; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011940:Receptor Aggregation / Q000187:drug effects / Q000502:physiology*; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects; D013347:Subcellular Fractions; D009435:Synaptic Transmission / Q000502:physiology
chemical_list: C490614:Ank3 protein, rat; D017487:Ankyrins; D002352:Carrier Proteins; D003902:Detergents; D008565:Membrane Proteins; D011963:Receptors, GABA-A; C073441:gephyrin; D005677:G(M1) Ganglioside; D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase