Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature.
Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.
Gil
Carles
C
Departament de Bioquímica i Biologia Molecular and Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain. carles.gil@uab.cat
Cubí
Roger
R
Blasi
Juan
J
Aguilera
José
J
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003902:Detergents; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D010938:Plant Oils / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011188:Potassium / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins / Q000032:analysis*; D012995:Solubility; D013570:Synaptic Membranes / Q000737:chemistry; D013574:Synaptosomes / Q000187:drug effects; D050827:Syntaxin 1 / Q000032:analysis; D013696:Temperature; D047392:beta-Cyclodextrins / Q000494:pharmacology
D003902:Detergents; D008565:Membrane Proteins; D010938:Plant Oils; D050600:SNARE Proteins; D050827:Syntaxin 1; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C017474:Lubrol WX; D011092:Polyethylene Glycols; C033084:polyethylene glycol oleyl ether; D011188:Potassium; D002118:Calcium