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ABSTRACT
Hydration and fluidity of lipid bilayers in different phase states were studied using fluorescent probes selectively located at the interface. The probe of hydration was a recently developed 3-hydroxyflavone derivative, which is highly sensitive to the environment, whereas the probe of fluidity was the diphenylhexatriene derivative, 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene. By variation of the cholesterol content and temperature in large unilamellar vesicles composed of sphingomyelin or dipalmitoylphosphatidlycholine, we generated different phases: gel, liquid ordered (raft), liquid crystalline, and liquid disordered (considered as liquid crystalline phase with cholesterol). For these four phases, the hydration increases in the following order: liquid ordered [much less than] gel [asymptotically =] liquid disordered < liquid crystalline. The membrane fluidity shows a somewhat different trend, namely liquid ordered [asymptotically =] gel < liquid disordered < liquid crystalline. Thus, gel and liquid ordered phases exhibit similar fluidity, whereas the last phase is significantly less hydrated. We expect that cholesterol due to its specific H-bonding interactions with lipids and its ability to fill the voids in lipid bilayers expels efficiently water molecules from the highly ordered gel phase to form the liquid ordered phase. In this study, the liquid ordered (raft) and gel phases are for the first time clearly distinguished by their strong difference in hydration.
INTRODUCTION
Cholesterol is a major constituent of mammalian plasma membranes that can induce domain formation (1). In this respect, microdomains rich in cholesterol and sphingomyelin, known as "lipid rafts", have received a huge attention because they are believed to be involved in regulation of cell functions such as signal transduction, lipid trafficking and membrane protein activity (2-5). Raft domains can also form in synthetic lipid vesicles and supported lipid bilayers containing saturated glycerophospholipids or sphingolipids and cholesterol (6-9). Rafts are characterized by a tight packing of the lipid saturated acyl chains (10,11). In biomembranes, rafts likely exist in a liquid-ordered state (L^sub o^), and behave like islands floating in a sea of loosely-packed domains of unsaturated glycerophospholipids being in a liquid disordered (fluid) state (L^sub d^). Comparing mixtures with and without cholesterol shows that cholesterol can promote phase separation (12,13) due to its favorable packing interactions with saturated lipids.
To better understand the mechanisms governing the cholesterol-induced domain formation, more information should be obtained on the molecular...