ARTICLE Ida C. Bonini ® Silvia S. Antollini Carlos Gutie´rrez-Merino ® Francisco J. Barrantes Sphingomyelin composition and physical asymmetries in native acetylcholine receptor-rich membranes Received: 8 January 2002 / Revised: 4 April 2002 /Accepted: 8 April 2002 / Published online: 14 June 2002 Ó EBSA 2002 Abstract Selective enzymatic hydrolysis, lipid composi- tional analyses, and fluorescence studies have been car- ried out on acetylcholine receptor (AChR)-rich membranes from Torpedinidae to investigate the to- pology of sphingomyelin (SM) in the native membrane and its relationship with the AChR protein. Controlled sphingomyelinase hydrolysis of native membranes showed that SM is predominantly (60%) localized in the outer half of the lipid bilayer. Differences were also observed in the distribution of SM fatty acid molecular species in the two bilayer leaflets. A fluorescent SM derivative {N-[10-(1-pyrenyl)decanoyl]sphingomyelin; Py-SM} was used to study protein-lipid interactions in the AChR-rich membrane and in affinity-purified Torpedo AChR reconstituted in liposomes made from Torpedo electrocyte lipid extracts. The efficiency of Fo¨rster resonance energy transfer (FRET) from the protein to the pyrenyl-labeled lipid as a function of ac- ceptor surface density was used to estimate distances and topography of the SM derivative relative to the protein. The dynamics of the lipid acyl chains were explored by measuring the thermal dependence of Py-SM excimer formation, sensitive to the fluidity of the membrane. Differences were observed in the concentration depen- dence of excimer/monomer pyrenyl fluorescence when measured by direct excitation of the probe as against under FRET conditions, indicating differences in the intermolecular collisional frequency of the fluorophores between bulk and protein-vicinal lipid environments, respectively. Py-SM exhibited a moderate selectivity for the protein-vicinal lipid domain, with a calculated relative affinity K r 0.55. Upon sphingomyelinase digestion of the membrane, FRET efficiency increased by about 50%, indicating that the resulting pyrenyl- ceramide species have higher affinity for the protein than the parental SM derivative. Keywords Sphingolipids ® Membrane lipids ® Fluorescence ® Energy transfer Introduction The major lipid components of biological membranes are phosphoglycerides, cholesterol, and sphingolipids. Sphingomyelins (N-acylsphingosine-1-phosphorylcho- line or ceramide-1-phosphorylcholine) (SMs), the sim- plest class of the sphingolipids, amount to about 10% of phospholipids of mammalian cell membranes. As with phosphatidylcholine (PC), the other choline-containing phospholipid, SMs occur mostly in the plasma mem- brane of the outer, exoplasmic leaflet of the bilayer (Barenholz and Thompson 1980; Koval and Pagano 1991). Aminophospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), on the other hand, predominate in the inner, cytoplasmic leaflet of the membrane (Devaux 1992). SM is also purported to play a key role in the so-called ‘‘SM cycle’’ (Hannun and Obeid 1995), in which SM-derived ceramide acts as a second messenger lipid in apoptosis. Another rapidly developing area in which the par- ticipation of SM appears to play a crucial role is that of cholesterol-SM microdomains or ‘‘rafts’’. These special lipid domains are postulated to result from the strong and preferential interactions between the two lipid spe- cies, resulting in lateral segregation in the plane of the membrane (Brown 1998; Jacobson and Dietrich 1999). Lipid rafts have been implicated to participate in pro- cesses such as cell signaling, and sorting and trafficking of some membrane proteins to the cell surface. In the case of the nicotinic acetylcholine receptor (AChR)-rich membranes obtained from the electric Eur Biophys J (2002) 31: 417–427 DOI 10.1007/s00249-002-0230-6 I.C. Bonini ® S.S. Antollini ® F.J. Barrantes (&) UNESCO Chair of Biophysics and Molecular Neurobiology/Instituto de Investigaciones Bioquı´micas de Bahı´a Blanca, 8000 Bahı´a Blanca, Argentina E-mail: rtfjb1@criba.edu.ar Tel.: +54-291-4861201 Fax: +54-291-4861200 C. Gutie´rrez-Merino Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de Extremadura, Spain