Preferential distribution of the fluorescent phospholipid probes NBD-phosphatidylcholine and rhodamine-phosphatidylethanolamine in the exofacial leaflet of acetylcholine receptor-rich membranes from Torpedo marmorata.
The distribution of the two fluorescent phospholipid analogs across acetylcholine receptor (AChR)-rich membranes from Torpedo marmorata has been studied by a combination of nonradiative fluorescence resonance energy transfer using fluorescent lipid probes and quenching of their fluorescence with Co2+ and 2,4,6-trinitrobenzenesulfonic acid. The fluorescent lipid analogs were supplied to the AChR-rich membrane or liposome suspension by simply injecting ethanol solutions of the probes into the medium. The efficiency of the fluorescence energy transfer between NBD-labeled phosphatidylcholine and rhodamine-labeled ethanolamine glycerophospholipids was measured in model membranes prepared in such a way that the probes could be targeted at the same or opposite halves of the bilayer, and the results were compared with those obtained for native AChR-rich membranes. It is shown that NBD-PC and Rho-PE can be efficiently (95%) incorporated into AChR-rich membranes and liposomes. On the basis of the comparison with model liposomes, the energy transfer experiments suggest a preferential exofacial location of the parental phospholipids in the native AChR-rich membrane. Fluorescence quenching with Co2+ and TNBS showed these two phospholipid analogs to be located predominantly in the outer leaflet of the bilayer in AChR-rich membranes. From the Co2+ quenching of the lipid analogs, it was also possible to calculate the surface potential of the outer leaflet of the membrane as being on the order of -15 mV.
Gutiérrez-Merino
C
C
Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sur, Bahía Blanca, Argentina.
Bonini de Romanelli
I C
IC
Pietrasanta
L I
LI
Barrantes
F J
FJ
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D010713:Phosphatidylcholines / Q000493:pharmacokinetics*; D010714:Phosphatidylethanolamines / Q000493:pharmacokinetics*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012235:Rhodamines / Q000493:pharmacokinetics*; D013050:Spectrometry, Fluorescence; D014101:Torpedo
C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011950:Receptors, Cholinergic; D012235:Rhodamines; C046703:lissamine-rhodamine-phosphatidylethanolamine; D003035:Cobalt; D009327:4-Chloro-7-nitrobenzofurazan