The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from I-d to I-o. can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca2+-ATPase (PMCA), in lipid rafts (10 phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present :study. Tryptophan intrinsic fluorescence increase, fluorescence,quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation,of PMCA embedded in lipid I-o phase is more compact than in lipid Id phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of 10 phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.