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

Abstract

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 l d to l o can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca 2+ -ATPase (PMCA), in lipid rafts (l o 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 l o phase is more compact than in lipid l d phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l o 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.

The Plasma Membrane Ca2+-ATPase Isoform 4 Is Localized in Lipid Rafts of Cerebellum Synaptic Plasma Membranes

“…1 and 3) may also account for the lower activity found in fraction 3 with respect to fraction 9. Our activity results about the corresponding decrease of PMCA activity with the increase of sphingomyelin and cholesterol content in reconstituted pig brain PMCA are in good agreement with those reported by Pang et al (49). The distribution pattern of PMCA isoforms was observed in SPM isolated not only from cerebellum but also from cerebrum, giving a more general than regional view on the process.…”

Spatial Relationship and Functional Relevance of Three Lipid Domain Populations at the Erythrocyte Surface

“…The membrane lipid order hypothesis is supported by (i) our present observation that SM/PC/chol-enriched domains were more disordered than the rest of the membrane; (ii) our previous observation that lipid domain order increases to a bigger extent than the bulk membrane order upon stimulation of calcium efflux by treatment of RBCs with EGTA [31]; and (iii) several studies in model membranes, although with sometimes conflicting information. For instance, PMCA activity is decreased in highly-ordered areas of liposomes made of PC/SM/chol [64] but is favored in highly ordered lens fiber lipids in comparison with disordered DOPC liposomes [65]. Besides membrane fluidity, SM/PC/chol-enriched domains could contribute to the regulation of PMCA activity through the reversible and controlled modulation of membrane:cytoskeleton anchorage upon deformation-induced calcium entry.…”

Rapid Turnover of Stereocilia Membrane Proteins: Evidence from the Trafficking and Mobility of Plasma Membrane Ca2+-ATPase 2

“…This may suggest an absence of PDZ proteins available for interacting with PMCA2wb in stereocilia. An alternative possibility for the selective retention of PMCA2 in stereocilia is through its association with stable lipid subdomains (or lipid rafts), because there is abundant evidence that many proteins are dynamically associated with caveolae, or raft domains, in plasma membranes, including PMCA (Schnitzer et al, 1995;Pang et al, 2005).…”