Dataset Information

Modulation of plasma membrane Ca2+-ATPase by neutral phospholipids: effect of the micelle-vesicle transition and the bilayer thickness.

ABSTRACT: The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca(2+) pump (PMCA). We found that Ca(2+)-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca(2+)-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Using static light scattering and fluorescence correlation spectroscopy, we monitored the changes in hydrodynamic radius of detergent/phosphatidylcholine particles during the micelle-vesicle transition. We found that, when PMCA is reconstituted in mixed micelles, neutral phospholipids increase the enzyme turnover. The biophysical changes associated with the transition from mixed micelles to bicelles increase the time of residence of the phosphorylated intermediate (EP), decreasing the enzyme turnover. Molecular dynamics simulations analysis of the interactions between PMCA and the phospholipid bilayer in which it is embedded show that in the 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer, charged residues of the protein are trapped in the hydrophobic core. Conversely, in the 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer, the overall hydrophobic-hydrophilic requirements of the protein surface are fulfilled the best, reducing the thermodynamic cost of exposing charged residues to the hydrophobic core. The apparent mismatch produced by a 1,2-dioleoyl-sn-glycero-3-phosphocholine thicker bilayer could be a structural foundation to explain its functional effect on PMCA.

SUBMITTER: Pignataro MF

PROVIDER: S-EPMC4358257 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Modulation of plasma membrane Ca2+-ATPase by neutral phospholipids: effect of the micelle-vesicle transition and the bilayer thickness.

Pignataro María Florencia MF Dodes-Traian Martín M MM González-Flecha F Luis FL Sica Mauricio M Mangialavori Irene C IC Rossi Juan Pablo F C JP

The Journal of biological chemistry 20150120 10

The effects of lipids on membrane proteins are likely to be complex and unique for each membrane protein. Here we studied different detergent/phosphatidylcholine reconstitution media and tested their effects on plasma membrane Ca(2+) pump (PMCA). We found that Ca(2+)-ATPase activity shows a biphasic behavior with respect to the detergent/phosphatidylcholine ratio. Moreover, the maximal Ca(2+)-ATPase activity largely depends on the length and the unsaturation degree of the hydrocarbon chain. Usin ...[more]

PMID: 25605721

Similar Datasets

Project description:Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8-10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8-10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid.

Project description:Mixtures of cationic and anionic surfactants often originate bilayer structures, such as vesicles and lamellar liquid crystals, that can be explored as model membranes for fundamental studies or as drug and gene nanocarriers. Here, we investigated the aggregation properties of two catanionic mixtures containing biomimetic surfactants derived from serine. The mixtures are designated as 12Ser/8-8Ser and 14Ser/10-10Ser, where mSer is a cationic, single-chained surfactant and n-nSer is an anionic, double-chained one (m and n being the C atoms in the alkyl chains). Our goal was to investigate the effects of total chain length and chain length asymmetry of the catanionic pair on the formation of catanionic vesicles, the vesicle properties and the vesicle/micelle transitions. Ocular observations, surface tension measurements, video-enhanced light microscopy, cryogenic scanning electron microscopy, dynamic and electrophoretic light scattering were used to monitor the self-assembly process and the aggregate properties. Catanionic vesicles were indeed found in both systems for molar fractions of cationic surfactant ≥0.40, always possessing positive zeta potentials (ζ = +35-50 mV), even for equimolar sample compositions. Furthermore, the 14Ser/10-10Ser vesicles were only found as single aggregates (i.e., without coexisting micelles) in a very narrow compositional range and as a bimodal population (average diameters of 80 and 300 nm). In contrast, the 12Ser/8-8Ser vesicles were found for a wider sample compositional range and as unimodal or bimodal populations, depending on the mixing ratio. The aggregate size, pH and zeta potential of the mixtures were further investigated. The unimodal 12Ser/8-8Ser vesicles (<DH> ≈ 250 nm, pH ≈ 7-8, ζ ≈ +32 mV and a cationic/anionic molar ratio of ≈2:1) are particularly promising for application as drug/gene nanocarriers. Both chain length asymmetry and total length play a key role in the aggregation features of the two systems. Molecular insights are provided by the main findings.

Project description:Using differential scanning calorimetry and lifetime analysis of trans-parinaric acid fluorescence, we have examined how cholesterol and cholesteryl phosphocholine (CholPC) affect gel-phase properties of palmitoyl ceramide (PCer) in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleyol-sn-glycero-3-phosphocholine (DOPC) bilayers. By 2H NMR, we also measured fluid-phase interactions among these lipids using deuterated analogs of POPC, PCer, and cholesterol. The PCer-rich gel phase in POPC bilayers (9:1 molar ratio of POPC to PCer) was partially and similarly dissolved (and thermostability decreased) by both cholesterol and CholPC (sterol was present equimolar to PCer, or in fourfold excess). In DOPC bilayers (4:1 DOPC/PCer molar ratio), CholPC was much more efficient in dissolving the PCer-rich gel phase when compared to cholesterol. This can be interpreted as indicating that PCer interaction with POPC was stronger than PCer interaction with DOPC. PCer-CholPC interactions were also more favored in DOPC bilayers compared to POPC bilayers. In the fluid POPC-rich phase, cholesterol increased the order of the acyl chain of d2-PCer much more than did CholPC. In DOPC-rich fluid bilayers, both cholesterol and CholPC increased d2-PCer acyl chain order, and the ordering induced by CholPC was more efficient in DOPC than in POPC bilayers. In fluid POPC bilayers, the ordering of 3-d1-cholesterol by PCer was weak. In summary, we found that in the gel phase, sterol effects on the PCer-rich gel phase were markedly influenced by the acyl chain composition of the fluid PC. The same was true for fluid-phase interactions involving the sterols. Our results further suggest that PCer did not display high affinity toward either of the sterols used. We conclude that the nature of unsaturated phospholipids (POPC versus DOPC) in bilayers has major effects on the properties of ceramide gel phases and on sterol-ceramide-phospholipid interactions in such complex bilayers.

Project description:The intracellular unbound drug concentration (Cu,cell) drives pharmacological and toxicological responses for targets inside cells. We have previously shown that Cu,cell is not always equal to extracellular unbound drug concentrations (e.g. plasma concentrations). However, the underlying mechanisms that drive cellular drug binding are poorly understood. The aim of this study was to evaluate drug binding to individual cellular components, such as neutral lipids (NL) and phospholipids (PL). The fibroblastic 3T3-L1 cell line was used as a cellular model. The cells were either used as wild-type (WT cells) or were treated with a differentiation cocktail that induced an adipocyte-like phenotype with large NL droplets (NL+PL+ cells), or treated with propranolol to induce accumulation of PL (PL+ cells). The three cell types were analysed for morphology, global protein expression, lipid content, cellular volume and lysosomal appearance. The cells were exposed to 23 drugs with diverse physicochemical properties, e.g., with regard to lipophilicity, charge, molecular weight and polar surface area. The cellular binding (fu,cell) and the total accumulation ratio (Kp) were measured and used to obtain the intracellular bioavailability (Fic)—defined as the unbound fraction of the drug concentration added to the cells that is available for intracellular target interaction. The NL+PL+ cells had an adipocyte-like morphology, whereas the PL+ were not visually distinguishable from WT cells. Analysis of the global proteome combined with pathway analysis supported the morphological appearances and confirmed the adipocyte-like phenotype of NL+PL+ cells and the normal morphology of PL+ cells. Fic was significantly altered in both treated cell types as compared to WT. A strong negative correlation between fu,cell and PL content in the cell homogenates was observed, whereas the increased NL content in the NL+PL+ cells did not increase binding further. The importance of PLs for drug binding was confirmed by affinities to beads coated with purified PLs. The NL+PL+ cells lacked acidic subcellular compartments (i.e., endo-lysosomal space), which further influenced the subcellular distribution of cationic drugs. In conclusion, our results suggest that PL content, but not NL content, is a major determinant of drug binding in cells, and that PL beads may constitute a simple alternative to more cumbersome cell distribution studies.

Dataset Information

Modulation of plasma membrane Ca2+-ATPase by neutral phospholipids: effect of the micelle-vesicle transition and the bilayer thickness.

Publications

Modulation of plasma membrane Ca2+-ATPase by neutral phospholipids: effect of the micelle-vesicle transition and the bilayer thickness.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets