Identification of human P2X1 receptor-interacting proteins reveals a role of the cytoskeleton in receptor regulation

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation.     

Disruption of lipid rafts inhibits P2X1 receptor-mediated currents and arterial vasoconstriction

P2X1 receptors for ATP are ligand-gated cation channels expressed on a range of smooth muscle preparations and blood platelets. The receptors appear to be clustered close to sympathetic nerve varicosities and mediate the underlying membrane potential changes and constriction following nerve stimulation in a range of arteries and resistance arterioles. In this study we have used discontinuous sucrose density gradients, Western blot analysis, and cholesterol measurements to show that recombinant and smooth muscle (rat tail artery, vas deferens, and bladder) P2X1 receptors are present in cholesterol-rich lipid rafts and co-localize with the lipid raft markers flotillin-1 and -2. Lipid rafts are specialized lipid membrane microdomains involved in signaling and trafficking. To determine whether lipid raft association was essential for P2X1 receptor channel function we used the cholesterol-depleting agent methyl-beta-cyclodextrin (10 mm for 1 h). This led to a redistribution of the P2X1 receptor throughout the sucrose gradient and reduced P2X1 receptor-mediated (alpha,beta-methylene ATP, 10 microm) currents in HEK293 cells by >90% and contractions of the rat tail artery by approximately 50%. However contractions evoked by potassium chloride (60 mm) were unaffected by methyl-beta-cyclodextrin and the inactive analogue alpha-cyclodextrin had no effect on P2X1 receptor-mediated currents or contractions. P2X1 receptors are subject to ongoing regulation by receptors and kinases, and the present results suggest that lipid rafts are an essential component in the maintenance of these localized signaling domains and play an important role in P2X1 receptor-mediated control of arteries.     

Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization

P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-β-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na⁺ and N-methyl-d-glucamine (NMDG⁺) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death.     

Differential sensitivity of human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts

ATP-stimulated P2X1 and ADP-stimulated P2Y1 receptors play important roles in platelet activation. An increase in intracellular Ca2+ represents a key signalling event coupled to both of these receptors, mediated via direct gating of Ca2+-permeable channels in the case of P2X1 and phospholipase-C-dependent Ca2+ mobilisation for P2Y1. We show that disruption of cholesterol-rich membrane lipid rafts reduces P2X1 receptor-mediated calcium increases by approximately 80%, while P2Y1 receptor-dependent Ca2+ release is unaffected. In contrast to artery, vas deferens, bladder smooth muscle, and recombinant expression in cell lines, where P2X1 receptors show almost exclusive association with lipid rafts, only approximately 20% of platelet P2X1 receptors are co-expressed with the lipid raft marker flotillin-2. We conclude that lipid rafts play a significant role in the regulation of P2X1 but not P2Y1 receptors in human platelets and that a reserve of non-functional P2X1 receptors may exist.     

The intracellular amino terminus plays a dominant role in desensitization of ATP-gated P2X receptor ion channels

P2X receptors show marked variations in the time-course of response to ATP application from rapidly desensitizing P2X1 receptors to relatively sustained P2X2 receptors. In this study we have used chimeras between human P2X1 and P2X2 receptors in combination with mutagenesis to address the contribution of the extracellular ligand binding loop, the transmembrane channel, and the intracellular regions to receptor time-course. Swapping either the extracellular loop or both transmembrane domains (TM1 and -2) between the P2X1 and P2X2 receptors had no effect on the time-course of ATP currents in the recipient receptor. These results suggest that the agonist binding and channel-forming portions of the receptor do not play a major role in the control of the time-course. In contrast replacing the amino terminus of the P2X1 receptor with that from the non-desensitizing P2X2 receptor (P2X1-2N) slowed desensitization, and the mirror chimera induced rapid desensitization in the P2X2-1N chimera. These reciprocal effects on time-course can be replicated by changing four variant amino acids just before the first transmembrane (TM1) segment. These pre-TM1 residues also had a dominant effect on chimeras where both TMs had been transferred; mutating the variant amino acids 21-23 to those found in the P2X2 receptor removed desensitization from the P2X1-2TM1/-2 chimera, and the reciprocal mutants induced rapid desensitization in the non-desensitizing P2X2-1TM1/-2 chimera. These results suggest that the intracellular amino terminus, in particular the region just before TM1, plays a dominant role in the regulation of the time-course of ATP evoked P2X receptor currents.     

Heat Shock Protein 90 Inhibitors Reduce Trafficking of ATP-gated P2X1 Receptors and Human Platelet Responsiveness

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70–85% by the selective HSP90 inhibitor geldanamycin (2 μm, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40–45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies.     

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation. Pascal Maurice and Ludovic Waeckel have contributed equally to this work.     

Characterization of the molecular interaction between caveolin-1 and the P2X receptors 4 and 7 in E10 mouse lung alveolar epithelial cells

P2X(4) and P2X(7) receptors are abundantly expressed in alveolar epithelial cells, and are thought to play a role in regulating fluid haemostasis. Here, we analyzed the expression and localization of the P2X(4)R, and characterized the interaction between Cav-1 and both P2X(4)R and P2X(7)R in the mouse alveolar epithelial cell line E10. Using the biotinylation assay, we found that only glycosylated P2X(4)R is exposed at the cell surface. Triton X-100 solubility experiments and sucrose gradient centrifugation revealed that P2X(4)R was partially localized in Cav-1 rich membrane fractions. Cholesterol depletion with Mbeta-CD displaced Cav-1 and P2X(4)R from the low-density to the high-density fractions. Suppression of Cav-1 protein expression using short hairpin RNAs resulted in a large reduction in P2X(4)R levels. Double immunofluorescence showed that P2X(4)R and Cav-1 partially colocalize in vitro. Using the GST pull-down assay, we showed that Cav-1 interacts in vitro with both P2X(4)R and P2X(7)R. Co-immunoprecipitation experiments confirmed the interaction between P2X(7)R and Cav-1. ATP stimulation increased the level of P2X(4)R in the lipid raft/caveolae fraction, whereas Cav-1 content remained constant. Our results support recent evidence that P2X receptors are present in both raft and non-raft compartments of the plasma membrane and thus exhibit variable ATP sensitivity.     

Lipid phosphate phosphatases 1 and 3 are localized in distinct lipid rafts

Lipid phosphate phosphatases (LPPs), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100-insoluble rafts, we here report that LPP1 is also associated with lipid rafts distinct from those harboring LPP3. We found that LPP1 was Triton X-100-soluble, but CHAPS-insoluble in LNCaP cells endogenously expressing LPP1 and several LPP1 cDNA-transfected cells including NIH3T3 fibroblasts. In addition to the non-ionic detergent insolubility, LPP1 further possessed several properties formulated for raft-localizing proteins as follows: first, the CHAPS-insolubility was resistant to the actin-disrupting drug cytochalasin D; second, the CHAPS-insoluble LPP1 floated in an Optiprep density gradient; third, the CHAPS insolubility of LPP1 was lost by cholesterol depletion; and finally, the subcellular distribution pattern of LPP1 exclusively overlapped with that of a raft marker, cholera toxin B subunit. Interestingly, confocal microscopic analysis showed that LPP1 was distributed to membrane compartments distinct from those of LPP3. Analysis using various LPP1/LPP3 chimeras revealed that their first extracellular regions determine the different Triton X-100 solubilities. These results indicate that LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions.     

Formation of carnosine-Cu(II) complexes prevents and reverts the inhibitory action of copper in P2X4 and P2X7 receptors

To further analyze the action of copper on brain synaptic mechanisms, the brain dipeptide carnosine (beta-alanyl-L-histidine) was tested in Xenopus laevis oocytes expressing the rat P2X4 or P2X7 receptors. Ten micromolar copper halved the currents evoked by ATP in both receptors; co-application of carnosine plus copper prevented the metal induced-inhibition with a median effective concentration of 12.1 +/- 3.9 and 12.0 +/- 5.5 microm for P2X4 and P2X7, respectively. Zinc potentiated only the P2X4 ATP-evoked currents; carnosine had no effect over this metal. The relative potency and selectivity of classical metal chelators to prevent the copper inhibition was compared between carnosine and penicillamine (PA), bathophenanthroline (BPh) or L-histidine (His). Their rank order of potency in P2X4 and P2X7 receptors was carnosine = PA = His > BPh > Glycine (Gly) and carnosine = BPh = His > PA > Gly, respectively. The potency to prevent the zinc-induced potentiation in the P2X4 receptor was BPh > PA > His; carnosine, Gly and beta-alanine were inactive. Whereas 1-100 microm carnosine or His alone did not modify the ATP-evoked currents, 10-100 microm PA augmented and 100 microm BPh decreased the ATP-evoked currents. Carnosine was able to revert the copper-induced inhibition restoring the maximal ATP gated current in a concentration-dependent manner. Electronic spectroscopy confirm the formation of carnosine-Cu(II) complexes, mechanism that can account for the prevention and reversal of the copper inhibition, revealing its potential in copper intoxication treatment.     

Lipid rafts regulate 2-arachidonoylglycerol metabolism and physiological activity in the striatum

Several G protein-associated receptors and synaptic proteins function within lipid rafts, which are subdomains of the plasma membranes that contain high concentrations of cholesterol. In this study we addressed the possible role of lipid rafts in the control of endocannabinoid system in striatal slices. Disruption of lipid rafts following cholesterol depletion with methyl-β-cyclodestrin (MCD) failed to affect synthesis and degradation of anandamide, while it caused a marked increase in the synthesis and concentration of 2-arachidonoylglycerol (2-AG), as well as in the binding activity of cannabinoid CB1 receptors. Surprisingly, endogenous 2-AG-mediated control of GABA transmission was not potentiated by MCD treatment and, in contrast, neither basal nor 3,5-Dihydroxyphenylglycine-stimulated 2-AG altered GABA synapses in cholesterol-depleted slices. Synaptic response to the cannabinoid CB1 receptor agonist HU210 was however intact in MCD-treated slices, indicating that reduced sensitivity of cannabinoid CB1 receptors does not explain why endogenous 2-AG is ineffective in inhibiting striatal GABA transmission after cholesterol depletion. Confocal microscopy analysis suggested that disruption of raft integrity by MCD might uncouple metabotropic glutamate 5-CB1 receptor interaction by altering the correct localization of both receptors in striatal neuron elements. In conclusion, our data indicate that disruption of raft integrity causes a complex alteration of the endocannabinoid signalling in the striatum.     

Transmembrane mutations to FcgammaRIIA alter its association with lipid rafts: implications for receptor signaling

Many immunoreceptors have been reported to associate with lipid rafts upon ligand binding. The way in which this association is regulated is still obscure. We investigated the roles for various domains of the human immunoreceptor FcgammaRIIA in regulating its association with lipid rafts by determining the resistance of unligated, or ligated and cross-linked, receptors to solubilization by the nonionic detergent Triton X-100, when expressed in RBL-2H3 cells. Deletion of the cytoplasmic domain, or destruction of the cytoplasmic palmitoylation site, had no effect on the association of the receptor with lipid rafts. A transmembrane mutant, A224S, lost the ability to associate with lipid rafts upon receptor cross-linking, whereas transmembrane mutants VA231-2MM and VVAL234-7GISF showed constitutive lipid raft association. Wild-type (WT) FcgammaRIIA and all transmembrane mutants activated Syk, regardless of their association with lipid rafts. WT FcgammaRIIA and mutants that associated with lipid rafts efficiently activated NF-kappaB, in an ERK-dependent manner. In contrast, WT FcgammaRIIA and the A224S mutant both presented efficient phagocytosis, while VA231-2MM and VVAL234-7GISF mutants presented lower phagocytosis, suggesting that phagocytosis may proceed independently of lipid raft association. These data identify the transmembrane domain of FcgammaRIIA as responsible for regulating its inducible association with lipid rafts and suggest that FcgammaRIIA-mediated responses, like NF-kappaB activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.     

Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland

The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X7 receptors were detected in these fractions. The location of the P2X7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-beta-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with alpha-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca2+]i) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A2 (PLA2). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. We conclude that P2X7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA2 in response to P2X7 receptor occupancy.     

Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Neuregulin-1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-beta 1a or the III-beta 1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-beta 1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.     

Signaling proteins in raft-like microdomains are essential for Ca2+ wave propagation in glial cells

The hypothesis that calcium signaling proteins segregate into lipid raft-like microdomains was tested in isolated membranes of rat oligodendrocyte progenitor (OP) cells and astrocytes using Triton X-100 solubilization and density gradient centrifugation. Western blot analysis of gradient fractions showed co-localization of caveolin-1 with proteins involved in the Ca2+ signaling cascade. These included agonist receptors, P2Y1, and M1, TRPC1, IP3R2, ryanodine receptor, as well as the G protein Galphaq and Homer. Membranes isolated from agonist-stimulated astrocytes showed an enhanced recruitment of phospholipase C (PLCbeta1), IP3R2 and protein kinase C (PKC-alpha) into lipid raft fractions. IP3R2, TRPC1 and Homer co-immunoprecipitated, suggesting protein-protein interactions. Disruption of rafts by cholesterol depletion using methyl-beta-cyclodextrin (beta-MCD) altered the distribution of caveolin-1 and GM1 to non-raft fractions with higher densities. beta-MCD-induced disruption of rafts inhibited agonist-evoked Ca2+ wave propagation in astrocytes and attenuated wave speeds. These results indicate that in glial cells, Ca2+ signaling proteins might exist in organized membrane microdomains, and these complexes may include proteins from different cellular membrane systems. Such an organization is essential for Ca2+ wave propagation.     

Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study

The epidermal growth factor (EGF) receptor partitions into lipid rafts made using a detergent-free method, but is extracted from low density fractions by Triton X-100. By screening several detergents, we identified Brij 98 as a detergent in which the EGF receptor is retained in detergent-resistant membrane fractions. To identify the difference in lipid composition between those rafts that harbored the EGF receptor (detergent-free and Brij 98-resistant) and those that did not (Triton X-100-resistant), we used multidimensional electrospray ionization mass spectrometry to perform a lipidomics study on these three raft preparations. Although all three raft preparations were similarly enriched in cholesterol, the EGF receptor-containing rafts contained more ethanolamine glycerophospholipids and less sphingomyelin than did the non-EGF receptor-containing Triton X-100 rafts. As a result, the detergent-free and Brij 98-resistant rafts exhibited a balance of inner and outer leaflet lipids, whereas the Triton X-100 rafts contained a preponderance of outer leaflet lipids. Furthermore, in all raft preparations, the outer leaflet phospholipid species were significantly different from those in the bulk membrane, whereas the inner leaflet lipids were quite similar to those found in the bulk membrane. These findings indicate that the EGF receptor is retained only in rafts that exhibit a lipid distribution compatible with a bilayer structure and that the selection of phospholipids for inclusion into rafts occurs mainly on the outer leaflet lipids.     

Ectodomain Movements of an ATP-gated Ion Channel (P2X2 Receptor) Probed by Disulfide Locking

The ectodomain of the P2X receptor is formed mainly from two- or three-stranded β-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the β-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures.     

Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cells

The P2X7 receptor has recently been described as a marker for lung alveolar epithelial type I cells. Here, we demonstrate both the expression of P2X7 protein and its partition into lipid rafts in the mouse lung alveolar epithelial cell line E10. A significant degree of colocalization was observed between P2X7 and the raft marker protein Caveolin-1; also, P2X7 protein was associated with caveolae. A marked reduction in P2X7 immunoreactivity was observed in lung sections prepared from Caveolin-1-knockout mice, indicating that Caveolin-1 expression was required for full expression of P2X7 protein. Indeed, suppression of Caveolin-1 protein expression in E10 cells using short hairpin RNAs resulted in a large reduction in P2X7 protein expression. Our data demonstrate a potential interaction between P2X7 protein and Caveolin-1 in lipid rafts, and provide a basis for further functional and biochemical studies to probe the physiologic significance of this interaction.     

Procyanidins can interact with Caco-2 cell membrane lipid rafts: Involvement of cholesterol

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-β-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol–Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex–cholesterol bondings. Procyanidin–lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.