Lipid Raft Composition Research Articles

Lipid Raft Alterations in Aged-Associated Neuropathologies.

Lipid rafts are membrane microdomains particularly enriched in cholesterol, sphingolipids and saturated fatty acids. These microstructures play a key role in a plethora of mechanisms involved in cell signaling, synapsis, cell-cell communication and cell survival. In the last years, increasing evidence indicate that lipid rafts may be altered in age-related neuropathologies, such as Alzheimer's disease and Parkinson disease even at asymptomatic stages. In particular, important changes in raft lipid composition are observed with the progression of these diseases, then inducing alterations in their physicochemical properties. Furthermore, these phenomena contribute to neuropathological events related to amyloidogenesis, aberrant protein aggregation and toxic cell signalling. In this review, we discuss some relevant data on the age-related molecular changes occurring in lipid rafts since the first stages of these neurodegenerative diseases. Further characterization of specific parameters associated with alterations of these microdomains may provide potential tools of diagnosis and prediction of these neuropathologies.

7-dehydrocholesterol efficiently supports Ret signaling in a mouse model of Smith-Opitz-Lemli syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a rare disorder of cholesterol synthesis. Affected individuals exhibit growth failure, intellectual disability and a broad spectrum of developmental malformations. Among them, renal agenesis or hypoplasia, decreased innervation of the gut, and ptosis are consistent with impaired Ret signaling. Ret is a receptor tyrosine kinase that achieves full activity when recruited to lipid rafts. Mice mutant for Ret are born with no kidneys and enteric neurons, and display sympathetic nervous system defects causing ptosis. Since cholesterol is a critical component of lipid rafts, here we tested the hypothesis of whether the cause of the above malformations found in SLOS is defective Ret signaling owing to improper lipid raft composition or function. No defects consistent with decreased Ret signaling were found in newborn Dhcr7−/− mice, or in Dhcr7−/− mice lacking one copy of Ret. Although kidneys from Dhcr7−/− mice showed a mild branching defect in vitro, GDNF was able to support survival and downstream signaling of sympathetic neurons. Consistently, GFRα1 correctly partitioned to lipid rafts in brain tissue. Finally, replacement experiments demonstrated that 7-DHC efficiently supports Ret signaling in vitro. Taken together, our findings do not support a role of Ret signaling in the pathogenesis of SLOS.

The Role of Lipid Rafts in the Localization and Function of the Chemosensory TRPA1 Channel

Lipid rafts are highly specialized membrane domains characterized by high concentrations of cholesterol, sphingolipids and gangliosides. This distinctive composition results in lateral phase separation and generation of a liquid-ordered domains accommodating various receptors and channels involved in numerous cellular processes. Recently, several members of the Transient Receptor Potential channels superfamily (TRPCs, TRPM8, and TRPV1) have been shown to locate mainly in the lipid rafts and that this compartmentalization modulates their activation properties. Yet, not much is known about localization and interactions of TRPA1 with the surrounding plasma membrane. Since TRPA1 can be gated by a large number of electrophilic and non-electrophilic compounds, as well as by physiological stimuli such as cold we hypothesize that its localization in the lipid rafts could be a crucial factor influencing the channel activity. Exploiting microscopy and biochemical approaches, this study provides evidence for TRPA1 localization in lipid rafts. TIRF microscopy experiments in HEK293T cells transfected with a mouse or human TRPA1 channel carrying a C-terminal mCherry tag indicate high co-localization rate between channel and the lipid raft maker cholera toxin subunit B. Density gradient centrifugation of Triton-X insoluble fractions confirmed co-expression of TRPA1 and the lipid raft marker flotillin-2 in the low density membrane fractions. Lipid raft disruption experiments further affirmed the cholesterol-related localization of the channel, shifting its localization into higher density gradients. Modification of lipid rafts composition by cholesterol depletion (methyl-b-cyclodextrin) or sphingolipids hydrolysis (sphingomyelinase) decreased the responses of TRPA1 to lipopolysaccharides, thymol, allyl isothiocyanate and cold. Taken together, these results indicate that TRPA1 is present in lipid rafts and this location is essential for its normal activation by different agonists.

Lipid rafts and raft-mediated supramolecular entities in the regulation of CD95 death receptor apoptotic signaling.

Membrane lipid rafts are highly ordered membrane domains enriched in cholesterol, sphingolipids and gangliosides that have the property to segregate and concentrate proteins. Lipid and protein composition of lipid rafts differs from that of the surrounding membrane, thus providing sorting platforms and hubs for signal transduction molecules, including CD95 death receptor-mediated signaling. CD95 can be recruited to rafts in a reversible way through S-palmitoylation following activation of cells with its physiological cognate ligand as well as with a wide variety of inducers, including several antitumor drugs through ligand-independent intracellular mechanisms. CD95 translocation to rafts can be modulated pharmacologically, thus becoming a target for the treatment of apoptosis-defective diseases, such as cancer. CD95-mediated signaling largely depends on protein-protein interactions, and the recruitment and concentration of CD95 and distinct downstream apoptotic molecules in membrane raft domains, forming raft-based supramolecular entities that act as hubs for apoptotic signaling molecules, favors the generation and amplification of apoptotic signals. Efficient CD95-mediated apoptosis involves CD95 and raft internalization, as well as the involvement of different subcellular organelles. In this review, we briefly summarize and discuss the involvement of lipid rafts in the regulation of CD95-mediated apoptosis that may provide a new avenue for cancer therapy.

Simulations Predict 18:0-22:6 Phosphatidylserine Drives α-Synuclein into the Liquid-Disordered Phase in Synaptic Vesicle Lipid Composition

Because α-Synuclein binds to anionic lipids and synaptic vesicles consist of 12% 18:0-22:6 phosphatidylserine (SDPS), we asked where this asymmetric lipid partitions, and how it affects α-Synuclein partitioning in a lipid raft system. Our coarse-grained molecular dynamics simulations of SDPS in a lipid raft composition show that 18:0-22:6 PS partitions to the liquid disordered phase (Ld). Additional simulations of lyso-18:0 and lyso-22:6 and subsequent energy fluctuations analysis demonstrate that SDPS partitioning to Ld is due to the 22:6 acyl-chain's greater affinity for Ld than the 18:0 chain's affinity for Lo. Furthermore, we show that α-synuclein's preference for anionic lipids drives it to join SDPS in the Ld phase. Knowing that the remainder of the synaptic vesicle composition is largely uncharged, we propose that SDPS will drive α-synuclein to Ld regions of the synaptic vesicle mixture.

Sterol carrier protein 2 regulates proximal tubule size in the Xenopus pronephric kidney by modulating lipid rafts

The kidney is a homeostatic organ required for waste excretion and reabsorption of water, salts and other macromolecules. To this end, a complex series of developmental steps ensures the formation of a correctly patterned and properly proportioned organ. While previous studies have mainly focused on the individual signaling pathways, the formation of higher order receptor complexes in lipid rafts is an equally important aspect. These membrane platforms are characterized by differences in local lipid and protein compositions. Indeed, the cells in the Xenopus pronephric kidney were positive for the lipid raft markers ganglioside GM1 and Caveolin-1. To specifically interfere with lipid raft function in vivo, we focused on the Sterol Carrier Protein 2 (scp2), a multifunctional protein that is an important player in remodeling lipid raft composition. In Xenopus, scp2 mRNA was strongly expressed in differentiated epithelial structures of the pronephric kidney. Knockdown of scp2 did not interfere with the patterning of the kidney along its proximo-distal axis, but dramatically decreased the size of the kidney, in particular the proximal tubules. This phenotype was accompanied by a reduction of lipid rafts, but was independent of the peroxisomal or transcriptional activities of scp2. Finally, disrupting lipid micro-domains by inhibiting cholesterol synthesis using Mevinolin phenocopied the defects seen in scp2 morphants. Together these data underscore the importance for localized signaling platforms in the proper formation of the Xenopus kidney.

Role of lipid rafts and GM1 in the segregation and processing of prion protein.

The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.

Lipid cell membrane composition: A novel therapeutic target in cancer.

7062 Background: Cell membrane cholesterol, more specifically, the lipid composition of lipid rafts is associated with regulation of trans-membrane receptors. Methods: We tested the hypothesis that...

Lipid raft abnormalities and subsequent protein trafficking effects in Niemann‐Pick type C1 (LB158)

Many proteins and lipid components are organized within membrane microdomains, known as lipid rafts (LR). LR are cholesterol‐ and sphingolipid‐rich membranes resistant to non‐ionic detergents. In Niemann‐Pick type C1 (NPC1), a neurovisceral lysosomal storage disease, entrapment of cholesterol and sphingolipids occurs and may lead to alterations in LR composition. We investigate the LR status in fibroblasts derived from a patient with NPC1 and analyze the trafficking profiles of a typical LR‐associated membrane protein, dipeptidylpeptidase IV (DPPIV).Sucrose density gradients of Triton X‐100 cellular lysates revealed a substantial shift of the LR‐marker flotillin2 in NPC fibroblasts from the floating LR fractions to the detergent‐soluble membranes. Notably, the cell surface expression of DPPIV increased significantly by 2‐fold in NPC1 cells, while the overall protein level of DPPIV remained constant. FACS analysis corroborated the increase of DPPIV at the cell surface. This accumulation is the result of delayed lipid rafts‐dependent endocytosis in NPC1 cells as assessed by (i) a significant reduction in the level of internalized biotin‐labeled DPPIV and (ii) imaging studies revealing a reduced localization of DPPIV with EEA‐1 in NPC1 cells. Strikingly, N‐butyl‐deoxynojirimycin (NB‐DNJ), an inhibitor of glucosylceramide synthase, restored significantly LR levels and endocytosis of DPPIV. We conclude that altered composition or reduced levels of LR constitute a potential mechanism in the pathogenesis of NPC1 and NB‐DNJ treatment restores a balanced membrane lipid composition and the associated biochemical anomalies.

Altered lipid composition in cortical lipid rafts occurs at early stages of sporadic Alzheimer's disease and facilitates APP/BACE1 interactions

The presence of lipid alterations in lipid rafts from the frontal cortex in late stages of Alzheimer's disease (AD) has been recently demonstrated. Here, we have isolated and analyzed the lipid composition of lipid rafts from different brain areas from control and AD subjects at initial neuropathologic stages. We have observed that frontal cortex lipid rafts are profoundly altered in AD brains from the earliest stages of AD, namely AD I/II. These changes in the lipid matrix of lipid rafts affected both lipid classes and fatty acids and were also detected in the entorhinal cortex, but not in the cerebellum from the same subjects. Paralleling these changes, lipid rafts from AD frontal and entorhinal cortices displayed higher anisotropy for environment-sensitive probes, indicating that lipid changes in AD lipid rafts increased membrane order and viscosity in these domains. The pathophysiological consequences of these alterations in the development and progression of AD were strengthened by the significant, and specific, accumulation of β-secretase within the lipid rafts of AD subjects even at the earliest stages. Our results provide a mechanistic connection between lipid alterations in these microdomains and amyloidogenic processing of amyloid precursor protein.

Quantitative Proteomics Analysis of Macrophage-Derived Lipid Rafts Reveals Induction of Autophagy Pathway at the Early Time of Francisella tularensis LVS Infection

Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host-pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early stages of microbe-host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection.

Lipid composition of membrane rafts, isolated with and without detergent, from the spleen of a mouse model of Gaucher disease

Biological membranes are composed of functionally relevant liquid-ordered and liquid-disordered domains that coexist. Within the liquid-ordered domains are low-density microdomains known as rafts with a unique lipid composition that is crucial for their structure and function. Lipid raft composition is altered in sphingolipid storage disorders, and here we determined the lipid composition using a detergent and detergent-free method in spleen tissue, the primary site of pathology, in a mouse model of the sphingolipid storage disorder, Gaucher disease. The accumulating lipid, glucosylceramide, was 30- and 50-fold elevated in the rafts with the detergent and detergent-free method, respectively. Secondary accumulation of di- and trihexosylceramide resided primarily in the rafts with both methods. The phospholipids distributed differently with more than half residing in the rafts with the detergent-free method and less than 10% with the detergent method, with the exception of the fully saturated species that were primarily in the rafts. Individual isoforms of sphingomyelin correlated with detergent-free extraction and more than half resided in the raft fractions. However, this correlation was not seen with the detergent extraction method as sphingomyelin species were spread across both the raft and non-raft domains. Therefore caution must be exercised when interpreting phospholipid distribution in raft domains as it differs considerably depending on the method of isolation. Importantly, both methods revealed the same lipid alterations in the raft domains in the spleen of the Gaucher disease mouse model highlighting that either method is appropriate to determine membrane lipid changes in the diseased state.

Characterization of rafts subtypes in monocytes of diabetic patients with and without neovascular complication

AbstractPurpose Diabetic retinopathy is a chronic inflammatory disease where the infiltration of plasma and neovascularization of the retina cause blindness. This disease affects millions of people in developed countries and early biological markers would better manage these patients.Methods For this purpose we purified monocytes, key players of inflammation, blood of healthy and sick people. On the surface of monocytes, there are dynamic structures called rafts membrane microdomains, which there are two subtypes: flat rafts and caveolae rafts. They correspond to dynamic assemblies and ordered cholesterol and sphingolipids. We isolated these rafts membranes of monocytes and studied protein AT2 (angiotensin receptor II) and gp91 (major membrane subunit of NADPH oxidase, producing enzyme superoxide anions), potential markers of the severity of diabetic retinopathy.Results Our results have shown a switch of cholesterol and sphingomyelin flat rafts to caveolae rafts. The analysis of the constituent proteins (flotillin and caveolin) and functional (AT2, gp91 and p47phox) have shown that these are mobile in the membrane in an inflammatory context. Subunits of NADPH oxidase seem to gather in caveolae rafts and more precisely when the enzyme is activated.Conclusion The proteins of interest and the switch composition of lipid rafts therefore would appear to be an indicator of the progression of diabetic retinopathy and it would be interesting to provide a mechanistic explanation for the evolution of this chronic vascular disease to consider potential therapeutic targets.

Interleukin-1β alters the sensitivity of cannabinoid CB1 receptors controlling glutamate transmission in the striatum

Proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1β (IL1β) regulate both excitatory and inhibitory synaptic transmission in the central nervous system. The interaction between IL1β and endocannabinoid system (ECS) is also emerging, based on the evidence that IL1β effects on striatal spontaneous excitatory and inhibitory postsynaptic currents are regulated by transient receptor potential vanilloid 1 (TRPV1) channels, members of the ECS. Furthermore, IL1β has also been shown to control the sensitivity of cannabinoid CB1 receptors controlling GABA transmission (CB1Rs(GABA)) in the striatum.To better detail the synaptic action of IL1β, and to clarify its complex interaction with the ECS, here we investigated the possible interplay between IL1β and CB1Rs controlling glutamate transmission (CB1Rs(glu)), other critical elements of the ECS. Our results show that the sensitivity of CB1Rs(glu) is fully blocked in the presence of IL1β in corticostriatal brain slices, and that the protein kinase C/TRPV1 pathway is involved in this effect. IL1β failed to modulate the sensitivity of glutamate synapses to the stimulation of GABAB receptors. We also provided evidence that IL1β–CB1Rs(GABA) but not IL1β–CB1Rs(glu) interaction is under the control of the brain-derived neurotrophic factor (BDNF)/trkB signaling and of lipid raft composition, because BDNF gene partial deletion, pharmacological blockade of trkB and membrane cholesterol removal with methyl-β-cyclodextrin all blocked IL1β-mediated inhibition of CB1Rs(GABA) but left unaltered the sensitivity of CB1Rs(glu) to this cytokine.Our results provide further evidence that synaptic transmission and the ECS are regulated by IL1β in the striatum.

Peroxisome proliferator‐activated receptor gamma‐coactivator‐1 alpha coordinates sphingolipid metabolism, lipid raft composition and myelin protein synthesis

Peroxisome proliferator-activated receptor gamma-coactivator-1 alpha (PGC1a) is involved in energy and lipid metabolism, and its loss leads to neurodegenerative changes in the striatum. Here we performed lipidomic analysis on brain extracts from PGC1a mutant and wild-type mice. We found increased phosphatidylcholine and decreased ceramides in the brain of PGC1a-deficient mice. An analysis of lipid raft fractions revealed increased ceramide, glucocylceramides and GM1 ganglioside in the PGC1a mutants. In the cerebellum, we observed a decrease in proteins associated with myelination, but were unable to detect any morphological abnormalities in compact myelin formation in PGC1a mutants compared with wild-type mice. Although PGC1a is involved in lipid biosynthesis, we concluded that altered lipid composition in the PGC1a mutant did not directly affect central nervous system myelin morphology.

Abstract 163: Metabolomic profiling reveals a protective modulation on fatty acid metabolism in colorectal cancer patients following consumption of freeze-dried black raspberries.

Abstract Metabolic reprogramming which refers to altered nutrient uptake and use is thought to be essential for rapid cancer cell proliferation. Accelerated phospholipid biosynthesis is another metabolic signature of cancer because proliferating cells have a significant need for membrane production. Fatty acids that compose the hydrophobic tails of membrane phospholipids can regulate gene transcription through enzyme-mediated pathways, e.g., cyclooxygenase, lipoxygenase, and changing lipid raft composition that affect receptor-mediated signalings. We previously showed that dietary intervention with freeze-dried black raspberries (BRBs) decreased cell proliferation in colorectal tumors and IL-8, a pro-inflammatory cytokine, in plasma in colorectal cancer patients. The goal of the current study was to determine if BRBs affect fatty acid metabolism which may contribute to their anti-proliferative and anti-inflammatory activities. Plasma samples were collected from 28 colorectal cancer patients before and after oral consumption of BRB powder (60g/day) for 1-to-9 wks for metabolomic profiling analysis. 421 biochemicals were analyzed using UHPLC/Gas chromatography and mass spectrometry. When data from all 28 patients were combined, the top 30 ranking biochemicals suggest that berry intervention led to alterations mostly in lipids, following by carbohydrates, amino acids, and cofactors and vitamins. Both monounsaturated fatty acids (MUFAs), e.g., eicosenoate (20:1n9 or 11), and polyunsaturated fatty acids (PUFAs), e.g., linoleate (18:2n6), arachidonate (20:4n6), were lower in the post plasma samples. PUFAs can be synthesized from linoleate and they also can be released by phospholipase A from phospholipid membrane. Therefore, berry intervention might alter activities of phospholipase A, elongases, and desaturases which in turn reduce levels of PUFAs. Alternatively, berry intervention increased secondary bile acids, e.g., glycodeoxycholate, produced by the action of enzymes existing in the microbial flora of the colonic environment, suggesting dietary BRBs could alter colonic microflora. Berry intervention associated alternations in bile acid metabolism could affect fat absorption and subsequently impact fatty acid metabolism. In conclusion, our results suggest that dietary berry consumption protectively modulates enzymes associated with fatty acid metabolism in the host as well as in gut microflora leading to decreased proliferation and inflammation in colorectal cancer patients. Supported by R01 CA148818 to L-S Wang Citation Format: Li-Shu Wang, Matthew Young, Chieh-Ti Kuo, Mark Arnold, Edward Martin, Christine Sardo, Gary Stoner. Metabolomic profiling reveals a protective modulation on fatty acid metabolism in colorectal cancer patients following consumption of freeze-dried black raspberries. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 163. doi:10.1158/1538-7445.AM2013-163

Colocalization of the ganglioside G(M1) and cholesterol detected by secondary ion mass spectrometry.

The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remain a major challenge. The concept of lipid rafts is widely invoked; however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of secondary ion mass spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano- and microscale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the colocalization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d > 3 μm); (3) nanometer-scale domains (d = 100 nm to 1 μm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.

Similar pyruvate kinase modifications in glioblastoma cells by 7β-hydroxycholesterol and glutamine withdrawal

Oxysterols possess anti-proliferative properties that may be used with much effect in the treatment of cancer. We have demonstrated previously that 7 beta-hydroxycholesterol (7b-HC) provokes both metabolic stress, as witnessed by AMPK activation, and changes in lipid raft composition in C6 glioblastoma cells. These observations suggested that glycolysis might have been changed. Here we will show that 7b-HC increases cell cycle time and that it changes the affinity of pyruvate kinase to its substrate, phosphoenol pyruvate. The latter effect is mimicked by glutamine withdrawal.

Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na⁺/H⁺ exchanger NHE3 is regulated by the Na⁺/H⁺ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO₂/HCO₃^- mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

Involvement of PDE2 in a Localized Regulation of the L-Type Ca2+ Channels by Progesterone

We have demonstrated that a nitric oxide production induced by stimulation of progesterone (P4) receptor suppresses cAMP-stimulated L-type Ca2+ currents (ICa,L) through an increase in cytosolic cGMP, which may be related to the sex difference of QT intervals and risks of arrhythmia. We here seek the underlying mechanisms on the regulation of ICa,L in patch-clamped guinea pig ventricular myocytes. An external application of P4 (100 nM) suppressed ICa,L about 30 % (100 nM P4; 58±4 %, n=13, without P4 (time control); 84±2 %, n=5). In the presence of a specific PDE2 inhibitor, EHNA (30 μM), P4 suppressed ICa,L to 84±5 % (n=7), which is the same extent with the time control. Pharmacological studies revealed that hydrolysis of cAMP by a cGMP-stimulated phosphodiesterase (PDE2) involves in the P4-induced ICa,L suppression. However the underlying mechanisms of the ICa,L suppression remain to be solved. Thus, we employed sucrose density gradient fractionation experiments to examine localization of the molecules in this system, which is dependent of lipid-raft composition. We found that substantial fraction of CaV1.2 and PDE2 clustered in caveolae fractions, but PDE3 clustered in extra-caveolae fractions. Furthermore, a proximity ligation assay (Olink) revealed that CaV1.2 interacted both with PDE2 and PDE3, but there was no interaction between PDE2 and PDE3. These results suggest that PDE2 and the L-type Ca2+ channel co-localizes at the caveolae for the non-genomic regulation of ICa,L by P4.