Role of lipid rafts in membrane delivery of renal epithelial Na+-K+-ATPase, thick ascending limb

2007 ◽  
Vol 292 (3) ◽  
pp. R1328-R1337 ◽  
Author(s):  
Pia Welker ◽  
Beate Geist ◽  
Jan-Henning Frühauf ◽  
Michele Salanova ◽  
David A. Groneberg ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Rat Kidney ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Thick Ascending Limb ◽  
Low Density ◽  
Α Subunit ◽  
Density Fraction ◽  
Short Term Exposure

Lipid rafts are cholesterol- and shingolipid-enriched membrane microdomains implicated in membrane signaling and trafficking. To assess renal epithelial raft functions through the characterization of their associated membrane proteins, we have isolated lipid rafts from rat kidney by sucrose gradient fractionation after detergent treatment. The low-density fraction was enriched in cholesterol, sphingolipid, and flotillin-1 known as lipid raft markers. Based on proteomic analysis of the low-density fraction, the protein with the highest significance score was the α-subunit of Na+-K+-ATPase (NKA), whose raft association was validated by simultaneous immunoblotting. The β-subunit of NKA was copurified from the low-density fraction. To test the role of lipid rafts in sorting and membrane delivery of renal-transporting epithelia, we have chosen to study thick ascending limb (TAL) epithelium for its high NKA activity and the property to be stimulated by antidiuretic hormone (ADH). Cultured rabbit TAL cells were studied. Cholesterol depletion and detergent extraction at warmth caused a shift of NKA to the higher-density fractions. Comparative preparations from blood monocytes revealed the absence of NKA from rafts in these nonpolarized cells. Short-term exposure of rabbit TAL cells to ADH (1 h) caused translocation and enhanced raft association of NKA via cAMP activation. Preceding cholesterol depletion prevented this effect. TAL-specific, glycosylphosphatidylinositol-anchored Tamm Horsfall protein was copurified with NKA in the same raft fraction, suggesting functional interference between these products. These results may have functional implications regarding the turnover, trafficking, and regulated surface expression of NKA as the major basolateral ion transporter of TAL.

scholarly journals Renal Na+-K+-Cl− cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent

2008 ◽  
Vol 295 (3) ◽  
pp. F789-F802 ◽  
Author(s):  
Pia Welker ◽  
Alexandra Böhlick ◽  
Kerim Mutig ◽  
Michele Salanova ◽  
Thomas Kahl ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Integral Membrane Protein ◽  
Surface Expression ◽  
Cholesterol Depletion ◽  
Xenopus Laevis Oocytes ◽  
Thick Ascending Limb ◽  
Short Term ◽  
Nacl Transport ◽  
Triton X

Apical bumetanide-sensitive Na+-K+-2Cl− cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40–70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na+-Cl− cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by 86Rb+ influx in Xenopus laevis oocytes was markedly reduced by methyl-β-cyclodextrin (MβCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MβCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.

scholarly journals Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds

2013 ◽  
Vol 16 (6) ◽  
pp. 1361-1371 ◽  
Author(s):  
Caroline Nothdurfter ◽  
Sascha Tanasic ◽  
Barbara Di Benedetto ◽  
Manfred Uhr ◽  
Eva-Maria Wagner ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Lipid Rafts ◽  
Gabaa Receptor ◽  
Lipid Raft ◽  
Tricyclic Antidepressant ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Receptor Modulation ◽  
Gabaa Receptor Subunits ◽  
Triton X

Abstract Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

scholarly journals Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

2008 ◽  
Vol 19 (10) ◽  
pp. 4366-4373 ◽  
Author(s):  
Xinxin Gao ◽  
Jin Zhang
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Lipid Rafts ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spatiotemporal Analysis ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Growth Factor Signaling

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.

Distribution and oligomeric association of splice forms of Na+-K+-ATPase regulatory γ-subunit in rat kidney

2002 ◽  
Vol 282 (3) ◽  
pp. F393-F407 ◽  
Author(s):  
Elena Arystarkhova ◽  
Randall K. Wetzel ◽  
Kathleen J. Sweadner
Keyword(s):  
Splice Variants ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Outer Medulla ◽  
Α Subunit ◽  
Proximal Tubule Cells ◽  
Γ Subunit ◽  
Outer Stripe ◽  
A Minor ◽  
Splice Forms

Renal Na+-K+-ATPase is associated with the γ-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, γa and γb. Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the α-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na+-K+-ATPase conformation. In the cortex, the proportion of the γb-subunit was markedly lower, and the γa-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the γb-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na+-K+-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the γb-subunit. Because different mechanisms maintain and regulate Na+ homeostasis in different nephron segments, the splice forms of the γ-subunit may have evolved to control the renal Na+ pump through pump properties, gene expression, or both.

A Critical Role for Membrane Sulfatide in Platelet Aggregation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 626-626
Author(s):  
Prasenjit Guchhait ◽  
Corie Shrimpton ◽  
Kochi Honke ◽  
Perumal Thiagarajan ◽  
Jose A. Lopez
Keyword(s):  
Platelet Aggregation ◽  
Lipid Rafts ◽  
Platelet Function ◽  
Critical Role ◽  
Membrane Microdomains ◽  
Systemic Lupus Erythematosis ◽  
Single Chain ◽  
Induce Platelet Aggregation ◽  
Adhesive Interactions

Abstract Sulfatide (galactocylceramide 3′-sulfate) is a sulfated glycosphingolipid expressed on the surfaces of erythrocytes, leukocytes, platelets and a variety other cells, that is known to interact with several cell adhesion molecules involved in hemostasis, including von Willebrand factor (VWF), laminin, thrombospondin, P-selectin and β2-glycoprotein I. Because these ligands are involved in many platelet adhesive interactions, we hypothesize that membrane sulfatide plays an important role in these processes. To examine this, we have cloned and purified a sulfatide-specific single-chain variable fragment (scFv) antibody from a phage-display library constructed from mRNA taken from the lymphocytes of patients with systemic lupus erythematosis. This scFv, PA38, specifically bound sulfatide, and did not react with the related sphingolipids cerebroside, ceramide, or sphingomyelin, or the phospholipids phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine. Using this tool, we examined the role of sulfatide in platelet function. We observed that PA38 dose-dependently (at 5 and 10 μg/ml) inhibited the aggregation of human platelets induced by either collagen or ADP. A control scFv produced in a similar manner had no effect. Furthermore, PA38 delayed platelet plug formation by 23 sec (with collagen-ADP agonist) and 46 sec (with collagen-epinephrine) in whole blood from normal human donors, as measured in a platelet function analyzer, PFA-100 (Dade Behring). Further, to verify that this was a sulfatide-specific effect, we compared collagen-induced platelet aggregation in normal mice to that of mice deficient in cerebroside sulfotransferase (CST)—a critical enzyme in the sulfatide synthetic pathway. The CST−/− mice fail to express sulfatide on the cell surface, and displayed defective platelet aggregation. Consistent with this, the PA38 also significantly inhibited collagen-induce platelet aggregation in wild-type mice. Given the importance of lipid rafts in signaling and adhesive processes, we looked for the localization of sulfatide in these membrane microdomains. Indeed, we found that sulfatide is enriched in lipid rafts suggesting a role for sulfatide in lipid-raft mediated events. Thus, we provide evidence for a key role of a membrane lipid, sulfatide in the adhesive interactions involved in platelet function. With one notable exception, the key adhesive roles in platelet-platelet interaction have all previously been assigned to proteins.

Modulation of Tissue Factor-Factor VIIa Signaling by Lipid Rafts and Caveolae.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1744-1744
Author(s):  
Vineet Awasthi ◽  
Samir Mandal ◽  
Veena Papanna ◽  
L. Vijaya Mohan Rao ◽  
Usha Pendurthi
Keyword(s):  
Cell Signaling ◽  
Lipid Rafts ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Factor Viia ◽  
Membrane Microdomains ◽  
Caveolin 1 ◽  
Intracellular Signaling Pathways ◽  
Plasma Membrane Microdomains

Abstract Tissue factor (TF) is a cellular receptor for clotting factor VIIa (VIIa) and the formation of TF-VIIa complexes on cell surfaces not only triggers the coagulation cascade but also transduces cell signaling via activation of protease-activated receptors (PARs), particularly PAR2. Although a number of recent studies provide valuable information on intracellular signaling pathways that are activated by TF-VIIa, the role of various cell surface components in mediating the interaction of TF-VIIa with PARs, and the subsequent signal transmittance are unknown. Unlike thrombin and trypsin, VIIa has to bind to its cellular receptor (TF) to activate PARs. The inability of TF-VIIa to effectively activate Ca2+ signaling and failure to desensitize the signaling to subsequently added trypsin suggest that the TF-VIIa is a poor activator of PAR2. Despite this, a number of studies have shown that VIIa is as effective as trypsin or PAR2 agonist peptide in activating intracellular signaling pathways and gene expression in cells expressing TF. Although the potential mechanism for this phenomenon is unknown, compartmentalization of TF, PAR2, and G-proteins in plasma membrane microdomains could facilitate a robust TF-VIIa-induced PAR2-mediated cell signaling. Although certain G-protein coupled receptors and G-proteins are known to be segregated into specialized membrane microdomains, lipid rafts and caveolae, little is known whether PARs are segregated into lipid rafts and caveolae, and how such segregation might influence their activation by TF-VIIa and the subsequent coupling to G-proteins. To obtain answers to some of these questions, in the present study, we have characterized TF and PAR2 distribution on tumor cell surfaces and investigated the role of lipid raft/caveolae in modulating the TF-VIIa signaling in tumor cells. Detergent extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF and PAR2 were distributed both in lipid rafts (low-density) and soluble fractions. Immunofluorescence confocal microscopy revealed that TF at the cell surface is localized in discrete plasma membrane microdomains, and colocalized with caveolin-1, a structural integral protein of caveolae, indicating caveolar localization of TF. Similar to TF, PAR2 also displayed significant punctuate staining and colocalization with caveloin-1. Further, a substantial fraction of TF and PAR2 was colocalized in caveolae. Disruption of lipid rafts/caveolae by ß-methyl cyclodextrin or filipin treatments reduced TF association with PAR2 in lipid rafts and caveolar fractions and impaired the TF-VIIa-induced cell signaling (PI hydrolysis and IL-8 gene expression). Additional studies showed that both mßCD and filipin treatments specifically impaired TF-VIIa cleavage of PAR2 and but had no significant effect on trypsin cleavage of PAR2. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling. In summary, the data presented herein demonstrate that TF localization at the cell membrane could influence different functions of TF differently. While caveolar localization of TF had no influence in propagating the procoagulant activity of TF, it is essential in supporting the TF-VIIa-induced cell signaling.

scholarly journals Dynamics of putative raft-associated proteins at the cell surface

2004 ◽  
Vol 165 (5) ◽  
pp. 735-746 ◽  
Author(s):  
Anne K. Kenworthy ◽  
Benjamin J. Nichols ◽  
Catha L. Remmert ◽  
Glenn M. Hendrix ◽  
Mukesh Kumar ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Long Range ◽  
Dominant Factor ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Biochemical Fractionation ◽  
Associated Proteins ◽  
Raft Domains

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (>4 μm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

Role for lipid rafts in regulating interleukin-2 receptor signaling

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1489-1497 ◽  
Author(s):  
Mina D. Marmor ◽  
Michael Julius
Keyword(s):  
Lipid Rafts ◽  
Interleukin 2 ◽  
Phosphatidyl Inositol ◽  
Membrane Microdomains ◽  
Lipid Microdomains ◽  
Interleukin 2 Receptor ◽  
Lipid Environment ◽  
Plasma Membrane Microdomains ◽  
Nonionic Detergents

Lipid rafts are plasma membrane microdomains characterized by a unique lipid environment enriched in gangliosides and cholesterol, leading to their insolubility in nonionic detergents. Many receptors are constitutively or inducibly localized in lipid rafts, which have been shown to function as platforms coordinating the induction of signaling pathways. In this report, the first evidence is provided for a role of these lipid microdomains in regulating interleukin-2 receptor (IL-2R) signaling. It is demonstrated that antibody- or ligand-mediated immobilization of components of lipid rafts, glycosyl-phosphatidyl-inositol–anchored proteins, and the GM1 ganglioside, respectively, inhibit IL-2–induced proliferation in T cells. IL-2Rα is shown to be constitutively enriched in rafts and further enriched in the presence of immobilized anti–Thy-1. In contrast, IL-2Rβ and IL-2Rγ, as well as JAK1 and JAK3, are found in soluble membrane fractions, and their localization is not altered by anti–Thy-1. IL-2–mediated heterotrimerization of IL-2R chains is shown to occur within soluble membrane fractions, exclusively, as is the activation of JAK1 and JAK3. As predicted by these results, the disruption of lipid raft integrity did not impair IL-2–induced signaling. Thus, the sequestration of IL-2Rα within lipid microdomains restricts its intermolecular interactions and regulates IL-2R signaling through impeding its association with IL-2Rβ and IL-2Rγ.

scholarly journals The voltage-gated sodium channel β2 subunit associates with lipid rafts by S-palmitoylation

2021 ◽  
Vol 134 (6) ◽  
Author(s):  
Eric Cortada ◽  
Robert Serradesanferm ◽  
Ramon Brugada ◽  
Marcel Verges
Keyword(s):  
Sodium Channel ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Cholesterol Depletion ◽  
Slight Reduction ◽  
Apical Surface ◽  
Voltage Gated Sodium Channel ◽  
Α Subunit ◽  
Voltage Gated ◽  
Partial Overlap

ABSTRACT The voltage-gated sodium channel is critical for cardiomyocyte function. It consists of a protein complex comprising a pore-forming α subunit and associated β subunits. In polarized Madin–Darby canine kidney cells, we show evidence by acyl-biotin exchange that β2 is S-acylated at Cys-182. Interestingly, we found that palmitoylation increases β2 association with detergent-resistant membranes. β2 localizes exclusively to the apical surface. However, depletion of plasma membrane cholesterol, or blocking intracellular cholesterol transport, caused mislocalization of β2, as well as of the non-palmitoylable C182S mutant, to the basolateral domain. Apical β2 did not undergo endocytosis and displayed limited diffusion within the plane of the membrane; such behavior suggests that, at least in part, it is cytoskeleton anchored. Upon acute cholesterol depletion, its mobility was greatly reduced, and a slight reduction was also measured as a result of lack of palmitoylation, supporting β2 association with cholesterol-rich lipid rafts. Indeed, lipid raft labeling confirmed a partial overlap with apical β2. Although β2 palmitoylation was not required to promote surface localization of the α subunit, our data suggest that it is likely implicated in lipid raft association and the polarized localization of β2.

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