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Keywords:

  • calcium;
  • calmodulin;
  • cholesterol;
  • lipid rafts;
  • plasma membrane Ca2+-ATPase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Spatial and temporal alterations in intracellular calcium [Ca2+]i play a pivotal role in a wide array of neuronal functions. Disruption in Ca2+ homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca2+-ATPase (PMCA) is a high affinity Ca2+ transporter that plays a crucial role in the termination of [Ca2+]i signals and in the maintenance of low [Ca2+]i essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or ‘lipid rafts’ in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca2+ signaling in the central nervous system.

Abbreviations used
CaM

calmodulin

CTXB

cholera toxin subunit B

LS

lovastatin

PMCA

plasma membrane Ca2+-ATPase

PVDF

polyvinylidene fluoride

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Spatial and temporal alterations in free intracellular Ca2+ concentration [Ca2+]i in neurons play a crucial role in key physiological processes such as neurotransmission, synaptic plasticity, signal transduction, and gene expression (Miller 1991; Berridge 1998, 2005). Disruption in the regulation of Ca2+ homeostasis has been implicated in neuronal dysfunction occurring in brain aging and in neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, epilepsy, and stroke (Choi 1995; Garcia and Strehler 1999; Sattler and Tymianski 2000; Mattson and Chan 2001). The plasma membrane Ca2+-ATPase (PMCA) is the primary Ca2+ extrusion system responsible for the transport of Ca2+ from the cytosol to the extracellular medium (Carafoli 1991; Garcia and Strehler 1999). In conjunction with the Na+/Ca2+ exchanger and sarco–endoplasmic reticulum Ca2+-ATPase, PMCA is responsible for the maintenance of tightly regulated levels of free intracellular Ca2+ (Brini and Carafoli 2000).

Mammalian PMCA is encoded by four genes giving rise to four different isoforms that differ quite significantly in their regulation by calmodulin (CaM), the primary Ca2+ sensor in cells (Strehler and Zacharias 2001). Binding of four Ca2+ ions to high affinity sites on apo-CaM triggers a conformational change in which the Ca2+-binding domains move farther apart, thus exposing hydrophobic residues on the central linker region (Babu et al. 1988; Ikura et al. 1992). CaM retains considerable structure in sodium dodecyl sulfate (SDS), and Ca2+-dependent conformational changes in the protein can be assessed by monitoring altered mobility in SDS–polyacrylamide gel electrophoresis (PAGE) (Klee et al. 1979; Zhang and Vogel 1994; Yao and Squier 1996). Calcium-activated CaM binds to an autoinhibitory domain on the C-terminal end of PMCA, dissociating it away from the active site, and resulting in several-fold stimulation of PMCA activity (Falchetto et al. 1992; Lushington et al. 2005). Recent in vitro studies have shown that PMCA is uniquely sensitive to the biophysical properties of its surrounding lipid environment (Pang et al. 2005; Duan et al. 2006; Tang et al. 2006). PMCA activity was found to be two- to threefold higher when the purified protein was reconstituted into ordered lipids such as eye lens fiber lipid, lens epithelial lipid, or the synthetic lipid dipalmitoylphosphatidylcholine compared with PMCA present in the disordered (fluid) lipid dioleoylphosphatidylcholine (Tang et al. 2006).

Lipid rafts are specialized microdomains in the plasma membrane known to serve as platforms for the assembly of protein complexes involved in signaling pathways (Simons and Toomre 2000; Grzybek et al. 2005). These membrane microdomains are characterized by their high content of cholesterol and sphingolipids (Brown and London 1998). It is believed that hydrogen bonding between the 3-OH group of cholesterol and the amide group of sphingolipids segregates these lipids away from the rest of the plasma membrane, resulting in the formation of tightly packed, ordered microdomains (Schroeder et al. 1994; Brown and London 1997, 1998). Given that PMCA activity is stimulated by the presence of ordered lipids in its surrounding membrane environment, it may be predicted that the naturally high degree of lipid order in neuronal rafts may be favorable for PMCA activity. Consistent with this possibility, PMCA was recently shown to be associated with lipid rafts isolated from synaptic membranes of porcine cerebellum (Sepulveda et al. 2006). The physiological significance of the raft-associated PMCA and its regulation in intact neurons, however, remain unclear.

The aim of the present study was twofold: (i) to determine and characterize the association of PMCA with lipid rafts in primary cultured neurons and (ii) to determine the effect of disruption of raft lipid environment on the properties of the raft-associated PMCA. Both biochemical and morphological evidence showed the partitioning of PMCA into rafts in preparations isolated from primary neurons and in intact cells. The raft-associated PMCA exhibited much higher activity than the non-raft PMCA, consistent with higher protein levels associated with these microdomains. Disruption of rafts by chronic depletion of cellular cholesterol dramatically decreased the activity of the raft-associated PMCA with no effect on the activity of PMCA excluded from these microdomains. The decrease in activity of the raft-PMCA was not due to alterations in its protein levels or the levels of its activator protein CaM. We propose that the ordered environment of rafts, maintained by high levels of cholesterol, enhance PMCA activity in vivo. The association of PMCA with lipid rafts therefore represents a novel CaM-independent mechanism for the regulation of its activity and, consequently, of Ca2+ signaling in the central nervous system.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Lovastatin (LS), mevalonate, ouabain, thapsigargin, oligomycin, ATP, and horseradish peroxidase-coupled cholera toxin subunit B (CTXB) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Bicinchoninic acid protein assay kit was obtained from Pierce (Rockford, IL, USA) and Protease Inhibitor Cocktail III was from Calbiochem (San Diego, CA, USA). Amplex Red kit for cholesterol measurement and gradient gels (8–16%) were purchased from Invitrogen (Carlsbad, CA, USA). The following sources were used for the various primary antibodies: anti-PMCA (Affinity Bioreagents, Golden, CO, USA), anti-CaM (Upstate Biotechnologies, Charlottesville, VA, USA), and anti-flotillin-1 and Thy-1 (BD Biosciences, San Jose, CA, USA). Isoform-specific PMCA antibodies were a gift from Dr E.E. Strehler, Mayo Clinic (Rochester, MN, USA) and NAP-22 antibodies were provided by Dr S. Maekawa, Kobe University (Kobe, Japan).

Cell culture

Dissociated cortical and hippocampal neuron cultures were established from 18-day-old Sprague–Dawley fetuses as described previously (Michaelis et al. 1994, 1998, 2005). Briefly, pups were delivered by cesarean section, while the dam was fully anesthetized with pentobarbital (0.1g/kg), and the brains recovered according to protocols approved by the institutional IACUC and in accordance with National Institutes of Health guidelines. The cortical lobes and hippocampi were dissected and cells dissociated by gentle trituration with trypsin. After the final precipitation step, neurons were re-suspended in fresh Dulbecco's modified Eagle's medium/F-12 with 10% fetal calf serum, and plated at densities ranging from 0.3–3 × 106 cells/dish on 35-mm tissue culture dishes coated with poly-d-lysine. After 24 h to allow cell attachment, the fetal calf serum-containing medium was replaced by a defined medium with Dulbecco's modified Eagle's medium/F-12 containing N2 supplements, potassium bicarbonate (15 mmol/L), and 20% glial conditioned medium. Cells were fed twice a week by replacing 1/3 of the medium with fresh medium. It was estimated that > 90% of the cells in the primary cultures were neurons, based on morphological characteristics and confirmation with immunofluorescent staining for glial fibrillary acidic protein and neuron-specific enolase.

Isolation of lipid rafts from primary neurons

Rafts were isolated from primary cortical neurons at day in vitro 6 in culture by previously described methods (Sargiacomo et al. 1993). Briefly, the medium was removed from the cell culture dishes, cells were washed twice with 200 mmol/L Tris–Cl, pH 7.4, and lysed with 3 mmol/L Tris–Cl, pH 8.0, containing a cocktail of protease inhibitors. The neuronal lysate was solubilized in a buffer containing 25 mmol/L Tris–Cl, 75 mmol/L NaCl, pH 7.5 (TNB) plus 1% (v/v) Brij 98, and incubated on ice for 30 min with mild vortexing. The suspension was mixed with an equal volume of 90% (w/v) sucrose solution in TNB, overlayed with a two-step gradient consisting of 35% (w/v) and 5% (w/v) sucrose in TNB. Centrifugation was performed for 18 h at 135 000 g at 4°C in a TLS-55 rotor in an OptimaTM MAX Ultracentrifuge (Beckman Coulter, Fullerton, CA, USA). Ten fractions (0.5 mL) were collected from the top to the bottom of the tube. Protein estimation was performed by the bicinchoninic acid method according to the manufacturer’s instructions.

Cholesterol and GM1 measurement and cholesterol depletion

Cholesterol was measured using Amplex Red kit according to the manufacturer’s instructions. GM1 ganglioside levels were determined by dot-blot using CTXB, known to have a very high affinity for this lipid. Briefly, 1 µg protein from each fraction was applied onto a polyvinylidene fluoride (PVDF) membrane, blocked with 5% milk, and exposed to CTXB (10 µmol/L) for 2 h (Hering et al. 2003). Color was developed by incubating the dot-blot in a mixture of 1.4 mmol/L 3,3′-diaminobenzidine tetrahydrochloride, 200 mmol/L nickel chloride, and 6.2 mmol/L H2O2.

Chronic depletion of cellular cholesterol was achieved by treating neurons with LS, an inhibitor of the enzyme HMG CoA reductase, the rate limiting step in cholesterol biosynthesis. It has been shown that LS when used by itself causes apoptosis in cells, however, the toxic effects of LS are abrogated by the addition of mevalonate, the product of HMG CoA reductase (Wang and Macaulay 1999), presumably by allowing the synthesis of other downstream non-steroidal derivatives of this pathway important for cell survival. Briefly, neurons were treated for 4 days with LS (2.5 µmol/L) and mevalonate (200 µmol/L) added to the culture medium on day in vitro 2 (Hao et al. 2001; Hering et al. 2003). The vehicle (ethanol) -treated and LS-treated cells were collected and lysed on day in vitro 6, protein concentration was measured and equal amounts of protein solubilized and loaded on sucrose density gradients for raft isolation as described above.

Measurement of PMCA activity

The activity of PMCA was determined by monitoring the generation of Pi from ATP as described previously (Zaidi et al. 1998). The activity buffer contained 25 mmol/L Tris–Cl, pH 7.4, 1 mmol/L ATP, 50 mmol/L KCl, 0.1 mmol/L ouabain, 4 mg/L oligomycin, 0.1 µmol/L thapsigargin, 200 μmol/L EGTA, and CaCl2 added to yield a final free Ca2+ concentration of 0.5 µmol/L. The final free Ca2+ concentration was calculated using the software calcium.com that calculates the multiple equilibria between all ligands in solution. PMCA activity measured in the absence of added CaM is referred to as basal activity and that in the presence of 340 nmol/L CaM as CaM-stimulated activity. After a 5 min pre-incubation, the reaction was started by the addition of ATP, continued for 30 min at 37°C, and stopped by addition of the Malachite Green dye (Lanzetta et al. 1979). The PMCA activity was defined as the Ca2+-activated ATP hydrolysis and expressed as nanomoles of Pi liberated per milligram of protein per minute, based on values from a standard curve.

Immunoblot analysis

Proteins were separated by SDS–PAGE on 8–16% gradient gels and transferred to methanol-activated PVDF membranes as described previously (Zaidi and Michaelis 1999; Zaidi et al. 2003). Non-specific interactions were blocked with 5% (w/v) milk for 1 h at 25°C and the membranes incubated overnight with the indicated concentrations of primary antibodies. Alkaline phosphatase-conjugated secondary antibodies (1 : 1000) were added for 2 h at 25°C. Immunoreactive bands were visualized by developing the immunoblots using the substrate 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium. Blots were scanned using an HP Scanjet 4470 scanner (Hewlett Packard, Palo Alto, CA, USA) and relative intensities of the immunoreactive bands quantified using Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA, USA).

Lipid raft patching in neurons

Exposure of hippocampal neurons to anti-Thy-1 antibody was used to induce patching of rafts according to previously described methods (Wong and Schlichter 2004). This was followed by labeling with anti-PMCA2 antibody. Briefly, hippocampal neurons were chilled on ice for 15 min and washed with ice-cold PBS supplemented with 0.7 mmol/L CaCl2 and 0.48 mmol/L MgCl2 (PBS-CM). Cells were subsequently incubated with anti-Thy-1 antibody (1 : 100) in 0.1% (w/v) BSA in PBS-CM for 1 h at 37°C, followed by Alexa Fluor 568 conjugated anti-mouse secondary antibody (1 : 200) for 1 h at 37°C. Neurons were then fixed with 4% (w/v) p-formaldehyde for 30 min on ice. After blocking with 5% (w/v) BSA, cells were labeled with anti-PMCA 2 antibody (1 : 500) and Alexa Fluor 488 conjugated anti-rabbit secondary antibody (1 : 500). Images were acquired on a Zeiss LSM-510 confocal microscope (Carl Zeiss, Thornwood, NY, USA) using fixed laser strength, pinhole size, and detector gain for all samples within a single experiment.

Data Analysis

All data are means ± SEM obtained from six experiments using raft preparations isolated from independent cultures. Statistical significance of differences between various samples was assessed using Student’s t-test for unpaired samples.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Characterization of lipid rafts isolated from primary cortical neurons

Lipid rafts can be isolated from membranes of different tissue and cell types based on their detergent insolubility at low temperature and low buoyant density after discontinuous sucrose gradient centrifugation. We isolated detergent-resistant membranes from neuronal lysates on the basis of their insolubility in 1% Brij 98, a non-ionic detergent known to preserve raft integrity even at physiological temperatures (Drevot et al. 2002; Lucero and Robbins 2004). After 18 h of centrifugation at 135 000 g, lipid rafts formed a floating band of white material at the interface of 5% and 35% sucrose. Ten fractions were collected from the gradient starting from the top to the bottom of the tube. Lipid rafts, identified by their enrichment in cholesterol and GM1 ganglioside, two major lipids that form the core of the raft membranes (Brown and London 2000), were found in the low density fractions 2–4, as shown in Figs 1a and b. Protein measurement in the various fractions showed a dual distribution. A small peak representing 18 ± 0.02% of the total protein (n = 6 preparations) was present in the low density fractions 2–4, whereas the larger peak corresponded to the high density fractions 6–10 (Fig. 1a), in accordance with earlier reports (Hering et al. 2003). We probed the density gradient fractions 2–10 for the GPI-anchored cell surface protein flotillin-1 and the cholesterol-binding protein NAP-22, positive markers for rafts (Bickel et al. 1997; Maekawa et al. 1999; Rivera-Milla et al. 2006). As expected, both proteins were highly enriched in the low density fractions corresponding to rafts (Fig. 1b). These results show that detergent-resistant, low density fractions isolated from primary cortical neurons exhibit the major properties of lipid rafts isolated from brain tissue.

image

Figure 1.  Distribution of protein, cholesterol, raft markers, and plasma membrane Ca2+-ATPase (PMCAs) in sucrose density gradient fractions isolated from primary cortical neurons. (a) Fractions 1–10, isolated from the sucrose density gradient were analyzed for cholesterol (•) monitored by the Amplex Red cholesterol assay kit and total protein (○) determined by the bicinchoninic acid assay. Data are mean ± SEM obtained from six experiments using different cultures. (b) Dot-blots and immunoblots to detect GM1 ganglioside and raft marker proteins, respectively. Typically, 1.0 µg of protein from fractions 2–10 were dot-blotted on PVDF membrane, blocked with 5% milk, and probed with peroxidase-labeled cholera toxin B subunit (10 µmol/L) to detect GM1 levels. For immunoblots, total protein from fractions 2–10 was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to PVDF membranes, and probed with the following antibodies: Anti-flotillin-1 (1 : 500), anti-NAP-22 (1 : 250), anti-pan PMCA (1 : 1000), and anti-calmodulin (CaM) (1 : 500) as described in the Materials and methods section. Protein amounts used for immunoblotting were: flotillin-1 (25 µg), NAP-22 (30 µg), PMCA (10 µg), and CaM (30 µg). To determine the mechanism underlying the altered electrophoretic mobility of CaM, neurons were lysed in presence of 5 mmol/L EDTA added to the lysis buffer, solubilization buffer, and the sucrose density gradient solutions. (c) Immunoblots to detect PMCA isoforms. Protein amount used for immunoblotting for each isoform was: 20 µg, and antibody concentration was 1 : 500 for PMCA 1, PMCA 2, PMCA 3, and PMCA 4. Representative blots from six independent experiments with similar results are shown.

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Partitioning of PMCA into neuronal rafts

Immunoblot analysis of the fractions from the sucrose density gradient centrifugation probed with a pan antibody known to recognize all 4 isoforms of PMCA showed that a substantial amount of PMCA was localized in fractions 2–4 (∼60% ± 3%, n = 6 preparations), the raft fractions, with lower levels present in the non-raft domains (∼40% ± 10%, n = 6 preparations) (Fig.1b). Examination of the density gradient fractions for the PMCA activator CaM showed that it was also distributed in both raft and non-raft domains of the neuronal membrane (34% ± 2.6% in rafts, n = 6 preparations) (Fig. 1b). Interestingly, the electrophoretic mobility of CaM associated with rafts (fractions 2–4) appeared to be higher compared with the pool excluded from these domains. Given that apo- and Ca2+-bound forms of CaM differ markedly in their structural conformations, and hence exhibit differential mobility in SDS–PAGE (Klee et al. 1979; Babu et al. 1988), it is possible that the faster moving raft-associated CaM may exist in a Ca2+-activated form. To test this possibility, we chelated Ca2+ in the neuronal lysates by adding 5 mmol/L EDTA to the lysis buffer, solubilization buffer, and the sucrose solutions used for raft isolation. Rafts isolated in the presence of EDTA were similar to the preparations isolated in its absence in terms of cholesterol profile, enrichment in raft markers, and the levels of PMCA (data not shown). However, addition of EDTA abolished the differential mobility of CaM in the raft versus the non-raft fractions, and CaM in all fractions moved uniformly on SDS–PAGE (Fig. 1b). Interestingly, addition of 5 mmol/L CaCl2 to the neuronal lysate and the various buffers used for raft isolation did not reverse the electrophoretic mobility of CaM, suggesting that the altered mobility of raft-localized CaM may not simply be due to conformational changes induced by Ca2+ binding but may be attributed to other potential mechanisms such as post-translational modifications, altered folding, and/or conformational changes induced in the protein due to its interaction with other proteins and/or lipids present in the raft environment.

Brain neurons are unique in that they express all four known isoforms of PMCA, each of which has unique characteristics, particularly those pertaining to regulation by Ca2+ and CaM (Elwess et al. 1997; Caride et al. 1999). In order to determine whether all PMCA isoforms are represented in neuronal rafts or a specific isoform is particularly abundant or exclusively present, we probed the fractions with isoform-specific antibodies. Like the pan protein, the PMCA isoforms, particularly PMCA 1, 2, and 3 appeared to be highly represented in the raft domains of nerve cells (Fig. 1c). Interestingly, the protein band separation pattern of the PMCA isoforms, particularly, that of PMCA 1 and PMCA 2, differed between the raft and non-raft fractions raising the possibility of the localization of different splice variants of these isoforms in specific subdomains of the plasma membrane.

Co-localization of PMCA2 with the raft marker Thy-1 in intact neurons

Having shown that PMCA is partitioned into rafts isolated from nerve cells, it was of interest to determine if PMCA could be visualized in raft-like domains in intact neurons. While the sizes of rafts have not been conclusively determined, a number of estimates place them below the resolution of light microcopy (Varma and Mayor 1998). Techniques to create ‘patches’ of lipid rafts using multivalent toxins and/or antibodies can make them large enough to be visualized by standard immunofluorescence microscopy and such approaches have been frequently used to complement biochemical studies. We patched rafts in the plasma membrane of intact hippocampal neurons using a monoclonal Thy-1 antibody that recognizes an external epitope of this protein. After labeling with anti-Thy-1 antibody and an anti-mouse secondary antibody, neurons were fixed and processed for immunolabeling with an antibody to the brain-specific isoform PMCA 2. Confocal microscopy showed that the patching procedure caused Thy-1 to distribute into discrete clusters on the neuronal plasma membrane of the cell bodies and throughout the neuritic network and exhibit a punctate appearance, shown in red (Fig. 2a). The PMCA 2 labeling appeared to be more uniformly spread across the plasma membrane of the cell body and extending into the neurites, as shown in green (Fig. 2b). In an overlay of the two images, PMCA 2 appeared to overlap with Thy-1 in discrete membrane patches shown in yellow (Fig. 2c). The co-localization of PMCA 2 with Thy-1 was not complete as there were regions, particularly in the cell body, where PMCA 2 immunoreactivity was evident, but no Thy-1 immunoreactivity was visible. Such PMCA distribution was probably related to the non-raft PMCA and/or the newly synthesized PMCA en route for delivery to the plasma membrane. Similar results were obtained when antibodies against PMCA 1 were used for the co-localization experiments suggesting that other PMCA isoforms were also co-localized with rafts in intact neurons (data not shown). Thus morphological observations in intact neurons complement our immunochemical studies showing the partitioning of PMCA into neuronal rafts.

image

Figure 2.  Co-localization of plasma membrane Ca2+-ATPase 2 (PMCA 2) with the raft marker protein Thy-1 in hippocampal neurons. Lipid raft patching and immunocytochemistry were performed on embryonic rat hippocampal neurons on day in vitro 7 in culture. Anti-Thy-1 (1 : 100) and goat anti-mouse secondary antibody conjugated to Alexa Fluor 568 (1 : 200) were used to patch and label lipid rafts in live neurons. After fixation and permeabilization, PMCA was labeled with anti-PMCA 2 (1 : 500) and a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1 : 500). (a) the immunoreactivity of Thy-1 shown in red, (b) the immunoreactivity of PMCA 2 shown in green, and (c) the regions where the two proteins overlap, in yellow (scale bars, 10 µm). Magnified images of the neuritic processes are shown in (d) and (e). Representative images from three independent experiments with similar results are shown.

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PMCA activity in raft and non-raft microdomains

Given that PMCA was distributed into both raft and non-raft microdomains, we sought to determine the functional characteristics of the two enzyme pools. PMCA activity was determined in each of the 10 density gradient fractions by previously described methods (Zaidi et al. 1998). The PMCA in the low buoyant density fractions 2–4 (raft pool) had considerably higher total activity than the pool present in the high density non-raft fractions (Fig. 3), consistent with the greater amount of PMCA protein present in those fractions (Fig. 1b). Addition of exogenous CaM stimulated PMCA activity only in fraction 3 (20% stimulation, p < 0.05, n = 6 experiments), with only a marginal stimulation observed in the other fractions.

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Figure 3.  Plasma membrane Ca2+-ATPase (PMCA) activity in the density gradient fractions. The sucrose density gradient fractions 1–10 were assayed for PMCA activity in the absence (○) and presence (•) of exogenous calmodulin (CaM) (340 nmol/L) as described in the Materials and methods section. Data are represented as means ± SEM from six independent experiments. Statistical evaluation of the data was performed by the Student’s t-test for unpaired samples. Both basal and CaM-stimulated PMCA activity in the raft fractions 2, 3, and 4 were higher when compared with the activity in fraction 1 (*p < 0.001). A 20% stimulation in PMCA activity was observed in fraction 3 upon the addition of CaM (**p < 0.05).

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Effect of cholesterol depletion on PMCA protein levels and enzyme activity

To determine if raft-associated PMCA is responsive to disruption in the lipid raft environment, we lowered the levels of cellular cholesterol. Neurons were treated with LS, an inhibitor of cholesterol biosynthesis, to chronically lower cholesterol in living cells without the harshness of cholesterol binding agents such as methyl-β-cyclodextrin that adversely affected cell viability of the neuronal cultures (data not shown). Primary cortical neurons were exposed to increasing doses of LS (1–10 µmol/L) for 1–5 days and cholesterol levels measured in the cellular lysates. The results showed a dose and time-dependent decrease in cholesterol levels, an ∼60% suppression was observed with 2.5 µmol/L treatment for 4 days, consistent with earlier results (Hering et al. 2003; Fortin et al. 2004). As no further decrease was observed at higher concentrations of LS or with increasing times of exposure, we used these experimental conditions for subsequent experiments. Next, we isolated rafts from vehicle-treated and LS-treated cells using the Brij 98 method as described above. All 10 fractions isolated from the density gradient showed a substantial decrease in cholesterol levels in the LS-treated neurons compared with the vehicle-treated control cells (Fig. 4a). However, the most dramatic decrease was evident in the low density fractions 1–4 where the cholesterol levels decreased by 72%, 73%, 55%, and 76%, respectively, when compared with similar fractions isolated from vehicle-treated cells. LS treatment also decreased total protein levels, particularly in the raft fractions (Fig. 4b). The loss of protein from rafts is consistent with earlier observations showing that some raft-associated proteins move out of these microdomains upon cholesterol depletion (Ilangumaran and Hoessli 1998; Magnani et al. 2004).

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Figure 4.  Effect of lovastatin (LS) on cholesterol and protein levels in neurons. Cortical neurons at day in vitro 2 were exposed to LS (2.5 µmol/L) for 4 days or to the vehicle ethanol. Control and LS-treated cells were lysed with 3 mmol/L Tris–Cl, pH 7.4, plus a protease inhibitor cocktail, the total protein was determined, and equal amounts of protein processed for raft isolation as described in the Materials and methods section. The sucrose density gradient fractions 1–10 from control (black bars) and LS-treated (grey bars) cells were assayed for (a) cholesterol, determined by the Amplex Red kit and (b) for total protein determined by the bicinchoninic acid method. Data are represented as means ± SEM from six experiments using different cultures. Statistical evaluation of the data was performed by the Student’s t-test for unpaired samples. *p < 0.01 and **p < 0.001 for the cholesterol data (a) and *p < 0.05 for the protein data (b).

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In order to determine if the significantly decreased membrane cholesterol affected the partitioning of PMCA in rafts, we compared PMCA levels in the control and LS-treated samples (Fig. 5a). Densitometric analysis of the PMCA immunoblots from six experiments showed no significant change in its protein levels in any of the fractions suggesting that cholesterol depletion did not influence its partitioning into rafts (Fig. 5b). Next, we examined the effect of cholesterol depletion on the levels of CaM associated with the raft and non-raft domains of the neuronal membrane. The results of CaM immunoblots showed a dramatic decline in its protein levels in rafts (fractions 2–5) but no significant change in the non-raft fractions. Densitometric analysis of six sets of immunoblots showed that there was a statistically significant decrease in CaM levels in fractions 2, 3, 4, and 5, which were lowered by 23.3% ± 10, 26.6% ± 8.6, 21.4% ± 5, and 18.3 ± 4%, respectively, compared with CaM levels in similar fractions from vehicle treated cells (*p < 0.05) (Fig. 5b). Levels of the raft marker proteins flotillin-1 and NAP-22 did not change with the LS-treatment suggesting that the ∼60% decrease in cholesterol did not disrupt the integrity of rafts and that the effect was rather specific for CaM.

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Figure 5.  Effect of cholesterol depletion on the levels of plasma membrane Ca2+-ATPase (PMCA), calmodulin (CaM), and other raft markers. (a) immunoblots to detect alterations in the levels of PMCA, CaM, and raft marker proteins and dot-blots to detect changes in GM1 levels. Fractions 2–9 from the control and lovastatin-treated neurons were probed with the following antibodies: anti-pan PMCA (1 : 1000), anti-CaM (1 : 500), anti-NAP-22 (1 : 250), and anti-flotillin-1 (1 : 500) as described in the Materials and methods section. Protein amounts used for immunoblotting were: PMCA (10 µg), CaM (30 µg), NAP-22 (30 µg), and flotillin-1 (25 µg). Dot-blots were performed by applying 1.0 µg of protein from fractions 2–9 on PVDF membrane, followed by blocking with 5% milk, and probing with peroxidase-labeled cholera toxin B subunit (10 µmol/L) to detect GM1 levels. Representative immunoblots and dot-blots from six independent experiments with similar results are shown. (b) Densitometric analyses of the levels of PMCA, CaM, and GM1 in fractions 2–9 isolated from control and cholesterol depleted neurons. Data are represented as means ± SEM from six experiments using independent cultures. Statistical evaluation of the data was performed by the Student’s t-test for unpaired samples (*p < 0.05 for CaM and *p < 0.005 and **p < 0.001 for GM1).

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To determine the effect of cholesterol depletion on the levels of GM1, the other major component of lipid rafts, dot-blots were performed by methods described earlier. The results of the densitometric analysis of six sets of dot-blots showed a substantial elevation in GM1 levels in all fractions, particularly in the raft fractions where GM1 is most abundant (*p < 0.005 and **p < 0.001) (Fig. 5b). Up-regulation of GM1 levels with the chronic decrease in cholesterol suggests potential cross-talk between the metabolic pathways regulating these two lipid components of rafts. Increased GM1 levels under conditions of lowered cholesterol may serve as an adaptive response induced in neurons to preserve the integrity of rafts.

To determine the effect of altered lipid raft composition on the functional characteristics of PMCA, we measured the enzyme activity in the various fractions isolated from control and LS-treated cells. The results showed a dramatic loss of total PMCA activity in the raft fractions 2, 3, and 4 in the LS-treated cells but not much change in the non-raft fractions (Fig. 6a). Decrease in PMCA activity was not due to a direct effect of LS on the calcium pump, because addition of 2.5 µmol/L LS to the assay mixture did not alter the values of the enzyme activity (data not shown). If LS-mediated PMCA inhibition was due to lowered levels of CaM in rafts, then one would predict that the addition of exogenous CaM might reverse the loss of activity. To test this possibility, we measured total PMCA activity in the presence of saturating concentrations of exogenous CaM (Fig. 6b). However, this manipulation did not restore PMCA activity in rafts from LS-treated neurons to that in controls, suggesting that reduced CaM levels were not directly responsible for the decline in PMCA activity. It is likely that alterations in the raft lipid environment by the marked decrease in cholesterol may affect PMCA directly and that the modulation of raft lipid composition may serve as a CaM-independent mechanism in the regulation of PMCA activity.

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Figure 6.  Effect of cholesterol depletion on plasma membrane Ca2+-ATPase (PMCA) activity. PMCA activity in fractions 1–10 isolated from control (black bars) and lovastatin-treated (grey bars) neurons was measured (a), in the absence of CaM, and (b) in the presence of 340 nmol/L CaM. Data are represented as means ± SEM from six experiments using independent cultures. Statistical evaluation of the data was performed by the Student’s t-test for unpaired samples (*p < 0.05 and **p < 0.01).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study shows for the first time that both PMCA and CaM are partitioned into lipid rafts in primary neurons. By taking advantage of the detergent insolubility and low buoyant density of these microdomains, we isolated rafts from cultured cortical neurons and showed the co-localization of PMCA with raft lipid markers cholesterol and GM1 and protein markers flotillin-1 and NAP-22 (Bickel et al. 1997; Naslavsky et al. 1997; Madore et al. 1999; Maekawa et al. 1999). Using intact hippocampal neurons, we also obtained morphological evidence showing that a substantial amount of PMCA was spatially segregated into lipid rafts, suggesting that such an association occurs in vivo. The partitioning of PMCA into raft and non-raft domains was corroborated by functional assays. The enzyme exhibited much higher total PMCA activity in the detergent insoluble and low buoyant density fractions compared with the pool in the non-raft domains, consistent with greater amounts of PMCA protein partitioned into rafts. Addition of exogenous CaM, however, stimulated PMCA activity only in fraction 3 with only marginal effects on PMCA in the other fractions. Low sensitivity or even a lack of sensitivity of PMCA to CaM in excitable tissue has been consistently observed by us and others and is believed to be either due to interaction with CaM constitutively bound to neuronal membranes and/or activation by acidic phospholipids known to render PMCA insensitive to CaM (Niggli et al. 1979; Missiaen et al. 1989; Zaidi et al. 1998; Levitan 1999a,b; Sepulveda et al. 2006).

A recent report showed the distribution of PMCA in rafts isolated from porcine cerebellum synaptic membranes (Sepulveda et al. 2006). Unlike the results in synaptic membranes showing the presence only of the isoform PMCA 4, our immunoblot analysis data shows the raft localization of all four PMCA isoforms. The discrepancy between the two studies may be attributed to distinct differences in the methodology used for raft isolation, such as detergent concentration, time of centrifugation, and the type of density gradient used. It seems quite likely that there are differences in the PMCA composition in rafts isolated from brain synaptic membranes compared with the entire plasma membrane of neurons as well as possible regional differences.

Given that the liquid ordered phase of rafts arises from the tight packing of interstitial spaces by cholesterol molecules (Brown and London 1997, 1998), cholesterol depletion is commonly used as a tool to disrupt the raft microenvironment. Rather than using acute treatments such as cholesterol binding or extracting agents, which we found to be damaging to cells, we chose to inhibit cholesterol biosynthesis. Additionally, chronic depletion of cellular cholesterol over the course of several days would allow cells to adapt to the altered environment and hence be a better indicator of its effects as they might occur in vivo. Reduced levels of cellular cholesterol are known to induce alterations in the lipid and protein composition of rafts, depending upon the cell type and the experimental conditions used for depletion (Ilangumaran and Hoessli 1998; Isshiki and Anderson 1999; Hao et al. 2004). In our hands, inhibition of cholesterol biosynthesis in cortical neurons lowered total protein levels, but had no significant effect on PMCA levels, suggesting that PMCA may not be associated with rafts by direct interaction with cholesterol. However, it cannot be overruled that the ∼40% residual cholesterol may be sufficient to anchor PMCA to rafts. Although raft-PMCA protein levels did not change with cholesterol depletion, a dramatic loss in enzyme activity was observed, suggesting that the cholesterol-rich, liquid ordered raft environment favored PMCA activity, in close agreement with in vitro studies reported earlier (Zhao et al. 2004; Pang et al. 2005; Tang et al. 2006). Our results are consistent with earlier studies showing a significant reduction in PMCA activity upon depletion of synaptic plasma membrane cholesterol by cholesterol oxidase, a manipulation that converts cholesterol to its oxidation product cholestenone (Wood et al. 1995). Cholesterol oxidation was accompanied by a decrease in synaptic plasma membrane interdigitation and increased membrane fluidity and biophysical changes that may reduce PMCA activity by lowering its mobility and/or causing protein aggregation (Cornea and Thomas 1994). Another interesting study showed that pharmacological tools used to lower cholesterol such as oxidosqualene cyclase inhibitors (for example compound A18666A) can directly intercalate into the membrane bliayer thus causing perturbation of the membrane structure and function (Cenedella et al. 2004). Although the exact mechanism of PMCA inhibition by LS-induced cholesterol depletion is not clear from the present study, a number of possibilities, such as perturbation of the membrane bilayer and consequent change in its molecular structure, alterations in thickness and fluidity, disruption in the order of raft lipid environment, loss of acidic phospholipids, and/or up-regulation of GM1 ganglioside, a known inhibitor of PMCA activity (Zhao et al. 2004), may underlie these observations.

There has been a long-standing interest in the role of rafts and caveolae (specialized forms of rafts with a distinct cave-like morphology and the presence of caveolin) in Ca2+ signaling (Isshiki and Anderson 1999, 2003). Not surprisingly, a number of Ca2+-regulating proteins, including an inositol trisphosphate receptor-like protein, growth associated protein, heterotrimeric G proteins, transient receptor potential-like protein channels, PMCA, and CaM have been found in caveolae in several non-neuronal cell types (Fujimoto 1993; Fujimoto et al. 1995; Schnitzer et al. 1995; Arni et al. 1998; Keshet et al. 1999; Ambudkar 2004). Caveolae are believed to be local sites for the orchestration of Ca2+-dependent signal transduction events (Isshiki et al. 1998, 2002a,b). The role of neuronal lipid rafts in Ca2+ signaling has been less appreciated given the lack of a distinct morphology, smaller size precluding detection by optical microscopy, and the transient nature of these microdomains. The findings that both PMCA and CaM are localized in neuronal lipid rafts suggests that raft domains may play an important role in local Ca2+ signaling in neurons.

The association of Ca2+ signaling proteins such as the PMCA and CaM with lipid rafts may have potential implications on the spatiotemporal distribution of these proteins in the plasma membrane. Given the high lateral mobility of rafts, association of PMCA and CaM with these microdomains may serve as a mechanism for their trafficking to various parts of the membrane in response to specific stimuli. Indeed, a recent report showed that ∼60% of PMCA 2 in hair stereocilia exhibits high mobility and is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface (diffusion constant 0.1–0.2 µm2/s), whereas the remaining pool was present in a relatively immobile fraction (Grati et al. 2006). On the other hand, many raft-associated proteins including the PMCA may be anchored to the underlying membrane cytoskeleton. Such an association may help stabilize the protein complexes of PMCA and its binding partners and consequently restrict lateral mobility. It is tempting to speculate that the differential distribution of PMCA in raft versus non-raft domains may serve to regulate its mobility, trafficking, and protein–protein interactions with its binding partners. Association with rafts may therefore serve as a novel mechanism not only in the regulation of PMCA activity by modulation of the cholesterol content but also in controlling its spatiotemporal distribution, consequently allowing the fine-tuning of local Ca2+ signaling.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr E.E. Strehler for providing the PMCA isoform-specific antibodies and Dr S. Maekawa for the NAP-22 antibody. This work was supported by NIH Grants RR-P20 17708, AG 12993 and General Research Funds and J.R. and Inez Jay Funds, University of Kansas, USA.

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