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Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic–density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its PDZ-binding sequence. Using the fluorescence recovery after photobleaching (FRAP) technique, we show that the lateral membrane mobility of the clustered PMCA4b is significantly lower than that of the non-clustered molecules. Disruption of the actin-based cytoskeleton by cytochalasin D resulted in increased cluster size. Our results suggest that PSD-95 promotes the formation of high-density PMCA4b microdomains in the plasma membrane and that the membrane cytoskeleton plays an important role in the regulation of this process.
Ca2+ is a ubiquitous cellular signal, hence its cytosolic concentration must be tightly controlled by specific channels, transporters and exchangers. The temporal and spatial aspects of Ca2+ signaling require specific sub-cellular arrangements of these Ca2+ handling proteins because an aberrantly located Ca2+ channel or transporter could change the nature of the Ca2+ signal and subsequently result in an abnormal biological response [1, 2].
The plasma membrane Ca2+ ATPases (PMCAs) are responsible for removing Ca2+ from the cell to the extracellular space. Thus, these proteins play an essential role in maintaining intracellular Ca2+ homeostasis. Four genes encode mammalian PMCAs (PMCA1-4) and with alternative splicing occurring at two separate sites over 20 different PMCA isoforms are generated. Although the need for this isoform diversity may be partly explained by the tissue-specific expression of many PMCAs, recent studies also suggest that different isoforms participate in localized Ca2+ signaling within specific membrane microdomains (for a recent review, see ).
Organizing PMCA molecules into discrete Ca2+ signaling microdomains or connecting them to other signaling pathways may be achieved through interaction with specific signaling and scaffolding proteins. A consensus sequence E-T/S-X-L/V for binding type-I PDZ (postsynaptic-density-95/discs large/zona occludens-1) domains was identified at the carboxyl terminus of the b-splice variants of all PMCA isoforms. Through this motif the “b” forms of PMCAs interact with members of the membrane-associated guanylate kinase (MAGUK) family, such as the synapse-associated proteins PSD-95/SAP90, SAP97/hDlg, SAP102, and PSD-93/chapsyn-110  or the Ca2+/CaM-dependent serine protein kinase CASK . In addition, PMCA2b (with the sequence ETSL at the C-terminus) interacts specifically with the Na+/H+ exchanger regulatory factor-2 (NHERF2) that may link the apically targeted PMCA2w/b via its ezrin/radixin/moesin (ERM)-binding domain to the actin cytoskeleton . On the other hand, PMCA4b with the sequence of ETSV at the C-terminus binds specifically to the PDZ domain of neuronal nitric oxide synthase (NOS-1) and down regulates NO production most likely due to a PMCA-mediated decrease of local [Ca2+] in the immediate vicinity of NOS-1 . While several proteins have been identified as interacting partners of the PMCA little is known about their involvement in targeting and specific membrane distribution of the pump molecules.
Here we show for the first time that the MAGUK family member PSD-95 increases the plasma membrane expression of PMCA4b and - similarly to other Ca2+ signaling molecules such as neurotransmitter receptors and channels - redistributes the calcium pump into clusters. We demonstrate that these clusters are restricted to specific regions of the plasma membrane, fenced by the underlying actin cytoskeleton. We also show that in the clustered state the lateral mobility of the PMCA molecules is significantly reduced. Our results suggest that PDZ domain directed clustering could associate the PMCA with complex macromolecular systems at specialized membrane regions such as the post-synaptic density or cell junctions.
Fugene 6 Transfection Reagent was obtained from Roche Applied Science. DMEM and OPTIMEM were obtained from Gibco. Monoclonal anti-PMCA antibody 5F10  was used at a dilution of 1:5,000 for immunoblotting and 1:100 for indirect immunofluorescence staining. Anti-PSD-95 was obtained from Zymed Laboratories and was used at 1:250 for immunoblotting and 1:330 for indirect immunofluorescence staining. TRITC-phalloidin was obtained from Sigma Chemical Co. Alexa Fluor 488, 594 and 633-conjugated goat anti-mouse IgG and anti-rabbit IgG were obtained from Invitrogen Corp. All other chemicals used were of reagent grade.
Plasmid pMM2-PMCA4b for expression of full-length human PMCA4x/b in mammalian cells has been described previously . GFP-PMCA4b encoding human PMCA4x/b fused at its NH2 terminus to GFP was generated by cloning an XhoI fragment carrying the full-length PMCA4x/b sequence into pEGFP-C2 (Clontech) as described . Construct GFP-PMCA4x/bct6 was similarly made by cloning a PCR-generated XhoI fragment encoding amino acids 2-1199 of hPMCA4x/b into pEGFP-C2. The PCR fragment was generated using primers HP4(400)Xho, 5'-GGG GGC TCG AGA ACG AAC CCA TCA GAC CGT GTC TTG CC-3' and HP4(Δ6)Xho, 5'-CCC CCT CGA GTC AAA CTG ATG TCT CTA GTC ACT GTA G-3' (XhoI sites underlined) and pMM2-PMCA4b as template. The mammalian expression construct for SAP90/PSD95 has been described  and was a kind gift from Morgan Sheng (Massachusetts Institute of Technology, Cambridge, MA) and Craig Garner (Stanford University School of Medicine, Stanford, CA).
COS-7 and Neuro-2A cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. Cells were kept at 37 °C, 5% CO2 in a humidified atmosphere. COS-7 and Neuro-2A cells were cultured to 80% confluency and transfected with plasmid DNA using FuGene 6 Transfection Reagent according to the manufacturer’s protocol.
For confocal imaging cells were grown on 8-well Nunc Lab-Tek Chambered Coverglass (Nalge Nunc International, Rochester, NY) previously coated with 0.03 mg/ml Vitrogen (Cohesion Technology, Palo Alto, CA). 24 to 48 hours after transfection, cells were rinsed with Dulbecco’s modified PBS (DPBS), fixed for 15 min at 37 °C in 4% paraformaldehyde and DPBS. After three washes with DPBS, cells were blocked and permeabilized for 1 h at room temperature in blocking buffer (DPBS containing 2 mg/mL bovine serum albumin, 1% fish gelatin, 0.1% Triton-X 100, and 5% goat serum). Samples were then incubated for 1 h at room temperature with primary antibodies (mouse monoclonal anti-PMCA antibody (5F10) and rabbit polyclonal anti-PSD-95 antibody). After three washes in DPBS, cells were incubated for 1 h at room temperature with Alexa-488, Alexa-594 or Alexa-633 conjugated goat anti-mouse or anti-rabbit secondary antibodies. In some cases, TRITC-phalloidin (0.1 µg/ml in DPBS) was added either alone or after incubation with secondary antibody.
Samples were studied with an Olympus IX-81 laser scanning confocal microscope and Fluoview FV500 (v4.1) software using an Olympus PLAPO 60x (1.4) oil-immersion objective (Olympus Europe GmbH, Hamburg, Germany).
For multichannel imaging, fluorescent dyes were imaged sequentially and the wavelength of light was collected. Each detection channel was set such that no detectable bleed-through occured between the different channels.
Raw images were deconvolved with ImageJ 1.36b (W. Rasband, National Institutes of Health, Bethesda, MD) using Iterative deconvolution plug-in (Bob Dougherty, Copyright 2005, OptiNav, Inc.) Theoretical estimate of the point-spread function was acquired for each of the channels at different wavelengths (PSF calculator, Scientific Volume Imaging B.V., The Netherlands). Pixel intensity profiles along ~ 10 µm segments of plasma membrane were assessed using Fluoview FV500 version 4.1 imaging software.
Cluster sizes in control and cytochalasin D-treated cells were determined by confocal imaging of basal optical sections and measuring the area of 500 clusters each using Fluoview FV500 (v4.1) imaging software.
Pearson’s correlation coefficients (r) were used to quantify the extent of co-localization between two labelings and were calculated with Image J 1.36b using the plug-in module Colocalization Finder (C. Laummoneire and J. Mutterer, Stasbourg, France). The minimum ratio for pixel intensity between the two channels was set to 0.4. Pearson’s correlation values represent the mean ± S.D. of calculations from ten cells of three independent experiments. Means and standard deviations were calculated using Microsoft Excel software (Microsoft, Seattle, WA).
COS-7 cells were cultured in DMEM on 8-well Nunc Lab-Tek Chambered Coverglass for 24h and transfected with DNA constructs (GFP-PMCA4b together or without PSD-95). Time lapse sequences were recorded with Fluoview Tiempo (v4.3) time course software at room temperature using an Olympus IX-81 laser scanning confocal microscope.
Fluorescence recovery after photobleaching (FRAP) experiments were performed 24 or 48 hours after transfection using the 488 nm line of an argon laser with a 60x 1.4 NA oil immersion objective. A defined segment of the ventral surface of the cell (circular spot with a diameter of about 2 µm) was photobleached by 3 iterations at full laser power (100% power) at 10x magnification. Pre- and post-bleach images were acquired every 3 seconds over a 5-minute time period at low laser intensity (3% power). Mean fluorescence intensity data from regions of interest (ROI) and non-bleached regions were collected using Fluoview FV500 (v4.1) imaging software. Data were imported into Microsoft Excel software, normalized to the pre-bleach intensities and corrected for acquisition bleaching for each time point caused by the fluorescence recording process using double normalization methods . The kinetics of recovery were determined by exponential fitting of the average data using the non-linear regression algorithm of PRISM software version 3.0 (Graphpad Software Inc., San Diego, California):
where Ai is the amplitude of each component, t is time, τi is the time constant of each component. The mobile fraction was calculated as:
where FE is the fluorescence intensity at the end of the FRAP experiment, Fi is the initial fluorescence intensity prior to bleach and F0 is the intensity immediately after the bleach.
Calculating mobile fractions and using exponential equations to approximate fluorescence recovery provides a relatively model-independent method of comparing different GFP-PMCA4b behaviors in clustered and non-clustered states .
Protein samples were electrophoresed on 7.5% acrylamide gels following Laemmli’s procedure  except that the sample buffer contained 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5 mM EDTA, 100 mM dithiothreitol and 125 mg/ml urea. The samples were subsequently electroblotted onto PVDF membranes and the blots were immunostained by antibodies 5F10 and anti-PSD-95 following standard Western blotting procedures  and using the ECL-Plus detection system (Amersham Biosciences).
Data are expressed as mean ± S.D. Statistical significance was evaluated using two tailed Student’s t-test. Statistical analyses were performed using Microsoft Excel software (Microsoft, Seattle, WA) and PRISM software version 3.0 (Graphpad Software Inc., San Diego, California).
To study the effect of PSD-95 on the sub-cellular localization of PMCA4b, we transiently transfected COS-7 cells with GFP-tagged PMCA4b with and without PSD-95 and examined optical sections at half depth of cells with confocal fluorescence microscopy. Plasma membrane expression of PMCA4b was assessed by calculating the proportion of cells that showed a detectable plasma membrane signal (Fig. 1E). One day after transfection and in the absence of PSD-95, GFP-PMCA4b localized almost exclusively in intracellular compartments showing perinuclear staining and a diffuse reticular pattern characteristic of the endoplasmic reticulum (Fig. 1A). As summarized in Fig. 1E, under these conditions only 14% of cells showed detectable plasma membrane localization of GFP-PMCA4b whereas two days after transfection, 75% of the cells showed a positive plasma membrane signal with a rather diffuse fluorescence pattern (Fig. 1B).
Co-expression of GFP-PMCA4b with PSD-95 greatly enhanced the plasma membrane expression of the PMCA (Fig. 1C and Fig. 1D); at one day post-transfection nearly 70% of the cells showed positive PMCA staining at the cell periphery and after two days plasma membrane localization was apparent in virtually all cells (Fig. 1E). These experiments suggested that PSD-95 facilitated recruitment of PMCA4b to the plasma membrane. The immunoblots shown in Fig. 1G demonstrate that co-expression with PSD-95 did not alter the expression level of the PMCA4b protein. We also note that the N-terminal GFP-tag did not alter the subcellular distribution of PMCA4b in the presence or absence of PSD-95 in these cells (for comparison see also Fig. 6).
Co-expression of the two proteins also altered the plasma membrane (PM) distribution of the PMCA. PM staining of PMCA4b without PSD-95 displayed a rather evenly distributed fluorescence pattern (Fig. 1B). By contrast, in the presence of PSD-95 the distribution of PMCA4b became discontinuous at the cell surface (Fig. 1D1). PSD-95 staining showed a similar distribution pattern with an obvious co-localization of the two proteins (Fig. 1D2). Fig. 1F shows the relative fluorescence intensity along the PM within the selected areas of the cells transfected with GFP-PMCA4b alone (F1) or together with PSD-95 (F2). The GFP signal of PMCA4b without PSD-95 revealed a homogenous membrane distribution (Fig 1F1). Examination of the GFP and the PSD-95 signals in the case of the co-expressed proteins showed clear overlap between the two proteins and a more structured distribution of GFP-PMCA4b. This indicated that the presence of PSD-95 resulted in a significant redistribution of PMCA4b at the plasma membrane compartment.
To examine further the effect of PSD-95 on the distribution of PMCA4b within the plasma membrane compartment in two dimensions we acquired confocal images at the ventral surface of the cell facing the coverslip (Fig. 2). These images confirm that the distribution of GFP-PMCA4b is rather diffuse at the plasma membrane when expressed without PSD-95 (Fig. 2A2). We used histogram analysis to summarize graphically the distribution of the fluorescence intensities of these sections (Fig. 2A1). The histogram shows a unimodal right skewed distribution which is a common distribution of data sets in biological systems. Transfection of PSD-95 alone in COS-7 cells also resulted in a diffuse localization of the protein both in the cytoplasm and in the plasma membrane (Fig. 2B) as previously described .
Co-expression of GFP-PMCA4b and PSD-95 resulted in a dramatic redistribution of both proteins into irregularly shaped plaque-like clusters (Fig. 2C2 and C3). Histogram analysis of the fluorescence intensity within the ventral surface of a co-transfected cell showed bimodal (dual-peaked) distribution (Fig. 2C1). The bimodality is due to a mixture of two data sets and indicates two populations of PMCA4b. The pixels with high intensity values (second peak) are confined to the areas of the plaque-like clusters of GFP-PMCA4b/PSD-95. The population of GFP-PMCA4b molecules represented by pixels with lower intensity values (first peak) is not limited to these regions; rather these molecules distribute homogeneously in the membrane.
The clustering efficiency of PMCA4b and PSD-95 (expressed as a percentage of the cluster-forming cells / cells co-expressing PSD-95 and PMCA4b) was greater than 90 % (93 ± 3.5 % [mean ± SD of three independent samples of 50 co-transfected cells each]), which is even higher than the previously described clustering efficiency of other proteins and PSD-95 [17–21].
It has been shown that PSD-95 interacts with PMCA4b directly via the PDZ-binding sequence , therefore we tested the role of the PDZ-interaction in PMCA clustering. We used a GFP-PMCA4b-ct6 mutant that lacks the last 6 residues corresponding to the PDZ binding sequence and is unable to bind PSD-95. Co-expression of GFP-PMCA4b-ct6 with PSD-95 did not result in the co-clustering of the two proteins (Fig. 2D2 and D3). Instead, in these cells, PSD-95 and GFP-PMCA4b-ct6 show the diffuse distribution characteristic of singly transfected cells (Fig. 2D1). Thus, formation of clusters depends crucially on the PDZ-interaction between PSD-95 and PMCA4b.
Because PSD-95 is a neuronal scaffolding protein we also analyzed the effect of PSD-95 on the expression pattern of PMCA4b using a neuroblastoma cell model. PMCA4b expressed alone in Neuro2A neuroblastoma cells exhibited diffuse distribution in the plasma membrane similarly to that seen in COS-7 cells (Fig. 3A). Formation of PMCA4b clusters could only be observed in PSD-95 and PMCA4b co-transfected Neuro2A cells (Fig. 3B1). However, comparing the distribution of GFP-PMCA4b and PSD-95 within the plasma membrane (Fig. 3B1 and B2) revealed only weak co-localization within the clusters, as indicated by the predominance of distinct red (PSD-95) and green (GFP-PMCA4b) patches (Fig. 3B3) and by the rather low Pearson’s co-localization coefficient r = 0.18 ± 0.07 (determined on 30 cells from 3 independent experiments). In contrast, in COS-7 cells the co-localization between the two proteins was significantly greater (Pearson’s correlation coefficient r = 0.58 ± 0.08; n=30 cells from 3 separate experiments). The weak co-localization in the clusters of Neuro2A cells suggests that although PSD-95 facilitates the recruitment of PMCA4b molecules into clusters, these PMCA4b clusters can persist without association with PSD-95.
Interactions with scaffolding proteins may restrict the PMCA diffusion in the plasma membrane. To test this, we studied the lateral mobility of GFP-PMCA4b in both the non-clustered and clustered state using FRAP in transfected COS-7 cells (Fig. 4). Recovery within the bleach region was normalized to the pre-bleach values using the “double normalization” method  to correct the acquisition bleaching. The recovery kinetic parameters were determined by exponential fitting of the average data using Equation 1 (see Materials and methods) (Fig. 4C). Fig. 4D shows the mobile fractions (Mf) calculated from the FRAP data using equation 2 (see Materials and methods). In the case of PMCA4b alone the mobile fraction was 0.81 ± 0.06 and the recovery could not be fitted well by a single exponential, requiring double-exponential fit (τ1= 5.43 s, τ2 = 42.97 s). The partial recovery and the need of multi-component equations to fit the data suggest that the movement of GFP-PMCA4b in the PM is not diffusion-limited.
In the presence of PSD-95 the FRAP data could also be fitted by the double exponential equation. The recovery was substantially decreased as indicated by a decrease in the mobile fraction (Mf = 0.57 ± 0.03) whereas the kinetics of recovery were only minimally affected; the time constants were only slightly different (τ1= 3.78 s, τ2 = 47.85 s) from those obtained in the singly transfected cells.
Taken together, the ~2-fold increase in the immobile fraction of GFP-PMCA4b (from ~19% to ~43%) shows that the interaction with PSD-95 immobilizes the PMCA4b molecules at the cell surface of COS-7 cells.
To study the dynamic behaviour of the PSD95/PMCA4b clusters in live COS-7 cells, time-lapse recordings were performed over a time period of 2 minutes. The GFP fluorescence was recorded in frames 5 seconds apart. In Fig. 5A time-lapse overlays demonstrate the movement of the clusters. This figure shows the first individual frame (A1) and the summed images of the first 7 (A2), 13 (A3), and 25 (A4) frames, respectively, as also explained in the scheme of Fig. 5B.
The summed images revealed that the movement of the clusters was restricted to a rather small region of the plasma membrane coralled by certain areas with a relatively low fluorescence signal. We followed the changes in the fluorescence intensities within the low signal area between the clusters and compared that to the recovery of the fluorescence signal of the bleached area within a cluster in the same cell (Fig. 5C). We found that the fluorescence intensities remained constant in the low signal area (marked in Fig. 5A) while the fluorescence of the bleached area increased continuously during the observation. This experiment demonstrates that the PMCA4b molecules in a cluster can not easily enter the low signal area while substantial movement of the molecules is permitted within the clustered area. This indicates that the movement of the clustered PMCA4b is constrained by relatively solid structures in proximity to the plasma membrane. The organization of the low signal areas displayed a mesh-like structure resembling the actin-based membrane skeleton meshwork.
The decreased translational diffusion of clustered PMCA4b and the characteristically confined movement of the clusters suggests that the actin cytoskeleton may influence the mobility of the clustered PMCA4b. To examine the connection between the PMCA4b and the actin cytoskeleton, we studied the localization of PMCA4b and F-actin visualized by TRITC-phalloidin at the bottom surface of COS-7 cells (Fig. 6A). No co-localization was observed of the PMCA4b clusters and the actin filaments. Phalloidin fluorescence was entirely absent in the areas of PMCA clusters; the PMCA4b signal was restricted to membrane sections surrounded by actin fibers. The typical arrangment of the PMCA clusters and the actin meshwork indicated that the PMCA in the clusters is fenced in by the actin cytoskeleton as also suggested by the experiment shown in Fig. 5. It is important to note that in these experiments we expressed the native PMCA4b (without the GFP-tag) indicating that the N-terminal GFP-tag used in the experiments above did not alter the clustering of PMCA4b. To examine further the effect of the actin-based membrane skeleton on the PMCA4b clusters, the F-actin depolymerizing agent, cytochalasin D (final concentration 2.5 µM) was added to the PMCA4b/PSD-95 expressing COS-7 cells. The fluorescent images in Fig. 6B show that the size of the PMCA4b clusters increased dramatically after treatment. The distribution of the cluster size is illustrated in the histograms of Fig. 6C. Disruption of the actin cytoskeleton resulted in the appearance of large clusters; the mean value of the cluster area increased by a factor of ~6. After cytochalasin D treatment about 80% of the clusters were greater than 1 µm2 compared to about 40% in cells without cytochalasin treatment. These data confirm that the intact actin-based membrane skeleton barrier causes corralling of the PMCA4b clusters. The disruption of the membrane cytoskeleton allows the PMCA molecules to move more freely and diffuse into larger areas in the PM.
In this study we identify a mechanism that controls the distribution of PMCA4b in the plasma membrane. We show that PSD-95, a neuronal scaffolding protein, can direct PMCA4b into well-defined clusters within the PM. Although an earlier report demonstrated that PMCA4b binds directly to PSD-95 via its PDZ binding sequence , the influence of this interaction on the subcellular localization of the pump had not yet been investigated. We observed the formation of PMCA clusters in COS-7 fibroblast and Neuro-2A neuroblastoma cells co-transfected with PMCA4b and PSD-95. In contrast, PMCA4b expressed alone distributed diffusely in the PM and no PMCA cluster formation was detected in the absence of PSD-95.
PSD-95 is known to induce clustering of several synaptic proteins such as potassium channels and neurotransmitter receptors [17–21]. The apparent “efficiency” with which PSD-95 induces clustering varies considerably among different proteins. In the case of PMCA4b, the clustering efficiency was very high, suggesting that the interaction with PSD-95 could be a very powerful tool to direct PMCA4b to specific membrane microdomains.
We also found that PSD-95 dramatically increased the surface expression of PMCA4b without affecting its overall level in the cell. This is in agreement with earlier studies that showed that PSD-95 facilitates the plasma membrane expression of several membrane proteins, mainly by suppressing their internalization (e.g. GLYT1 glycine transporter-1 , N-methyl-D-aspartate (NMDA) receptors , β1-adrenergic receptors  and K+ channels [26, 27]. Whether a similar mechanism applies for PMCA4b remains to be determined.
Clearly, the PDZ-binding C-terminal sequence of PMCA4b is essential for PSD-95 mediated clustering of the PMCA, as deletion of this sequence abolished the effects of PSD-95. This fits previous studies showing the importance of the interaction with the PDZ domains of PSD-95 for the surface expression of other partner proteins [21, 23, 24, 26]. In Neuro-2A neuroblastoma cells PSD-95 appears to be needed for recruitment of PMCA4b molecules into clusters but not necessary for keeping them together. According to a recent theory membrane protein clustering can be explained by self-organization of proteins based on simple physical principles . Weak homophilic interactions between the proteins and crowding-induced repulsion are deemed to be enough to induce protein clustering within the plasma membrane. It is known that PMCA4b can form homo-oligomers via its C-terminal region and that oligomerization of PMCA4b occurs spontaneously at higher enzyme concentration [29, 30]. Thus, it is conceivable that PSD-95-induced assembly increases the local PMCA concentration in certain PM microdomains favoring oligomerization and stabilization of the clusters. Interactions between the PMCA molecules could then explain the persistence of the clusters without the presence of PSD-95 that we observed in Neuro-2A cells.
The membrane skeleton fence model proposes that the actin meshwork under the plasma membrane forms compartment boundaries restricting the lateral movement of membrane proteins [31–33]. We found that depolymerization of the actin cytoskeleton by cytochalasin D treatment increased the PMCA4b cluster areas. This supports the idea that actin filaments play an important role in maintaining the PMCA cluster separation. A similar dependence on the actin cytoskeleton has been recently described for the clustering of voltagegated potassium 2.1 (Kv2.1) channels; i.e. the disruption of the actin cytoskeleton also significantly increased the cluster size [34, 35].
Co-expression of PSD-95 increased the level of Kv1.4 voltage-gated potassium channel in raft fractions in rat brain membranes . Recently, compartmentalized distribution of PMCA4 in pig cerebellum has been detected in lipid rafts from synaptic membrane . The raft association was explained as a result of an intrinsic property of the PMCA4 isoform or the existence of partner proteins that could promote this association. One possibility is that PSD-95 (or other PDZ protein) induced clustering facilitates raft association of PMCA4b.
We investigated the mobility of GFP-PMCA4b by using the FRAP technique in living COS-7 cells. For clustered GFP-PMCA4b the fluorescence recovery was incomplete, and the data points were fitted by a multi-component exponential equation. Both the complex nature of the curve and the lack of a complete recovery after photobleaching can be explained by the presence of multiple populations of molecules with different diffusion rates, or by the existence of monomeric and oligomeric forms of the protein [38, 39]. Histogram analysis of the confocal images showed that two populations (clustered and non-clustered) of PMCA4b molecules exist in the plasma membrane of COS-7 cells co-transfected with PSD-95 (Fig. 2). A complex analysis (stimulated emission depletion (STED) microscopy, quantitative biochemistry, FRAP, and simulations) of the dynamics of syntaxin 1 clusters suggested that the molecules arranged in clusters and the ones moving freely between the clusters are in equilibrium with each other . The exchange between the clustered and the free pool follows complex kinetics because the release of the molecules from the clusters depends on their location within the clustered microdomain: the inner molecules are released more slowly than the molecules located at the rim of the clusters. The dynamic equilibrium between the clustered and the freely diffusing molecules and the complex kinetics between the pools resulted in a relatively large mobile fraction in spite of a comparatively small fraction of freely diffusing syntaxin 1 molecules. A similar relationship between the fraction of PMCA4b molecules existing in the clustered state and PMCA4b molecules diffusing freely between the clusters can explain the characteristics of the FRAP recovery curves we observed in our system.
PMCA4b can interact with multiple scaffolding and signaling proteins besides PSD-95 and these associations may also limit the lateral mobility of individual PMCA4b molecules. Organization of the PMCA into clusters results in a higher concentration of the enzyme in certain areas of the plasma membrane. This may influence the Ca2+ signal produced within specific Ca2+ microdomains. It has been observed that many ion channels and receptor proteins are spatially organized in clusters [40–43]. Such spatial clustering of Ca2+ signaling molecules results in regulatory effects within membrane microdomains that increase the flexibility of the Ca2+-mediated signals . In the case of PMCA4b, its specific membrane distribution allows it to create functional interactions and regulate various signaling pathways in local microdomains. In T-cells, where the predominant isoform is PMCA4b, PMCA and the Ca2+ release-activated Ca2+ (CRAC) channel communicate through a local microdomain . The close physical coupling between PMCA and the CRAC channel enables the cell to regulate local Ca2+ signals independently of changes in global calcium concentration. In the heart, PMCA4b specifically co-localizes with the syntrophin-dystrophin protein complex which organizes functional signaling complexes at the cytoskeleton-plasma membrane . Within this complex PMCA interacts with α-1 syntrophin and nitric oxide synthase (NOS-1). PMCA4b and α-1 syntrophin negatively regulate the NOS-1 activity, which has significant implications for nitric-oxide-regulated signaling pathways. These examples underline the importance of clustering and specific membrane localization for the function of PMCA4b in local Ca2+ control. The PSD-95 mediated membrane retention and clustering of PMCA4b described here may thus be essential for the physiological role of the pump in specific cells and tissues. Investigations of the consequences of a disruption of the PSD-95-PMCA4b interaction for the development of disease will generate much interest in the future.
We thank Krisztina Lór for her skillful technical assistance, and Dr. Steven DeMarco for construction of the GFP-PMCA4x/bct6 plasmid.
This work was supported in part by grants from the Hungarian Academy of Sciences (OTKA K49476 and ETT 042/2006 to AE), a Hungarian Academy of Sciences Postdoctoral Fellowship (OTKA D48496 to RP), a Bolyai Janos Research Fellowship (BO/00585/5 to KP) and by a grant from the National Institutes of Health (NS51769 to EES).
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