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First published online 8 December 2005
doi: 10.1242/jcs.02706
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Research Article |
1 Institute of Microbiology and Immunology, National Yang-Ming University, 155 Sec. 2 Linong Street, Taipei 112, Taiwan
2 Department of Life Science, National Yang-Ming University, 155 Sec. 2 Linong Street, Taipei 112, Taiwan
3 Institute of Biophotonics Engineering, National Yang-Ming University, 155 Sec. 2 Linong Street, Taipei 112, Taiwan
4 Department of Surgery, Veteran General Hospital, 201 Sec. 2 Shih-Pai Road, Taipei 112, Taiwan
* Author for correspondence (e-mail: linch{at}ym.edu.tw)
Accepted 21 September 2005
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Summary |
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-subunits to the aberrant apical location. Such apical targeting is mediated via the indirect transcytosis pathway. Cells containing apical Na+/K+-ATPase appear to be defective in maintaining the ionic gradient across the plasma membrane and in executing hepatic activities that are dependent upon the ionic homeostasis such as canalicular excretion.
Key words: Glycosylation, Protein targeting, Na+/K+-ATPase, Liver
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Introduction |
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; Mostov et al., 2003; Rodriguez-Boulan et al., 2005). The protein sorting events result in the formation and maintenance of the individual specific composition of the apical and basolateral domains, which are essential for many cellular functions executed by the polarized epithelial cells (Nelson and Yeaman, 2001). Defects in protein sorting/trafficking are often associated with diseases (Altschuler et al., 2003).
Integral membrane proteins destined to either apical or basolateral membrane may undergo direct sorting from the trans-Golgi network (TGN) (Rodriguez-Boulan et al., 2005; Simons and Wandinger-Ness, 1990) or selected retention and/or degradation that results in differential distributions at one surface domain or the other. In hepatocytes, a third pathway (indirect apical targeting) exists that delivers certain basolateral proteins to the apical membrane by a transcytosis route (Hubbard, 1991). Various signals or cell machineries have been shown to be involved in the protein homing process, including sorting motifs on the cargo molecules (Corbeil et al., 1992), glycosyl-phosphatidylinositol (GPI) anchorage (Brown et al., 1989; Lisanti et al., 1988), inclusions in the lipid rafts or lipid microdomains (Simons and Ikonen, 1997) and the interactions with proteins that are being specifically sorted.
Apart from these relatively well-recognized sorting mechanisms, the role played by protein glycosylation, especially N-glycans, in regulating protein targeting remain somewhat controversial. In addition to the generally accepted role of protein glycosylation in modulating protein degradation and metabolism (Helenius and Aebi, 2004), some studies have demonstrated a direct apical sorting effect of N-glycans in some epithelial cells (Gut et al., 1998; Scheiffele et al., 1995); however, there is also contradictory evidence to this finding (Laughery et al., 2003; Su et al., 1999).
In this study, we investigated sorting of the Na+/K+-ATPase in the polarized hepatic cells. Na+/K+-ATPase plays an important role in keeping ionic imbalance or homoeostasis across the plasma membrane (Skou and Esmann, 1992). Most Na+/K+-ATPase in polarized epithelia are present on the basolateral membrane (Blitzer and Boyer, 1978); apical presences have only been found in retinal pigment epithelia and choroid plexus epithelia (Gundersen et al., 1991; Rizzolo, 1999). Na+/K+-ATPase is a transmembrane enzyme that consists of a large catalytic -subunit, and a small type II membrane glycoprotein ß-subunit (Lingrel, 1992). Which subunit dominates the sorting of the heterodimer is not well understood, and may be different depending on the cell types. Some reports have shown direct basolateral sorting by signals at the fourth transmembrane domain of the -subunit (Dunbar et al., 2000), whereas other studies suggest that integration, metabolism, and routing of the -subunit are actively modulated by the ß-subunit (Geering, 2001; Hasler et al., 1998; Jaunin et al., 1993). In the retinal pigment epithelial cell, the apical distribution of Na+/K+-ATPase seems to be due to the association of -subunit with ankyrin (Gundersen et al., 1991; Nelson and Veshnock, 1987), whereas in MDCK cells, the expression and basolateral localization of Na+/K+-ATPase are related to plasma membrane contact or the interaction between neighboring cells (Shoshani et al., 2005). Little is known regarding the sorting of Na+/K+-ATPase in polarized hepatic cells.
We report here that deglycosylation treatments of the heavily glycosylated Na+/K+-ATPase ß-subunit by either pharmacological treatment or site-directed mutagenesis treatment are able to effectively cause this basolateral integral membrane protein to be transported to the apical domain in well-differentiated and polarized hepatic cells. The wrongly targeted ß-subunit could still bind to the catalytic -subunit, causing a proportion of the Na+/K+-ATPase proteins to be incorrectly targeted to the apical membrane. Such defects in sorting of the Na+/K+-ATPase appear to confer a functional deficiency on these hepatic cells.
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). In these `well-polarized' hepatic cells, membrane proteins were correctly transported to their specific membrane domains. Na+/K+-ATPase, and E-cadherin and CD147/CE 9 (Bartles et al., 1985; Spring et al., 1997) were present mainly on the basolateral membrane (Fig. 1A, arrowheads). The bile canaliculi were therefore stained red (arrows) in the color-merged panels. On the other hand, dipeptidylpeptidase IV (DPPIV) was present predominantly on the apical domain (Abbott et al., 1994), making the bile canaliculi in the color-merged panel appear yellow (arrows). Treating Hep G2 cells with 20 µM tunicamycin over the last 24 hours of the 3 day culture period caused a redistribution of Na+/K+-ATPase from the native basolateral localization to the apical bile canaliculi domain (Fig. 1B, double arrowheads), but had little effect on the distribution of E-cadherin, CD147, or DPPIV (Fig. 1C). The degree of apical targeting was determined by examining the presence (from the gray-scale image) of the membrane glycoproteins of interest in the bile canaliculus. A total of 500 bile canaliculi were counted; three separate experiments were included in the statistics. Viability of the cells was not significantly affected by the drug exposure (data not shown).
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Different degrees of apical sorting by Na+/K+-ATPase were noticed when the Hep G2 cells were treated with various glycosylation inhibitors, including tunicamycin (TM), 1-deoxymannojirimycin (DMJ), 1-deoxynojirimycin (DNJ) and kifunensine (KIF). In the control conditions (CTL), the ß-subunit of Na+/K+-ATPase was heavily glycosylated; major bands were concentrated at around 50 kDa (***ß, Fig. 1E). Adding 20 µM TM, 1 mM DNJ, 0.2 mM KIF or 1 mM DMJ to Hep G2 cells for 24 hours resulted in a significant decline in the fully glycosylated proteins (in the cases of TM, DMJ and KIF), or an increase in the intermediately glycosylated proteins (**ß, in the cases of DMJ, DNJ and KIF), or the core proteins (*ß, in the cases of TM and KIF).
Localization studies revealed that 82%, 50%, 48% or 18% of the bile canaliculi (or apical domains) contained Na+/K+-ATPase ß-subunit following TM, DMJ, DNJ or KIF treatments, respectively (Fig. 1F). Note that exposure to KIF appeared to cause the most profound deglycosylation of Na+/K+-ATPase ß-subunit (with some degradation, arrow, Fig. 1E) (see also Elbein et al., 1990), yet the effect on apical targeting was relatively minor compared with other deglycosylation compounds. The nature underlying this discrepancy was unclear. We could not rule out the possibility that KIF might affect the apical targeting process undergone by the deglycosylated ß-subunits.
Deglycosylation by site-directed mutagenesis also caused apical targeting of the Na+/K+-ATPaseß-subunit
In addition to the pharmacological experiments, which are often associated with non-specific drug effects, we performed a series of site-directed mutagenesis experiments to specifically address the effects of protein glycosylation in sorting the Na+/K+-ATPase ß-subunit. Asn124 and Asn240 (the two N-linked glycosylation residues of the ß-subunit) or both were changed to glutamines (N124Q, N240Q or N124/240Q, respectively); a FLAG tag was added to the mutated gene for detection and biochemical purification. Hep G2 cells transfected with mock solution, the wild-type, N124Q, N240Q, or N124/240Q ß-subunit were subjected to western blot analyses (Fig. 2A) and localization studies (Fig. 2B). The exogenous wild-type Na+/K+-ATPase ß-subunit contained fully glycosylated proteins (***ß), intermediates (**ß) and a small amount of core protein (***ß). Tunicamycin treatment (TM) resulted in a decrease in the glycosylated forms and a corresponding increase in the core protein. Hep G2 cells transfected with N124Q Na+/K+-ATPase ß-subunit contained intermediates and core proteins but very little fully glycosylated form; after the deglycosylation by tunicamycin, only the core protein was left. In the N240Q experiments, intermediate forms were more abundant than the core protein in the control cells; after deglycosylation the core proteins became the predominant form, but there was still a significant amount of intermediates left. N124/240Q contained only core proteins before or after tunicamycin treatment. These results suggested that the addition of carbohydrates to Asn124 and Asn240 accounted for the major protein glycosylation in Na+/K+-ATPase ß-subunit in hepatic cells, and that glycosylation at Asn124 was more important than that at Asn240.
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Deglycosylated Na+/K+-ATPase ß-subunit could still interact with the -subunit
We wondered if the deglycosylated ß-subunit could still interact or form a heterodimer with the -subunit. Immunoprecipitation experiments (Fig. 3A) demonstrated that the immunoprecipitates pulled down by either the anti--subunit or anti-ß-subunit antibodies contained both -subunit and ß-subunit proteins (mainly the fully glycosylated form ***ß and intermediately glycosylated form **ß) in control conditions. Deglycosylation of the ß-subunit by tunicamycin treatment did not interfere with its interaction with the -subunit as evidenced by the presence of both - and ß-subunits (especially the appearance of ß-subunit core proteins, *ß) in the individually prepared immunoprecipitates. Similar observations were made when the deglycosylated ß-subunit mutants were applied (Fig. 3B). The immunoprecipitate isolated by the anti-FLAG antibody, which contained proteins interacting with the exogenous FLAG-tagged wild type or each of the three deglycosylated ß-subunit mutants, all contained the endogenous -subunit protein. Similarly, the immunoprecipitate prepared by the anti--subunit antibody also contained the exogenous ß-subunit protein, present at molecular masses corresponding to their degrees of glycosylation (Fig. 2A). Co-transfected EYFP-tagged -tubulin was used as the transfection control.
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-subunit and deglycosylated ß-subunits suggested that the -subunit (although not a glycoprotein itself) could be targeted to the apical domain by the deglycosylation treatments. This possibility was tested by the localization studies (Fig. 3C-D). In control Hep G2 cells, both - and ß-subunits were present only along the basolateral membrane (arrowheads, Fig. 3C CTL). After tunicamycin treatment, a significant amount of both - and ß-subunits exhibited apical targeting (double arrowheads). In Hep G2 cells transfected with EGFP-tagged wild-type (WT) ß-subunit, the exogenous ß-subunit (EGFP-ß, or the green channel in the color-merged panel, Fig. 3D) were found mainly along the basolateral membrane, together with the endogenous -subunit (blue). The apical domain appeared red with F-actin staining. In N124Q-transfected cells, some mutated ß-subunit protein was incorrectly sorted to the apical domain (double arrowheads), accompanied by the presence -subunits at that aberrant localization. Similar observations were made in N240Q- and N124/240Q-transfected cells (data not shown).
The rate of metabolism was similar when the deglycosylated Na+/K+-ATPase ß-subunits were compared with wild-type proteins
To test if the rate of protein metabolism was significantly higher in the deglycosylated ß-subunits than in the wild type, we conducted a series of `pulse-chase' experiments using [35S]methionine labeling to measure the rates of protein degradation. As shown in the autoradiograph (Fig. 4A) and the corresponding normalization plot (Fig. 4B), we found no significant difference between the wild-type ß-subunits and the three deglycosylated mutant proteins tested. The co-transfected EYFP-tagged -tubulin was used as an internal control for transfection efficiency in Hep G2 cells, and because of its long metabolic half-life, as a reference to normalize the data taken at different time points (Fig. 4B).
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Aberrant apical targeting of Na+/K+-ATPase perturbed the maintenance of ionic homeostasis in polarized hepatic cells
To address the functional consequence of the aberrant apical targeting of Na+/K+-ATPase, we performed a series of live cell experiments to test the maintenance of ionic homeostasis. Intracellular Na+ concentrations ([Na+]i) of the well-polarized Hep G2 cells were continuously monitored before, during and after washout of the solution containing high sodium (300 mM NaCl for 10 seconds) (Fig. 6). In the experiments using non-ratio Sodium Green to measure [Na+]i (Fig. 5A), we found that the control cells (CTL) were able to keep [Na+]i within a very limited range during the high-Na+ challenge process, except for a brief fluorescence intensity downturn immediately after the addition of high-Na+ solution (the reason for the deflection is unknown but might be due to morphogenesis of the cell in the transient hyperosmotic environment). On the other hand, cells treated with tunicamycin (TM) for 12 hours, or with ouabain (a Na+/K+-ATPase inhibitor) for 10 minutes exhibited an apparent and progressive increase in [Na+]i in response to the elevated extracellular Na+ concentration. Treating Hep G2 cells with both tunicamycin and ouabain did not cause any further increase in [Na+]i during the high-Na+ challenge when compared with ouabain treatment alone. This finding argues that the tunicamycin effects are `confined' by the ouabain effects, suggesting strongly that the defective ionic homeostasis observed in tunicamycin-treated cells is probably due to defects in sodium excretion, rather than mechanisms such as an unregulated increase of sodium influx.
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Bile canalicular secretion by the transcytotic transport was inhibited by the deglycosylation treatments
Although the Hep G2 cells containing the deglycosylated apical Na+/K+-ATPase ß-subunit were alive, some of their functions were defective. We and other labs have previously demonstrated that polarized Hep G2 cells were capable of excreting FDA from the culture medium to the bile canaliculi through a transcytotic transport pathway, whereas 3 kDa dextran enters the bile canaliculi via permeable tight junctions in a process termed `paracellular transport' (Lian et al., 1999). When applying these functional assays to the deglycosylation experiments (Fig. 7A), we found that about half of the bile canaliculi in the control Hep G2 cells were loaded with the fluorescent dye (arrows, or the yellow bile canaliculi in the color-merged panel) after adding FDA to the culture media for 30 minutes. The canalicular excretion of FDA was profoundly reduced by tunicamycin treatment; in the drug-treated Hep G2 cells, only about 20% of the bile canaliculi were capable of excreting FDA, compared with 48% in control cells (P<0.05 by Student's t-test; filled bars, Fig. 7C). On the other hand, the entrance of 3 kDa dextran to the bile canaliculi via the paracellular transport pathway was not significantly affected by the drug treatment (Fig. 7B). An hour after adding 3 kDa dextran to the culture media, 52% and 45% of the bile canaliculi were found to contain the dextran markers in the control and tunicamycin-treated cells, respectively. Similar conclusions were drawn from the functional studies using cells transfected with deglycosylated mutants of ß-subunit (data not shown).
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; Hunziker et al., 1991; Matter et al., 1992; Mostov et al., 1986; Rodriguez-Boulan et al., 2005), whereas the physical properties of the protein itself (e.g. GPI anchorage or associations with lipids) appear to mediate selective apical sorting (Brown and Rose, 1992; Lisanti et al., 1989; Shvartsman et al., 2003). It is generally accepted that the basolateral sorting signals are predominant over the apical ones; the latter are revealed only when the former are ablated. Some studies also suggest that N-glycan is an active apical sorting signal, as the carbohydrate moieties may be recognized by (lectin-like) receptors present along the apical sorting pathway. Based on this general view, it is surprising to find in this study that the removal of N-glycan from a basolateral membrane protein results in its apical targeting.
The basolateral sorting signals for the Na+/K+-ATPase ß-subunit, if present as the conventional views predict, should remain intact even after the deglycosylation treatments used here. Yet, rather than stay in the basolateral membrane, the N-glycan-depleted Na+/K+-ATPase ß-subunit was found to travel from the basolateral membrane to the apical domain via the conventional `transcytosis' or indirect transport (Fig. 5) that is typically used for sorting apical resident proteins in hepatic cells. There is no evidence indicating the involvement of direct apical pathway (such as the sorting route for canalicular ABC transporter) (Kipp and Arias, 2000) for transporting deglycosylated ß-subunits. The unusual journey taken by the deglycosylated membrane protein involves selective internalization from the basolateral membrane, sorting among the endosomal compartments, transportation within the vesicular compartments and reincorporation to the apical membrane. How the removal of N-linked glycosylation in the ß-subunit triggers or regulates these processes is unknown; however, it is intriguing to consider the possibility that removing the carbohydrates may reveal new recognition epitopes or change the physical properties of the glycoprotein, and thereby facilitate its interactions with the apical sorting machinery (Huet et al., 2003).
Our results argue against the view that the apical presence of the Na+/K+-ATPase ß-subunit may be due to selective retention of the glycoprotein which is randomly targeted to both apical and basolateral domains. First, the aberrant apical sorting was only found in the deglycosylated Na+/K+-ATPase ß-subunit, but not in two other basolateral membrane proteins (E-cadherin and CD147) or the apical marker DPPIV; this specificity is unlikely to be accounted for by relatively non-specific control mechanisms such as protein degradation. Secondly, we found that the rates of protein degradation are almost indistinguishable between the wild type and the deglycosylated ß-subunit mutants (Fig. 4) (see also Beggah et al., 1997). This finding is not unprecedented, there are several examples demonstrating that deglycosylation has no obvious effect on metabolism, targeting, molecular complex formation or the function of the glycoprotein (Ackermann and Geering, 1990; Laughery et al., 2003; Zamofing et al., 1988). Thirdly, although KIF treatment induced the most significant ß-subunit degradation, this drug is least effective in causing apical sorting of the ß-subunit compared with the other glycosylation inhibitors tested (Fig. 1C,D). Although the mechanisms underlying the lack of apical presence of the ß-subunit upon KIF treatment are unknown, we argued that the KIF-mediated deglycosylation did cause apical targeting of the ß-subunit; however, the majority of the apical ß-subunit was further degraded perhaps because of other KIF effects. Interestingly, KIF has been shown to cause little glycoprotein degradation in many cell types (Belbeoc'h et al., 2003; Wang and Androlewicz, 2000). The profound ß-subunit degradation by KIF observed here might be related to its apical targeting effect in our hepatic cell model. Taken together, our results favor the notion that the N-linked carbohydrates of the Na+/K+-ATPase ß-subunit may play an active role in modulating the glycoprotein sorting process, and this modulation is not mediated though the regulation of protein degradation.
The interactions between the - and ß-subunit of Na+/K+-ATPase is an intriguing cell biology topic that involves a range of different and somewhat controversial views. With respect to the basolateral residence of this ion pump, there are reports suggesting the fourth transmembrane domain of -subunit is the active basolateral sorting signal (Muth et al., 1998), whereas other studies have demonstrated a role played by the ß-subunit in guiding the transport of component (Hasler et al., 1998; Laughery et al., 2003). The results shown in Fig. 3 support the latter notion: namely, when the deglycosylated Na+/K+-ATPase ß-subunit is ectopically delivered to the apical domain, there are interactions between the deglycosylated ß-subunit and -subunit (which is not a glycoprotein and therefore should not be directly affected by the deglycosylation treatments applied) that appear to override the native sorting signals and transport the heterodimer to the apical membrane. The simplest explanation of our results is that the Na+/K+-ATPase ß-subunit can bind to the -subunit and actively regulate its sorting after biogenesis. Such an interaction between the - and ß-subunits may occur transiently or only along certain steps of the sorting process; the functional -subunits may exist on the plasma membrane dissociated from the ß-subunit (Laughery et al., 2003).
Given a fixed amount of Na+/K+-ATPase -subunit in a cell, and no evidence of increased protein synthesis following the deglycosylation treatments, the apical sorting of the deglycosylated ß-subunits is likely to reduce the presence of catalytic -subunits at the native basolateral membrane because a proportion of -subunits have been translocated to the apical domain, where the very different physicochemical environment is able to render the apical catalytic enzymes inactive (Devonald et al., 2003; Koivisto et al., 2001). It is therefore reasoned that polarized hepatic cells containing apical Na+/K+-ATPase may experience defects in maintaining enough Na+/K+-ATPase to maintain ionic homeostasis. Indeed, this is what we have observed here. After the deglycosylation treatments by either drugs or mutagenesis procedures, the cells appear to maintain a relatively high [Na+]i (21-35 mM, Fig. 6C) compared with the control cells (8-19 mM), and their adjustments to the transient high-Na+ influxes are less effective (Fig. 6A,B). Furthermore, such ionic homeostatic defects may lead to the reduction in biliary secretion (as indicated by the decreased transcytosis transport of FDA, Fig. 7A) because the sodium gradient across the plasma membrane plays a role in driving canalicular secretion (Burwen et al., 1992). On the other hand, the paracellular transport, which is governed by tight junction permeability, is not affected by the deglycosylation treatments (Fig. 7) (see also Ihrke et al., 1993).
Defects in the trafficking of membrane proteins in polarized epithelial cells are often associated with diseases, including cystic fibrosis, Liddle's syndrome, nephrogenic diabetes insipidus and Dubin-Johnson syndrome (Altschuler et al., 2003). Apical mislocation of Na+/K+-ATPase is associated with human polycystic kidney disease (Ogborn et al., 1993; Wilson, 1997; Wilson et al., 1991). The results shown in this report suggest that there is a more complex targeting role for the N-glycans of Na+/K+-ATPase (and other membrane glycoproteins) than has been previously thought.
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). The primary antibodies included those made against FLAG M2 and -tubulin (from Sigma), Na+/K+-ATPase -subunit (from Affinity Bioreagents), Na+/K+-ATPase ß-subunit and CD147 (from BD Bioscience), calreticulin (ER marker, from Abcam, Cambridge, UK). The fluorophore-conjugated secondary antibodies were all from Jackson Immuno Research (West Grove, PA). Rhodamine-phalloidin (Molecular Probes) was used for F-actin staining at a concentration of 1 U/ml. The stained samples were observed under a confocal microscope (TCS-SP2, Leica Microsystem, Germany).
Immunoprecipitation and western blot analysis
For immunoprecipitation (IP) experiments, Hep G2 cells were transfected using FuGENE 6 transfection Reagent (Roche), and lysed after 48 hour incubations with NET buffer (150 mM NaCl, 0.5% NP-40, 50 mM Tris, 1 mM EDTA and 1% Triton X-100, supplemented with protease inhibitors). The soluble proteins were collected after centrifugation at 12,000 g at 4°C for 30 minutes, and incubated with 40 µl protein A-agarose beads coated with antibodies of interest. The resulting immunoprecipitates were separated by 10.5% SDS-PAGE and transfected to a nitrocellulose membrane (Bio-Rad). After confirmation of the presence of proteins by Ponceau-S staining, standard western blotting procedures were performed (Lian et al., 1999). The blotting signal was detected by SuperSignal Chemiluminescent substrate (Pierce) and recorded by Hyperfilm (Amersham-Pharmacia).
Mutagenesis of the Na+/K+-ATPase ß-subunit
Point mutations of Na+/K+-ATPase ß-subunit was created by the PCR overlapping method using the human sequence as the template. Asn124 and Asn240 were replaced by Gln (resulting in two single mutants N124Q and N240Q, and one double mutant N124/240Q). The constructed cDNA was transfer to the donor vector (pDNR-dual) of the Creator system (Clontech Laboratories) by fusion PCR, with or without the incorporation of FLAG or EGFP tags at the N-terminus of the polypeptide chain. Plasmids were transformed to E. coli (DH5) for amplification and purified by NucleoBond plasmid purification kits (Clontech).
Protein degradation assay
Metabolism of the wild-type or mutant Na+/K+-ATPase ß-subunits were determined by a pulse-chase assay after radio-isotope labeling (Rajasekaran et al., 2001). After transfection, Hep G2 cells were grown for 48 hours and starved in Met- and Cys-free DMEM containing 1% dialyzed FBS for 2 hours at 37°C before metabolic labeling with 2 mCi/ml trans-[35S]methionine (Amersham-Pharmacia) for 15 minutes at 37°C. The cells were washed twice with PBS and chased with isotope-free culture medium for the periods indicated, before the IP experiments. The resulting autoradiograph was scanned and analyzed with Phoretix 1D standard software (NonLinear Dynamics), and quantified by densitometry analysis.
Kinetic tracing of Na+/K+-ATPase ß-subunits in living cells
Kinetic tracing of Na+/K+-ATPase ß-subunits in living cells was performed as previously described (Ihrke et al., 1993) with some modifications. Briefly, 3 day cultured Hep G2 cells were incubated with 0.1 µg/ml anti-Na+/K+-ATPase antibodies at 4°C for 15 minutes. Cells were washed twice with cold serum-free medium then incubated with complete medium at 37°C for the time period indicated. Cells were then fixed and subjected to IF staining.
Intracellular Na+ measurements
The intracellular sodium concentration [Na+]i of the living cells was monitored by Sodium Green and SBFI-AM (Molecular Probes). The loading of dye was facilitated by adding 0.02% Pluronic F-127 to the culture medium at 37°C for 60 minutes. Emission fluorescence of SBFI at 510 nm by dual excitation at 340/380 nm (using Hamamatsu monochromater) was recorded and analyzed with Acqua-Cosmos software (Hamamatsu) equipped with documented ratio algorithms (Gilon and Henquin, 1993). Calibration of SBFI-AM fluorescence was performed by adding cells with known concentrations of Na+ in the presence of 10 µg/ml of Na+ ionophore gramicidin D (Harootunian et al., 1989). The high-Na+ challenge was performed using PBS supplemented with 300 mM NaCl, pH 7.2. The challenge period was 10 seconds.
Biliary secretion assay
Biliary secretions through transcytosis or paracellular transport route were measured by loading the culture medium with Fluorescein diacetate (FDA, 5 µg/ml at 37°C for 30 minutes), or 3 kDa FITC-dextran (5 µg/ml at 37°C for 1 hour) as previously described (Lian et al., 1999). The bile canaliculi were visualized by Rhodamine-phalloidin staining. The percentage of bile canaliculi that contained excreted FDA (indicating competent transcytosis transport activity) or 3 kDa FITC-dextran (indicating competent paracellular transport activity) were calculated. The microscopy was done on a Leica DMIRBE inverted fluorescence microscope equipped with a cool-CCD (ORCA-1394, Hamamatsu, Japan) and MetaMorph imaging system (Universal Imaging, West Chester, PA).
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) and tunicamycin-treated cells (
). The bile canaliculi were recognized by F-actin staining; those also positively stained for Na+/K+-ATPase ß-subunit were identified. At least 500 bile canaliculi obtained from approximately 20 microscopic images were included in the calculation. Results shown are the means ± s.d. of three independent experiments. Bars, 10 µm.
