Instituto de Investigaciones Médicas Alfredo Lanari and Departamento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, 1427 Buenos Aires, Argentina
Submitted 12 July 2002 ; accepted in final form 14 April 2003
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ABSTRACT |
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CFTR; K562 cells; leukemia cells; ion channels; glibenclamide; DIDS; PCR; immunohistochemistry
Previous studies in K562 cells reported the activity of Cl- (50), Ca, and Na channels (29), mechanosensitive cation channels (43), and a Ca-dependent Na channel regulated by the actin cytoskeleton (32). K562 cells also express cystic fibrosis transmembrane regulator (CFTR) mRNA transcripts (51) and P-glycoprotein (50). According to this, we recently reported (3) in K562 cells the presence of an anionic channel with an outward rectification (ORCC), a channel regulated by the CFTR (21, 37).
Although it is not known at what stage of development these channels first appear in blood cells, ion channel activity is involved in the mechanism of action of antileukemic agents that induce differentiation in K562 cells (20). Aberrant expression of ion channels has been detected in tumors of nonhematopoietic and hematopoietic origins (41). In other blood cell types, ion channels are related to T cell activation, activation of leukocytes by inflammatory stimuli, and cell volume regulation (10, 25). These findings stress the importance for their role in cell transformation and therapeutic potential value (41).
The CFTR, a member of the ABC superfamily of transporters, is a cAMP-activated chloride channel (34, 37). It was proposed that CFTR conducts or regulates ATP movement (see also Ref. 7), maintaining cellular anion secretory properties (14). CFTR also regulates other ion channels, including ORCC and epithelial Na channels (37; reviewed in Ref. 21). CFTR enhances apoptosis due to regulation of the Cl-/HCO3- exchanger (4). Mutations in the CFTR gene cause cystic fibrosis, a genetic disease characterized by defective functions of Cl- and other types of channels (2).
By providing abundant amounts of cells, the K562 line can serve as a useful model for cells representing the different stages of maturation and also for testing agents that induce terminal differentiation (28) or as target for the development of antileukemic agents (20). Our long-term goals include defining channels as markers for various stages of cell differentiation, evaluating the regulatory mechanisms of channel expression, and exploring possible functional correlates of different patterns of ion channel expression. Taking into account all these considerations, we found it of interest to determine whether in K562 cells, which may become resistant to cytostatic drugs and are capable of differentiation, ABC-type proteins can be expressed and whether they have a role as ion channels. The patch-clamping studies and the RT-PCR and Western blot experiments demonstrated the presence in K562 cells of a cAMP-stimulated anion channel with characteristics consistent with the presence of a functional CFTR.
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MATERIALS AND METHODS |
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RT-PCR. Total RNA was isolated using a SV Total RNA isolation system (Promega). Reverse transcription was performed for 60 min on 5 µg of total RNA from K562 cells by using moloney murine leukemia virus reverse transcriptase, oligo (dT)15 primer, and 400 µM of each deoxyribonucleotide triphosphate (dNTP) at 42°C. PCR (30 cycles) at 94°C for 60 s, 58°C for 60 s, and 72°C for 60 s, followed by a final extension of 10 min at 72°C, was carried out using 5 µM of two specific oligonucleotide primers coding for nucleotides 43544374 and 46504630, respectively, of CFTR exon 24 (COOH terminus) sequence (sense 5'-ACTATTGCCAGGAAGCCATT-3', antisense 5'-CACCGGAACTCAAGCAAGTG-3'). The human colon carcinoma cell line T84 was used as positive control (8, 47) and was processed as above. Control experiments were performed without the addition of reverse transcriptase to evaluate the absence of genomic DNA amplification.
Western blot analysis. K562 or T84 cells were washed with cold phosphate-buffered saline (PBS) and scraped in a small volume of lysis buffer (50 mM HEPES, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, and 1 mM DTT). Nuclei and unbroken cells were removed with centrifugation (6,000 g for 15 min at 4°C). Total protein in each sample was quantified using the bicinchoninic acid assay (BCA Protein, Pierce Chemical).
For Western blot studies, 100 µg of total proteins from K562 cells and 50 µg of total proteins from T84 cells were dissolved in loading buffer (4% sodium dodecil sulfate, 0.125 M Tris·HCl, pH 6.8, 0.2 M dithiothreitol, 0.02% bromophenol blue, and 20% glycerol). The preparation was heated to 90°C for 2 min, resolved on 7.5% polyacrylamide gel, and electro-transferred onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia Biotech, UK). These membranes were blocked with 1% (wt/vol) bovine serum albumin in PBS for 30 min at room temperature and incubated overnight with a mouse monoclonal antibody (Genzyme) against the R domain (MAb 13-1) of human CFTR diluted 1:1,000. Antibody specificity was demonstrated previously by Gregory et al. (16). Membranes were then washed with PBS-Tween 0.1% and incubated 1 h at room temperature with a goat anti-mouse IgG (Sigma Chemical) (1:1,000) conjugated with alkaline phosphatase. Filters were washed and revealed with BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) (Promega).
Immunoperoxidase studies. A few drops of the K562 cell suspension were placed on a slide and fixed in methanol. Imprints were rinsed in PBS and post-fixed permeabilized with absolute acetone for 30 min at -20°C. The cells were incubated in 1% BSA in PBS for 1 h at room temperature to quench nonspecific binding sites. The imprints were incubated for 1 h at 37°C with a mouse monoclonal primary antibody against R domain (MAb 13-1) of human CFTR (1:50 dilution) in a wet chamber. The samples were washed with PBS and incubated with an anti-IgG biotinylated anti-mouse antibody dilution 1:100 for 2 h at room temperature in a wet chamber. Staining was conducted with R.T.U. Vectastain kit (Vector Laboratories). Labeling was visualized by reaction with DAB (diaminobenzidine tetrahydrochloride) and counterstained with hematoxilin.
The samples were mounted on aqueous medium and observed under light microscopy. Non-immune mouse IgG without primary antibody was used as a negative control.
Electrical recordings. The activity of ion channels in K562 cells
was recorded in the cell-detached (inside-out) configuration with a standard
patch-clamp technology by using an amplifier Warner PC501 (Warner Instruments)
with a 10 G feedback resistor. Electrical signals were filtered at 2
KHz and digitized at 6 KHz using PCLAMP v.6 software (Axon Instruments, Union
City, CA).
Forskolin (20 µM) was used to induce channel activation under cell-attached conditions, the patch was excised, and 100 µM cAMP and 1 mM Mg-ATP were added to the bath to prevent channel rundown or inactivation (27, 37). Channel activity remained steady for 2030 min.
Currents and potentials are described by the physiological convention such that negative potentials mean negative on the cytoplasmic side and inward current (plotted downward in the deflections) corresponds to the outward anion flow. All experiments were done at room temperature (2024°C).
In this study, 67% of the patches were multichannel patches and 33% were single-channel patches (n = 29). Event-duration and open probability histograms were constructed from records in which only a single open level was observed. Exponential functions were fitted using a least-squares routine of pCLAMP6. Single-channel records were further filtered at 750 Hz for display purposes only.
Solutions. Pipette and bath solutions contained (in mM) 140 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, and 10 HEPES (pH 7.4). For anion substitution experiments, NaCl in the bath was replaced by either Na-gluconate or NaF. The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Sigma was used at a final concentration of 10 µg/ml. Forskolin and glibenclamide from Alomone Labs (Jerusalem, Israel) and Sigma were diluted in DMSO and added directly to the chamber containing the cells in solution. DIDS from Sigma was dissolved in DMSO at a concentration of 20 mM and kept refrigerated until use. The final concentration of DMSO per se used did not affect ion channel activity (trials in our laboratory, with up to 0.2% DMSO on various cell types, data not shown).
The monoclonal antibody MAb 13-1 was directly diluted to 1:100 times in the bath solution from a stock solution (292 µg/ml). An inactivated antibody was obtained by preheating the CFTR antibody for 30 min at 100°C (22).
Calculations. To calculate the anion perm-selectivity ratio under
F/Cl or gluconate/Cl asymmetrical conditions, current amplitudes were best
fitted to the Goldman-Hodgkin-Katz equation
(11)
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Data were expressed as means ± SD (n = number of patches analyzed). For data comparisons, the Student's t-test was performed. Differences were considered statistically significant when P < 0.05.
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RESULTS |
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Immunolocalization of CFTR in K562 cell membranes. Further
determination of CFTR expression in K562 cells was conducted by Western blot
analysis of the membrane proteins. Figure
1B shows blots of K562 cells. The membranes were
incubated with a monoclonal antibody generated against exon 13 (R domain). A
band of 160 kDa was observed that corresponds to mature CFTR. In
addition, another band of a lower molecular weight was evident in K562 cells
representing an immature, deglycosylated form of CFTR. This is in agreement
with previous results indicating that the immunoreactive bands in the 140- to
180-kDa regions represent various forms of the CFTR protein
(35). The 160-kDa band was
present in T84 cells used as positive control. No bands were detected either
in the absence of immune mouse serum or when the primary antibody was omitted
(data not shown). Immunoperoxidase cytochemical labeling with the anti-CFTR
antibody showed strong labeling of the plasma membrane of K562 cells
(Fig. 2A). Control
samples with nonimmune mouse IgG showed a very low staining
(Fig. 2B) (4
experiments).
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Electrical activity of K562 cells. Forskolin (20 µM), an adenylyl cyclase activator, was used to induce channel activation under cell-attached conditions. Channel activity appeared 23 min after addition of the drug to the extracellular solution. After channel activation, the patch was excised and 100 µM cAMP and 1 mM Mg-ATP were added to the bath to prevent channel rundown or inactivation (27, 37). Channel activity remained steady for 2030 min.
Forskolin-induced Cl- channel activity showed linear inward and outward unitary currents in symmetrical Cl- solutions (Fig. 3). The linear current-voltage (I-V) relationship indicated a single-channel slope conductance of 11.2 ± 2.1 pS (n = 10), illustrating the nonrectifying properties of the channel. Bathing Na+ replacement with N-methyl-D-glucamine (NMDG) did not change the reversal potential (Er): 140 NaCl (control), 1.9 ± 0.8 mV (n = 10); and NMDG, -0.29 ± 0.7 mV (n = 3, P = 0.3), indicating that the channel activity was carried by the Cl- ions. Forskolin addition under cell-attached conditions was sufficient to maintain a high level of activation even after excision. Thus further addition of PKA and ATP to excised patches would result in no change in the already active channel function. The experimental protocol was as follows: forskolin (20 µM) was used to induce channel activation under cell-attached configuration in otherwise quiescent patches. Ion channel activity became apparent 23 min after the addition of the drug to the extracellular solution. After that, the patch was excised and the catalytic subunity of PKA (10 µg/ml) and 1 mM Mg-ATP were added to the bath. Channel activity remained steady for 2030 min. Figure 4 shows one such experiment in which ion channel activity in patches held at 80 and 60 mV was not affected by PKA and ATP added to the cytoplasmic side of the patch. In six experiments, a comparison of the open probability (Po) values for single channels obtained in the presence or absence of PKA plus ATP (0.44 ± 0.09 vs. 0.36 ± 0.09, n = 6; P < 0.3) indicated no differences for addition of the enzyme.
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To further demonstrate the anionic selectivity of this channel, anion substitution experiments were also performed. NaCl (140 mM) in the bath was replaced by either 140 mM Na-gluconate or 140 mM NaF. All other constituents of the solution were similar; thus substitution solutions contained 12 mM residual Cl-. Figure 5A shows single channels recorded in excised, inside-out patches held at different positive and negative potentials with either Cl- or gluconate on the intracellular side (Fig. 4A). The I-V relationship determined in these substitutions experiments with gluconate or F- are shown in Fig. 5B. Currents could be observed, although they were smaller at negative potentials and of higher amplitude at positive potentials (see DISCUSSION). The shift in the Er to more negative potentials is indicative of a lower permeability for either gluconate or F- compared with Cl-. The Er were 140 NaCl (control); 1.9 ± 0.8 mV (n = 10); 140 NaF, -28.3 ± 4.6 mV (n = 4); and 140 Na-gluconate, -27.8 ± 7.2 mV (n = 6).
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Effect of CFTR channel blockers on Cl- channel of K562 cells. To determine whether CFTR was indeed involved in the forskolin-induced Cl- channel currents, the MAb 13-1 was added to the cytoplasmic side of the patch to determine whether channel activity was affected. Previous studies indicate that active MAb 13-1 selectively blocks CFTR channel activity (22). In three experiments, addition of active MAb 13-1 (2.92 µg/ml), but not heat-inactivated MAb 13-1, decreased Cl- channel activity in excised patches of K562 cells (Fig. 6). Figure 6 (right) also shows histograms of current amplitude in control, heat-inactivated, and active MAb 13-1. In the presence of the active antibody, the open level distribution is shifted towards the closed level (C).
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The effect of the CFTR blocker glibenclamide was also assessed. Figure 7A shows a representative record in which ion channel activity was reduced by glibenclamide (100 µM) added to the cytoplasmic side of the patch. Glibenclamide had no effect on single-channel amplitude but increased the frequency of open-to-closed transitions. This effect was not markedly dependent on the membrane potential (Fig. 7B,n = 6). The effect of the drug was also observed in the open and closed-time histograms (Fig. 8). A minimum resolution of 1 ms was imposed on the data by ignoring all events shorter than this time (12). Addition of glibenclamide caused a reduction in the open time constant (Fig. 8, left) from 23.2 ± 7.1 to 2.1 ± 1.4 ms (n = 7) for control and test solution, respectively (P < 0.01). No effect on the closed time constant was observed: 39.3 ± 16.3 vs. 42.0 ± 22.6 ms (n = 7, P = 0.2). The effect of different glibenclamide concentrations was also assessed (25, 50, and 100 µM), indicating that the decrease in Po was concentration dependent, with a half-maximum inhibition (Ki) of 42.3 ± 4.6 µM (n = 3, SD).
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The effect of DIDS, another CFTR blocker (26, 33, 40), was also assessed in inside-out patches with stable channel activity (Fig. 9). As with glibenclamide, addition of DIDS (200 µM) had no effect on single-channel amplitude but decreased Po from 0.55 ± 0.23 to 0.13 ± 0.09 for control and test solutions, respectively (n = 4, P < 0.01). The effect of DIDS was irreversible. Even when the patch was washed several times with control solution, the Po did not recover (Fig. 9) with a value of 0.09 ± 0.01 not different from that obtained in the presence of the drug (n = 4).
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DISCUSSION |
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Our data indicate that forskolin addition under cell-attached conditions was sufficient to maintain a high level of activation even after excision. Thus further addition of PKA and ATP to excised patches would result in no change in the already active channel function. This, however, does not indicate the lack of PKA responsiveness; rather, the effect of supramaximal concentration of forskolin was sufficient to fully activate the channels. The lack of effect of added PKA in K562 cells is most consistent with experimental conditions under which rundown of forskolin-stimulated CFTR activity is very slow or practically absent in the excised patches without PKA.
CFTR is normally activated in cells by cAMP signaling pathways. The cAMP-dependent PKA phosphorylation event activates the channel, which in turn can be inactivated by phosphatases (1; see Ref. 17 for a review). Thus it is possible that PKA was not required in our experiments because little phosphatase activity was present in the patches. This interpretation was raised by Sørensen and Larsen (40), who reported that no rundown of CFTR channel activity in inside-out patches was noted in the presence of ATP after removing cAMP-dependent protein kinase from the solution. The same interpretation is given by Luo et al. (27) in patches from transfected Chinese hamster ovarian cells.
Another possibility to be considered is that after the excision of the patch, the addition of cAMP could indeed activate PKA bound to the membrane. This is consistent with Bradbury (6), who shows a physical association between CFTR and PKA and that in T84 cells PKA subunits are cytosolic (RI) and membrane bound (RII). Earlier evidence has also shown that PKA is found in the membranes of red blood cells (see Ref. 42).
The Cl- channels described in this report had a single-channel
conductance and an anion perm-selectivity consistent with CFTR. Conductance
values reported for CFTR have a range of 615 pS, and subconductance
states have been observed under certain experimental conditions
(15,
46). Some variations in the
reported conductance and kinetics are the consequence of variable parameters,
including recording conditions, species-dependence, and ATP concentrations.
Differences between wild-type and transfected CFTR have also been observed
(13,
14,
23,
27,
36,
39,
52,
54). In this study, the
gluconate/Cl and F/Cl permeability ratios for these channels were studied
under bi-ionic conditions. Cl- replacement by either F-
or gluconate shifted the Er to more negative potentials.
Both ionic substitutions exhibited relative perm-selectivity ratios of
0.3. Similar perm-selectivity ratios for these anions were described by
others for CFTR expressed either endogenously or heterologously in a number of
cell types (reviewed in Ref.
31). In fibroblasts infected
with a retrovirus expressing CFTR, for example, F- stimulated
open-state probability of CFTR channels in the presence of ATP after
phosphorylation (5). When
gluconate or F- were the main anions in the bath, the current
amplitude was smaller at negative potentials and greater at positive
potentials (Fig. 5). This is
consistent with previous findings in Chinese hamster ovarian cells
(45) expressing wild-type CFTR
when bathing Cl- was replaced by F-. In our own study,
the higher current amplitude at positive potentials when Cl- was
replaced as the main anion could be explained if we had subconductance states,
with lower amplitude current levels being more evident in the control
solution. Although interesting, this possibility is unlikely because we did
not observe subconductance levels under any of our experimental
conditions.
CFTR channels are characterized by their sensitivity to blocking by several agents, including the sulfonylurea hypoglycemic agent glibenclamide and its congener tolbutamide (reviewed in Refs. 19, 53). The blocking effect of glibenclamide in excised membrane patches seen in the present study had a Ki value of 42 µM, in agreement with earlier observations (13, 14, 36, 39). These studies suggest an open-channel blockade and the drug gaining access to its binding site in the channel from the intracellular side of the membrane (see also Ref. 1). Open time constant of channels exposed to glibenclamide decreased, in agreement with data from Schultz et al. (36) in mouse L cells and those of Sheppard et al. (39) obtained in mammalian epithelial cells. Our results showed a weak voltage dependence for the glibenclamide effect, with the effect being more evident at negative potentials (Fig. 7B). We also found that DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches, supporting previous results by Sørensen et al. (40) in the luminal membrane of frog skin exocrine gland acini. Linsdell and Hanrahan (26) also reported that, when added to the inside, DIDS blocked whole CFTR currents recorded from excised patches of transfected Chinese hamster ovarian cells. Similar results were obtained by Reddy and Quinton (33) in the human sweat duct.
Active transcription translation of the CFTR gene product is detectable in
a variety of epithelial and nonepithelial cells
(49,
51). These observations of
CFTR widespread distribution suggest a housekeeping gene
(51), implying a great range
of functions in many human blood cells, including possible regulatory roles in
the secretion of antibodies and cytokines by lymphocytes in response to
extracellular signals (9,
30,
44) and regulation of
lipopolysaccharide and interferon--induced macrophage activation
(18). In K562 cells, CFTR and
other channels could be involved in various cell functions, including control
of the membrane potential and cell differentiation and proliferation
(10,
24,
48). Previous studies
demonstrated that ion channels have specific functions in K562 cells. Thus
Sheehy (38) found an increment
in ion channel activity in human lymphocytes by lymphokines. A selective
upregulation of human ether-a-go-go-related gene (HERG) K channels has also
been reported in neoplastic hematopoietic cells, including K562 cells,
providing a marker and target for potential therapeutics
(41). In conclusion, our data
demonstrated the presence of a cAMP-activated, nonrectifying anion channel
highly selective for Cl- over Na+ and with gluconate/Cl
and F/Cl perm-selectivity ratios of about 0.3. This is most consistent with
the presence of a functional CFTR that was confirmed by RT-PCR and
immunohistochemical analysis. Further studies will be required to assess the
role(s) of a functional CFTR in K562 cells, including cell functions such as
differentiation and proliferation.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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