| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Departments of 1Physiology and Biophysics and 2Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada
Submitted 20 February 2007 ; accepted in final form 22 June 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
potassium channel; volume regulation; mammary gland
In common with other epithelial cell types, those of the mammary gland would be predicted to possess volume-sensitive transport mechanisms that would permit them to respond dynamically to changes in the osmolarity of their external milieu and enable the cell to remain at its optimal volume. Indeed, it has been proposed that mammary epithelial cells will experience changes in volume as a result of variations in hydration status secondary to metabolism, and also due to milk containing high concentrations of impermeable solutes such as lactose (45, 46). Additionally, the K+ concentration of milk is actually severalfold higher than that of plasma, suggesting that some mechanism for K+ secretion must be present in mammary epithelial cells. Indeed, one classic proposed model predicts that the apical membrane of these cells must possess K+ channels (27). However, little is known about the molecular nature of these channels. Because cell swelling and volume regulatory mechanisms are often associated with increased activity of K+ channels, we were interested in determining whether there was an apically located, volume-activated K+ channel in human mammary epithelial cells that could potentially help the cells respond to changes in volume.
To date, investigations into fundamental aspects of mammary epithelial physiology have utilized mouse (5, 6) and bovine (41) cell lines. Such studies have determined that fluid secretion across the apical membrane of murine mammary epithelial cells could be stimulated by inhibition of the epithelial Na+ channel or activation of the cystic fibrosis transmembrane conductance regulator Cl– channel (5) or Ca2+-dependent Cl– channels coupled to P2Y2 purinoceptors (6). More recently, the work of Quesnell et al. (41) demonstrated that the electrolyte composition at the apical membrane of bovine mammary epithelial cells has profound effects on the distribution of the tight junction protein occludin. Thus these models demonstrate the complex and dynamic nature of transepithelial transport across the mammary epithelial barrier. However, there is a considerable paucity of data concerning mammary epithelial ion transport, in particular in relation to K+ channels.
The MCF-7 cell line is derived from a human mammary adenocarcinoma and retains many characteristics of differentiated mammary epithelium, such as the ability to form a tight epithelial monolayer (11) and retaining the estrogen receptor (48). Furthermore, they also possess the capacity to form domes (48), indicative of transepithelial fluid movement. MCF-7 cells are widely used as a model to investigate various aspects of the signaling cascades involved in breast cancer progression, particularly to investigate the role of prolactin and estrogen in breast tumorgenesis (13, 15). However, little attention has been paid to their fundamental properties as epithelial cells. Previous studies have demonstrated the expression of a number of K+ channels in MCF-7 cells, including the large-conductance Ca2+-activated K+ channel (KCNMA1; Ref. 9), voltage-gated K+ (Kv) 1.1 (KCNA1; Ref. 39), Kv1.3 (KCNA3; Ref. 1), human EAG (KCNH1), and human intermediate-conductance Ca2+-activated K+ (KCNN4; Ref. 40), several of which appear to play a role in the progression through the cell cycle and cell proliferation. Additionally, MCF-7 cells are known to express the cystic fibrosis transmembrane conductance regulator Cl– channel (16) and the epithelial Na+ channel (11). However, the roles of these channels in the transport properties of these cells have not been described.
In the present study, we have investigated whether K+ channels play a role in cell volume regulation in MCF-7 cells. In addition to identifying the KCNQ1 K+ channel as an essential player in mediating the response to hyposmotic stress, we additionally localize the expression of this channel to the apical membrane, where it would be ideally located to both respond to changes in extracellular fluid composition, and potentially mediate transepithelial K+ conductance.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
N-KCNQ1; Ref. 21) was then generated within the pIRES2-EGFP vector by site-directed mutagenesis, using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Initially, site-directed mutagenesis was used to introduce a new start codon within the first membrane-spanning region of KCNQ1, just downstream of a newly introduced NheI site. The mutagenesis primers used for this reaction were 5'-CCGGCTGGAAATGCTTCGGCTAGCACTTCATGGACTTCCTCATCGTCCTGGTC-3' and 5'-GACCAGGACGATGAGGAAGTCCATGAAGTGCTAGCCGAAGCATTTCCAGCCGG-3'. The region encoding the truncated protein was then removed using NheI and SmaI and subcloned into the multicloning region of freshly prepared pIRES2-EGFP. For visualization in immunocytochemistry experiments, an NH2-terminal FLAG-tagged version of full-length KCNQ1 was prepared by subcloning into the pFLAG-CMV vector (Sigma, Oakville, ON, Canada) using EcoRI and Cfp9I restriction sites. Cell culture. The human mammary epithelial cell line MCF-7 (obtained from the American Type Culture Collection, Rockville, MD) was cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 10 µM nonessential amino acids, 0.1 g/ml human insulin (Sigma-Aldrich), and a mixture of penicillin (100 U/ml) and streptomycin (100 U/ml) (all from Gibco BRL, Burlington, ON, Canada). The culture medium was renewed every 2 days, and cells were incubated at 37°C in humidified 5% CO2-95% air. For RNA and protein extraction, cells were cultured on 100-mm-diameter Falcon culture dishes (Becton Dickinson, Franklin Lanes, NJ).
For measurement of cell volume and patch-clamp experiments, dissociated MCF-7 cells were plated on glass coverslips between 4 and 7 h before the experiment.
To obtain polarized MCF-7 cell monolayers, cells were seeded on Snapwell cell culture supports (Corning Costar, Cambridge, MA). Once confluent (
5 days growth), cell monolayers were fixed and prepared for immunocytochemistry (see below). In some experiments, confluent cell monolayers were transfected with 390 ng/ml pFLAG-CMV-KCNQ1 plasmid DNA using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada), following the manufacturer's instructions, and fixed for immunocytochemistry 44 h posttransfection.
For overexpression studies, MCF-7 cells were transfected with either full-length (pIRES2-EGFP-KCNQ1) or truncated (pIRES2-EGFP-N-KCNQ1) plasmid DNA (390 and 242 ng/ml, respectively) using Lipfectamine 2000. The negative controls for these experiments consisted of MCF-7 cells transfected with the pIRES2-EGFP vector alone. Forty-eight hours after transfection, cells were re-dissociated and plated on cover slips for volume measurements or patch-clamp recordings.
For patch-clamp experiments, baby hamster kidney (BHK) cells were cultured, as previously described (26). One day after seeding, BHK cells were transfected with 330 ng/ml pIRES2-EGFP-KCNQ1 plasmid DNA plus 670 ng/ml pIRES1-CD8-KCNE3 plasmid DNA or with 1 µg/ml pIRES2-EGFP-KCNQ1 plasmid DNA alone using Lipofectamine 2000. Transiently transfected cells could be identified by fluorescence microscopy within 24 h and were used for patch-clamp recording 1–3 days after transfection. BHK cells were also transfected with 670 ng/ml pIRES1-CD8-KCNE1 plasmid DNA for use as a positive control for the RT-polymerase chain reaction (PCR) for KCNE1 cDNA.
Cell volume experiments.
MCF-7 cells plated on glass cover slips between 4 and 7 h earlier were continuously superfused at a rate of 1 ml/min using a peristaltic pump (Gilson, Middleton, WI). Cells were initially placed in an isosmotic solution (in mM: 75 NaCl, 2.5 KCl, 1.2 CaCl2, 0.5 MgCl2, 5 glucose, 10 HEPES, 150 mannitol; pH to 7.4). After at least 5 min in the isosmotic solution, this was changed to a hyposmotic solution (in mM: 75 NaCl, 2.5 KCl, 1.2 CaCl2, 0.5 MgCl2, 5 glucose, 10 HEPES; pH to 7.4) containing either the K+ channel blocker of interest or vehicle alone (control cells). After 20 min in the hyposmotic solution, cells were returned to the isosmotic solution to confirm that they could return to their original size (results not shown). The osmolarity of solutions was measured using a freezing point osmometer (model 110, Fiske Associates, Norwood, MA) and was determined as 171.8 ± 0.92 mosM (n = 18) for the hyposmotic solution, and 329.9 ± 1.04 mosM (n = 18) for the isosmotic solution.
Cells were imaged used a Zeiss Axioskop FS microscope (Zeiss, Oberkochen, Germany) employing a x63 objective (Zeiss). Images of singles cells with well-defined cell membranes were captured every 60 s using a charge-coupled device camera and imaging software (Pixelink camera and Pixelink Capture SE software). Captured images were then analyzed using ImageJ software (NIH, version 1.24 available at http://rsb.info.nih.gov/ij/), which permitted their area to be determined. Since it was assumed that the cells were spherical, the radius (R) was calculated from R = 
(area/
), the volume (V) from V = 4/3 x x r3, and cell volume was then normalized to that at time t = 0 (28). The percent regulatory volume decrease (RVD) was also calculated from 100[1 – (Vmin – 1)/(Vmax – 1)], where Vmax is the peak volume in hyposmotic solution, and Vmin is the volume before returning to isosmotic solution, again normalized to that at t = 0.
To image EGFP fluorescence, a 75-W Xenon lamp (LUDL Electronic Products, Hawthorne, NY) was used in combination with appropriate filters (XF100, 475 nm excitation, 535 nm emission, dichroic >505 nm; Omega Optical, Brattleboro, VT).
RNA extraction. Total RNA was extracted from MCF-7 cells and also from BHK cells transfected with pIRES1-CD8-KCNE1 using the TRIzol reagent (Gibco BRL). RNA was DNase treated with RQ1 RNase-free DNase (Promega, Madison, WI), and 2-µg DNase-treated RNA were then reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen) in the presence of 5 mM dNTP (Invitrogen) and 1 µM oligo(dT) (Amersham Pharmacia, Baie d'Urfe, PQ, Canada) to produce cDNA.
PCR. After reverse transcription, PCR was performed to amplify DNA fragments. Custom primers were obtained from Invitrogen, and reactions were performed using primer pairs at 10 µM with 2.5 units Taq polymerase (MBI Fermentas, Burlington, ON, Canada), 25 mM MgCl2, and 5 mM dNTP in a total reaction volume of 25 µl. The primers and amplification conditions used (denaturation/annealing/extension/cycle number/expected product size in base pairs) were as follows: KCNQ1 5'-CACCATCGAGCAGTATGCCGC-3' and 5'-CATCGCGTCCTTCTCAGCCA-3' (94°C 1 min/63°C 1 min/72°C 0.5 min/40 cycles/436 bp); KCNE1 5'-TGGTACTGGGATTCTTCGGC-3' and 5'-AGGAAGGTGTGTGTTGGGTTG-3' (95°C 1 min/56°C 1 min/72°C 1 min/35 cycles/224 bp); KCNE2 5'-GCTGAGGCTTGTGTGCAACC-3' and 5'-GGATGGTGGCCTTCGATTC-3' (95°C 1 min/58°C 1 min/72°C 2 min/35 cycles/449 bp); KCNE3 5'-ACCAATGGAACGGAGACCTG-3' and 5'-ACTACGCTTGTCCACTTTGCG-3' (95°C 1 min/58°C 1 min/72°C 2 min/35 cycles/258 bp). Primers and amplification conditions for KCNQ1 were taken from Bernard et al. (4), whereas those for KCNE1, KCNE2, and KCNE3 were from Lan et al. (23). PCR products were visualized by loading a 12-µl sample on a 1.5% agarose gel containing 250 µg/l ethidium bromide, alongside a 100-bp DNA ladder (Gibco BRL). To confirm the identity of the amplified PCR fragments, the product was isolated from the gel using the QIAquick gel extraction kit (Qiagen, Mississauga, ON, Canada) and sequenced using a commercial sequencing facility (DalGEN Microbial Genomics Centre, Dalhousie University, Halifax, NS, Canada).
Western blotting. Cells were removed with a cell scraper and spun down, and the pellet was resuspended in a lysis buffer containing 10% SDS and 15 mg/ml dithiothreitol. Complete protease inhibitor (Roche Applied Science, Indianapolis, IN) was added, and the mixture sonicated. Total protein (50 µg) was run on a 7.5% polyacrylamide gel and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Immunoblotting for KCNQ1 was performed using a rabbit anti-human KCNQ1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1 in 500 dilution, followed by incubation with horseradish peroxide-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) at 1 in 5,000 dilution. Detection was carried out using the ECL Plus kit, following manufacturer's instructions (Amersham Pharmacia).
Immunolocalization. To prepare inserts for microscopy, confluent cell monolayers on Snapwell cell culture supports were rinsed five times with PBS, and the membrane was removed from its support with a razor blade and mounted on a microscope slide. Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) and permeabilized by adding PBS containing 0.3% Triton X-100 (Sigma-Aldrich) for 15 min. Before incubation with the appropriate antibodies, 10% goat serum was added to the inserts for 1 h to block nonspecific binding. MCF-7 cells were then coincubated with rabbit anti-human KCNQ1 (Chemicon International, Temecula, CA) at 1 in 100 dilution with either mouse anti-human zona occludens-1 (ZO-1; Clontech) at 1 in 200 dilution or mouse anti-human Na-K-ATPase (Upstate Biotechnology, Lake Placid, NY) at 1 in 200 dilution. FLAG-KCNQ1-transfected monolayers were incubated with Cy3-conjugated mouse anti-FLAG (Sigma-Aldrich) at 1 in 1,000. In all cases, primary antibodies were prepared in PBS containing 0.3% Triton X-100 and 2% goat serum, and cells were incubated with antibody overnight at 4°C in a humid chamber. Inserts were then washed with PBS before application of the appropriate secondary antibody, prepared in PBS (containing 2% goat serum) for 2 h at room temperature. For endogenous KCNQ1, the secondary antibody was Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) at 1 in 1,000, while the secondary antibodies used to label ZO-1 and Na-K-ATPase were goat anti-mouse Alexa Fluor 594 IgG (Invitrogen) used at 1 in 200. Experimental controls, in which the primary antibodies were omitted, were completed in parallel.
Immunolocalized proteins were visualized using a META LSM 510 laser scanning confocal microscope (Carl Zeiss Canada, North York, ON, Canada). Z-stack images were collected by capturing 1-µm optical slices at predetermined focal points. Subsequently, z- and x-axis images were produced from the captured Z-stacks using LSM Image Browser release 3.2 software (Carl Zeiss Canada).
Electrophysiological recording. Membrane currents were recorded using the whole cell configuration of the patch-clamp technique. Currents were recorded using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), low-pass filtered at 2 kHz using an eight-pole Bessel filter, digitized at 5 kHz, and analyzed using pCLAMP9 software (Molecular Devices). Standard pipette solution for BHK cells contained the following (in mM): 150 potassium-gluconate, 5 K2ATP, 1 MgCl2, 5 EGTA, and 10 HEPES. The same pipette solution was used for MCF-7 cells, except that EGTA was omitted. Hyposmotic extracellular (bath) solution contained the following (in mM): 75 sodium-gluconate, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES; this solution was supplemented by addition of 150 mM D-mannitol to obtain isosmotic bath solution. The pH values of the pipette and bath solutions were adjusted to 7.3 using KOH and NaOH, respectively. Cells were maintained at a resting membrane potential of –60 mV. To obtain current-voltage relationships, the membrane potential was pulsed between –80 mV and +100 mV (at 20-mV increments) for 500 ms and thereafter repolarized to –40 mV for tail current recordings. Pulses were applied at a frequency of 0.2 Hz. Recordings were made at room temperature.
Statistics. Differences between groups were tested for using an analysis of variance, followed by Student's t-test to investigate for differences between the control and treated group, with significance determined as P < 0.05. In electrophysiological experiments, a paired t-test was employed, and differences were considered significant at P < 0.05.
Chemicals. 293B and XE991 were obtained from Tocris (Ellisville, MO), while all other chemicals were from Sigma-Aldrich.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
21°C; n = 21), cells at both temperatures underwent cell swelling followed by an RVD response (Fig. 1B); however, those at 37°C reached a higher maximum fold increase in volume compared with those at room temperature (1.70 ± 0.05 or 70% vs. 1.60 ± 0.03 or 60%), although this did not reach statistical significance. The rate of onset of cell swelling and the RVD response both appeared somewhat slower at room temperature. Since RVD is governed by the activity of cell membrane channels and transporters, it is not surprising that this phenomenon is temperature dependent. However, since there was a large and apparent cell swelling and RVD response at room temperature, remaining experiments were performed at room temperature to maintain consistency with patch-clamping experiments (see below). At the end of each experiment, cells were switched back to isosmotic solution and allowed to recover to confirm their viability (results not shown).
|
|
Since the voltage-gated, outwardly rectifying K+ channel KCNQ1 is reportedly expressed in many epithelial cells (10, 17, 22, 29) and has also been implicated in volume regulation (18, 24), we also investigated the ability of the KCNQ1 inhibitors chromonol 293B (7) and XE991 (50) to influence RVD. Both 293B (50 µM, n = 11) and XE991 (50 µM, n = 10) abolished the ability of MCF-7 cells to recover from a hyposmotic stress (Fig. 2, C, D, and E), implicating a role for KCNQ1 in this process.
Confirming a role for KCNQ1 in the RVD response of MCF-7 cells.
To confirm the involvement of KCNQ1 in RVD, we also investigated its potential role using nonpharmacological means. To this end, we examined the ability of MCF-7 cells transfected with the dominant-negative N-KCNQ1 construct (n = 8) to undergo RVD compared with cells transfected with the pIRES-EGFP vector alone (n = 6). As can been seen in Fig. 3A, transfection with N-KCNQ1 inhibits MCF-7 cells from undergoing RVD,1 whereas RVD was apparently unaffected in vector-only transfected cells (Fig. 3B). The percent RVD was statistically significantly inhibited in the presence of the dominant-negative KCNQ1 construct, confirming the importance of KCNQ1 to the RVD response of MCF-7 cells (Fig. 3B). Figure 3B also shows that, when MCF-7 cells were transfected with a full-length KCNQ1 construct to overexpress this protein (n = 8), there was no apparent difference in the ability of the overexpressing cells to undergo RVD compared with mock-transfected MCF-7 cells (n = 6). When we additionally investigated the effects of 293B (50 µM) on cells transfected with the N-KCNQ1 construct, there was no significant difference in the RVD response, with or without the presence of the chromanol (n = 4; Fig. 3B), suggesting that dominant-negative transfection and 293B were affecting the same K+ conductance.
|
|
70 kDa) was detected (n = 3 passages of MCF-7 cells). Localization of KCNQ1 in polarized MCF-7 monolayers. To investigate the cellular distribution of KCNQ1 protein in MCF-7 cells, confluent monolayers of cells grown on cell culture supports were incubated with an anti-KCNQ1 antibody concurrent with an antibody against either the Na-K-ATPase to mark the basolateral membrane, or the tight junction marker ZO-1. Figure 5A shows a Z-axis projection of immunofluorescence for Na-K-ATPase (shown in green), showing little KCNQ1 staining (red) at the level of the cell where Na-K-ATPase staining is strongest. When this costaining is examined along its X-axis using an image reconstructed from optical slices (Fig. 5B), it becomes apparent that the KCNQ1 is located at the apical aspect of the MCF-7 cells, whereas the Na-K-ATPase is found along the basolateral membrane. Figure 5, C and D, demonstrates costaining for KCNQ1 (red) using another polarization marker, ZO-1 (green). ZO-1 can be seen strictly localized to the tight junctions, with KCNQ1 staining again apical. Finally, when MCF-7 cells were transfected with the pFLAG-CMV-KCNQ1 construct, anti-FLAG immunofluorescence was clearly present exclusively at the apical membrane (Fig. 5, E and F), confirming the results obtained for immunolocalization of the endogenous KCNQ1 protein.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Maintenance of a constant cell volume is critical if the cell is to function normally, since even small perturbations may upset the activity of a plethora of intracellular events (25). Therefore, cells possess the capacity to respond to changes in the osmolarity of their environment by activating a set of membrane transporters that will provide the required gain or loss of osmolytes and water, leading to a net regulatory volume increase or RVD (reviewed in Ref. 25). RVD is achieved principally by the activation of volume-regulated K+ and Cl– channels, activated following an osmotic challenge, and that permit the cell to return to its optimal volume. Using a number of nonselective and selective K+ channel inhibitors, we first established a role for K+ channels in mediating the RVD response of MCF-7 cells and then determined that, of these K+ channels, KCNQ1 appears to be crucially important in this RVD. Using both pharmacological (293B and XE991) and molecular (heterologous expression of the dominant-negative N-KCNQ1 construct) means, we demonstrate that inhibition of KCNQ1 activity essentially abolishes the ability of MCF-7 cells to undergo RVD (Figs. 2 and 3). While the osmotic stress applied in these experiments is not one that mammary epithelial cells would see under normal physiological conditions, large, nonphysiological changes were necessary to measure cell volume accurately using our system. However, we believe the same processes are underlying RVD in response to both physiological and nonphysiological changes in osmolarity. We conclude that KCNQ1 channel activity is essential to allow these cells to regulate their intracellular volume in response to a decrease in extracellular fluid osmolarity, suggesting a physiological role for KCNQ1 in regulating mammary epithelial cell volume.
KCNQ1 is a member of the voltage-gated, outwardly rectifying KCNQ family (42), members of which play a number of important functions, as demonstrated by the extensive human pathologies arising from mutations of their genes (reviewed in Ref. 20). KCNQ1 coassembles with the smaller accessory subunit minK (encoded by KCNE1) to form the slow, voltage-gated cardiac channel IKs (3, 44), mutations of which result in long QT syndrome or Jervell, Lange-Nielsen cardioauditory syndrome, characterized by a prolonged QT interval and congenital bilateral deafness (36). However, KCNQ1 can also coassemble with other accessory subunits (KCNE2–5) to produce functional channels in a number of nonexcitable tissues, including gastric parietal cells (19) and the secretory epithelia of the colon (22) and airway (10, 17, 29). In the present study, we demonstrate KCNQ1 expression in MCF-7 cells at both the mRNA and protein level (Fig. 4) and also detect mRNA for KCNE1, KCNE2, and KCNE3 (Fig. 4). Since MCF-7 cells possess KCNE1 and KCNE3, it is possible that KCNQ1 may coassemble with either (or both) subunits to form functional K+ channels in MCF-7 cells. While both KCNQ1/E1 and KCNQ1/E3 coassembly has been proposed in other epithelial cell types (10, 17, 22, 29, 34, 38, 51), this is the first time KCNQ1 and KCNE expression has been reported in mammary epithelial cells. Coassembly of KCNE2 with KCNQ1 has been demonstrated to modify KCNQ1 activity using heterologous coexpression systems, producing a voltage-independent "leak" channel (49). There is little information to date concerning the physiological role of KCNQ1/KCNE2 in native cells, although a recent report indicates it may be important for gastric acid secretion from parietal cells (43).
KCNQ1 has been suggested to play an important role in volume regulation in a number of cell types (18, 24), including isolated rat liver (24) and cultured rat hepatocytes (23). In epithelial cells, the ability to perform vectoral transepithelial movement of salt and water is essential and requires tight control over cell volume, and changes in volume have been demonstrated to affect the activity of a number of epithelial K+ channels (reviewed in Ref. 25). Lock and Valverde (28) reported that the KCNQ1/KCNE1 inhibitor clofilium inhibited RVD in murine tracheal epithelial cells, while cells from KCNE1 knockout mice had a reduced RVD response. Similarly, renal proximal tubules from KCNE1 knockout mouse demonstrate impaired RVD (34), thereby implicating the KCNQ1/KCNE1 complex in RVD in epithelial cells.
The requirement for KCNQ1 activity to allow MCF-7 cells to undergo RVD suggests that KCNQ1-mediated K+ currents may be augmented by cell swelling in these cells. Indeed, small but significant 293B-sensitive whole cell currents were observed in MCF-7 cells under hypotonic, but not isotonic, conditions (Fig. 6). Unfortunately, we were unable to demonstrate directly that these small currents were activated by hypotonic cell swelling. To investigate the cell volume sensitivity of KCNQ1-containing K+ currents in mammalian cells, we, therefore, moved to a heterologous expression system. As described above, we consider it most likely that KCNQ1 may coassemble with KCNE1 and/or KCNE3 in MCF-7 cells to form functional K+ channels. Indeed, we found that coassembly of KCNQ1 and KCNE3 in BHK cells led to the expression of a volume-sensitive, 293B-inhibited K+ current (Fig. 7). Recently, our laboratory showed that K+ channels formed by coassembly of KCNQ1 and KCNE1 in these same cells are also stimulated by extracellular hyposmotic solutions (35). Lan et al. (23) concluded that a KCNQ1/KCNE3 complex mediates a significant proportion of a volume-activated K+ conductance in rat hepatocytes. Therefore, current activation by cell swelling appears to be a common feature of KCNQ1/KCNE1 and KCNQ1/KCNE3 K+ channels expressed in mammalian cells, suggesting that the channel proteins themselves may mediate volume sensitivity. Indeed, KCNQ1 expressed alone exhibits similar sensitivity to extracellular hyposmolarity (Fig. 7B). Thus KCNQ1 together with KCNE1 or KCNE3 (or both) may contribute to the activation of membrane K+ currents in MCF-7 cells exposed to hyposmotic solutions.
KCNQ1 protein (whether endogenously and heterologously expressed) is localized to the apical membrane of polarized MCF-7 cell monolayers (Fig. 5). KCNQ1 has been localized to both the apical and basolateral membranes of epithelial cells, possibly reflecting its specific physiological role. For example, in gastric parietal cells, KCNQ1 is found in the apical (luminal) membrane and has been proposed as a pathway for the luminal K+ recycling necessary for gastric H+ secretion to occur (17). Similarly, KCNQ1 is found in the apical membrane of inner ear vestibular cells, where it is involved in secretion of K+ from dark cells into the K+-rich endolymph (38). KCNQ1 is found basolaterally in the jejunum and colon (51) and has been identified electrophysiologically as a basolateral conductance in murine (17) and human airway epithelial cells (10, 30). Coassembly of KCNQ1/KCNE3 results in a constitutively open channel (33), thought to underlie a cAMP-regulated K+ conductance in secretory epithelia (30), where its activity would be prosecretory, since exit of K+ across the basolateral membrane will hyperpolarize the cell and result in increased anion secretion via apical membrane Cl– channels. Recently, the localization of KCNQ1 throughout the mouse kidney has been described, with both apical and basolateral protein distribution being described, dependent on the cell type (52). Thus KCNQ1 appears to have cell-specific roles in the kidney, depending on whether K+ absorption or secretion is required.
Within the mammary epithelium, the requirement for an apically located K+ channel has long been proposed (27), since the final K+ concentration of milk is considerably higher than that of plasma (47). Our findings that KCNQ1 is expressed apically when MCF-7 cells are cultured as a polarized monolayer suggest that KCNQ1 could be a potential candidate channel to mediate apical K+ secretion from the mammary epithelium. Furthermore, in terms of mammary disease, gross fibrocystic breast disease is the most frequent benign breast pathology (32), affecting at least 7% of women, although estimates have reached as high as 40–60% (32). Breast cysts can be classified according to their electrolyte composition (12), with type I breast cysts being defined as secretory and containing increased levels of K+ and low Na+ and Cl– (2), while type II cysts are transudative and exhibit electrolyte concentrations similar to serum (2). Although frequently considered benign, several epidemiological studies have reported that gross fibrocystic breast disease may lead to an increased risk of developing breast cancer, in particular when the cysts are of the type I (high K+ concentration) variety (8, 31), although this remains somewhat controversial (14). Regardless of their potential association with breast cancer, breast cysts represent an important example of inappropriate ion and fluid transport by mammary epithelial cells and provide further evidence that investigations into the nature and subcellular localization of mammary epithelial K+ channels are long overdue.
In conclusion, we demonstrate that KCNQ1 and a number of its accessory subunits are expressed in the human mammary epithelial cell line, MCF-7, suggesting for the first time that this class of K+ channels may play a role in mammary epithelial physiology. In addition, our work points to two possible roles for these K+ channels in mammary epithelia: namely, K+ secretion and volume regulation. KCNQ1 protein is located exclusively in the apical membrane when these cells are cultured under conditions that induce their polarization. Thus it may be that KCNQ1 is a potential candidate channel to mediate K+ secretion across the apical aspect of mammary epithelial cells. To date, very few studies have been undertaken to investigate the transepithelial transport proteins of mammary epithelial cells, and this is the first to focus exclusively on a specific K+ channel. We additionally determine that KCNQ1 plays an important role in volume regulation in these cells, since impairing channel activity absolutely abolishes their ability to RVD. We cannot conclude definitively whether KCNQ1/KCNE1 or KCNQ1/KCNE3 underlies the volume-sensitive current in these cells; however, KCNQ1 could clearly play an important role in mediating the recovery in response to changes in cell volume in the mammary epithelium.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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.
1 Of the MCF-7 cells transfected with the N-KCNQ1 construct and which demonstrated GFP expression (detectable green fluorescence), 8 of 12 failed to RVD, while 4 of 12 appeared unaffected by the transfection and were undistinguishable from control cells. We interpret this to mean that, in 4 of 12 cells, the level of N-KCNQ1 expression was insufficient to change the phenotype of the cells; consequently, these cells have not been included in our data analysis and interpretation. 
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Angeli A, Bradlow HL, Bodian CA, Chasalow FI, Dogliotti L, Haagensen DE. Criteria for classifying breast cyst fluids. Ann NY Acad Sci 586: 49–52, 1990.[Web of Science][Medline]
3. Barhanin J, Lesage F, Guillemare E, Fink M, Lazdunski M, Romey G. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature 384: 78–80, 1996.[CrossRef][Medline]
4. Bernard K, Bogliolo S, Soriani O, Ehrenfeld J. Modulation of calcium-dependent chloride secretion by basolateral SK4-like channels in a human bronchial cell line. J Membr Biol 196: 15–31, 2003.[CrossRef][Web of Science][Medline]
5. Blaug S, Hybiske K, Cohn J, Firestone GL, Machen TE, Miller SS. ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia. Am J Physiol Cell Physiol 281: C633–C648, 2001.
6. Blaug S, Rymer J, Jalickee S, Miller SS. P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia. Am J Physiol Cell Physiol 284: C897–C909, 2003.
7. Bleich M, Briel M, Busch AE, Lang HJ, Gerlach U, Gogelein H, Greger R, Kunzelmann K. KVLQT channels are inhibited by the K+ channel blocker 293B. Pflügers Arch 434: 499–501, 1997.[CrossRef][Web of Science][Medline]
8. Bundred NJ, West RR, Dowd JO, Mansel RE, Hughes LE. Is there an increased risk of breast cancer in women who have had a breast cyst aspirated? Br J Cancer 64: 953–955, 1991.[Web of Science][Medline]
9. Coiret G, Matifat F, Hague F, Ouadid-Ahidouch H. 17-Beta-estradiol activates maxi-K channels through a non-genomic pathway in human breast cancer cells. FEBS Lett 579: 2995–3000, 2005.[CrossRef][Web of Science][Medline]
10. Cowley EA, Linsdell P. Characterization of basolateral K+ channels underlying anion secretion in the human airway cell line Calu-3. J Physiol 538: 747–757, 2002.
11. Cowley EA, Linsdell P, Osmond A. Dexamethasone-enhanced sodium absorption in the human mammary epithelial cell line, MCF-7 (Abstract). FASEB J 20: A794, 2006.
12. Dogliotti L, Orlandi F, Caraci P, Puligheddu B, Torta N, Angeli A. Biochemistry of breast cyst fluid. An approach to understanding intercellular communication in the terminal duct lobular units. Ann NY Acad Sci 586: 17–28, 1990.[Web of Science][Medline]
13. Dupont J, Le Roith D. Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects. Mol Pathol 54: 149–154, 2001.
14. Ebbs SR, Bates T. Breast cyst type does not predict the natural history of cystic disease or breast cancer. Br J Surg 75: 702–704, 1988.[CrossRef][Web of Science][Medline]
15. Farooqui M, Geng ZH, Stephenson EJ, Zaveri N, Yee D, Gupta K. Naloxone acts as an antagonist of estrogen receptor activity in MCF-7 cells. Mol Cancer Ther 5: 611–620, 2006.
16. Flezar M, Heisler S. P2-purinergic receptors in human breast cancer tumor cells: coupling of intracellular calcium signaling to anion secretion. J Pharmacol Exp Ther 265: 1499–1510, 1993.
17. Grahammer F, Warth R, Barhanin J, Bleich M, Hug MJ. The small conductance K+ channel, KCNQ1: expression, function, and subunit composition in murine trachea. J Biol Chem 276: 42268–42275, 2001.
18. Grunnet M, Jespersen T, MacAulay N, Jorgensen NK, Schmitt N, Pongs O, Olesen SP, Klaerke DA. KCNQ1 channels sense small changes in cell volume. J Physiol 549: 419–427, 2003.
19. Heitzmann D, Grahammer F, van Hahn T, Schmitt-Graff A, Romeo E, Nitschke R, Gerlach U, Lang HJ, Verrey F, Barhanin J, Warth R. Heteromeric KCNE2/KCNQ1 potassium channels in the luminal membrane of gastric parietal cells. J Physiol 561: 547–557, 2004.
20. Jentsch TJ. Neuronal KCNQ potassium channels: physiology and role in disease. Nat Rev Neurosci 1: 21–30, 2000.[CrossRef][Web of Science][Medline]
21. Jiang M, Tseng-Crank J, Tseng GN. Suppression of slow delayed rectified current by a truncated isoform of KvLQT1 cloned from normal human heart. J Biol Chem 272: 24109–24112,1997.
22. Kunzelmann K, Hubner M, Schreiber R, Levy-Holzman R, Garty H, Bleich M, Warth R, Slavik M, von Hahn T, Greger R. Cloning and function of the rat colonic epithelial K+ channel KVLQT1. J Membr Biol 179: 155–164, 2001.[CrossRef][Web of Science][Medline]
23. Lan WZ, Abbas H, Lemay AM, Briggs MM, Hill CE. Electrophysiological and molecular identification of hepatocellular volume-activated K+ channels. Biochim Biophys Acta 1668: 223–233, 2005.[Medline]
24. Lan WZ, Wang PY, Hill CE. Modulation of hepatocellular swelling-activated K+ currents by phosphoinositide pathway-dependent protein kinase C. Am J Physiol Cell Physiol 291: C93–C103, 2006.
25. Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, Haussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247–306, 1998.
26. Linsdell P, Gong X. Multiple inhibitory effects of Au(CN)(2-) ions on cystic fibrosis transmembrane conductance regulator Cl(-) channel currents. J Physiol 540: 29–38, 2002.
27. Linzell JL, Peaker M. Intracellular concentrations of sodium, potassium and chloride in the lactating mammary gland and their relation to the secretory mechanism. J Physiol 216: 683–700, 1971.
28. Lock H, Valverde MA. Contribution of the IsK (MinK) potassium channel subunit to regulatory volume decrease in murine tracheal epithelial cells. J Biol Chem 275: 34849–34852, 2000.
29. MacVinish LJ, Guo Y, Dixon AK, Murrell-Lagnado RD, Cuthbert AW. XE991 reveals differences in K+ channels regulating chloride secretion in murine airway and colonic epithelium. Mol Pharmacol 60: 753–760, 2001.
30. Mall M, Wissner A, Schreiber R, Kuehr J, Seydewitz HH, Brandis M, Greger R, Kunzelmann K. Role of K(V)LQT1 in cyclic adenosine monophosphate-mediated Cl– secretion in human airway epithelia. Am J Respir Cell Mol Biol 23: 283–289, 2000.
31. Mannello F, Malatetesta M, Gazzanelli G. Breast cancer in women with palpable breast cysts. Lancet 354: 677–678, 1999.[Web of Science][Medline]
32. Mannello F, Tonti GAM, Papa S. Human gross cyst breast disease and cystic fluid: bio-molecular, morphological, and clinical studies. Breast Cancer Res Treat 97: 115–129, 2006.[CrossRef][Web of Science][Medline]
33. Melman YF, Domemech A, de la Luna S, McDonald TV. Structural determinants of KvLQT1 control by the KCNE family of proteins. J Biol Chem 276: 6439–6444, 2001.
34. Millar ID, Hartley JA, Haigh C, Grace AA, While SJ, Kibble JD, Robson L. Volume regulation is defective in renal proximal tubule cells isolated from KCNE1 knockout mice. Exp Physiol 89: 173–180, 2004.
35. Missan S, Linsdell P, McDonald TF. Role of kinases and G-proteins in the hyposmotic stimulation of cardiac I(Ks). Biochim Biophys Acta 1758: 1641–1652, 2006.[Medline]
36. Moss AJ, Kass RS. Long QT syndrome: from channels to cardiac arrhythmias. J Clin Invest 115: 2018–2024, 2005.[CrossRef][Web of Science][Medline]
37. Neville MC. The physiological basis of milk secretion. Ann NY Acad Sci 586: 1–11, 1990.[CrossRef][Web of Science][Medline]
38. Nicolas M, Dememes D, Martin A, Kuperschmid S, Barhanin J. KCNQ1/KCNE1 potassium channels in mammalian vestibular dark cells. Hear Res 153: 132–145, 2001.[CrossRef][Web of Science][Medline]
39. Ouadid-Ahidouch H, Chaussage F, Roudbaraki M, Slomianny C, Dewailly E, Delcourt P, Prevarskaya N. KV1.1 K(+) channels identification in human breast carcinoma cells: involvement in cell proliferation. Biochem Biophys Res Commun 278: 272–277, 2000.[CrossRef][Web of Science][Medline]
40. Ouadid-Ahidouch H, Roudbaraki M, Delcourt P, Ahidouch A, Joury N, Prevarskaya N. Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression. Am J Physiol Cell Physiol 287: C125–C134, 2004.
41. Quesnell RR, Erickson J, Schultz BD. Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium. Am J Physiol Cell Physiol 292: C305–C318, 2007.
42. Robbins J. KCNQ potassium channels: physiology, pathophysiology, and pharmacology. Pharmacol Ther 90: 1–19, 2001.[CrossRef][Web of Science][Medline]
43. Roepke TK, Anantharam A, Kirchoff P, Busque SM, Young JB, Geibel JP, Lerner DJ, Abbott GW. The KCNE2 potassium channel ancillary subunit is essential for gastric acid secretion. J Biol Chem 281: 23740–23747, 2006.
44. Sanguinetti MC, Curran ME, Zou A, Shen J, Spector PS, Atkinson DL, Keating MT. Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel. Nature 384: 80–83, 1996.[CrossRef][Medline]
45. Shennan DB, Grant ACG, Gow IF. The effect of hyposmotic and isomotic cell swelling on the intracellular [Ca2+] in lactating rat mammary acinal cells. Mol Cell Biochem 233: 91–97, 2002.[CrossRef][Web of Science][Medline]
46. Shennan DB, Gow IF. Volume-activated K+ (Rb+) efflux in lactating rat mammary tissue. Biochim Biophys Acta 1509: 420–428, 2000.[Medline]
47. Shennan DB, Peaker M. Transport of milk constituents by the mammary gland. Physiol Rev 80: 925–951, 2000.
48. Soule HD, Vazquez J, Long A, Albert S, Brennan M. A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 51: 1409–1416, 1973.[Web of Science][Medline]
49. Tinel N, Diochot S, Borsotto M, Lazdunski M, Barhanin J. KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel. EMBO J 19: 6326–6330, 2000.[CrossRef][Web of Science][Medline]
50. Wang HS, Brown BS, McKinnon D, Cohen I. Molecular basis for differential sensitivity of KCNQ and I(Ks) channels to the cognitive enhancer XE991. Mol Pharmacol 57: 1218–1222, 2000.
51. Warth R, Garcia Alzamora M, Kim JK, Zdebik A, Nitschke R, Bleich M, Gerlach U, Barhanin J, Kim SJ. The role of KCNQ1/KCNE1 K(+) channels in intestine and pancreas: lessons from the KCNE1 knockout mouse. Pflügers Arch 443: 822–888, 2002.[CrossRef][Web of Science][Medline]
52. Zheng W, Verlander JW, Lynch IJ, Cash MN, Shao J, Stow LR, Cain BD, Weiner ID, Wall SM, Wingo CS. Cellular distribution of the potassium channel, KCNQ1, in normal mouse kidney. Am J Physiol Renal Physiol 292: F456–F466, 2007.
This article has been cited by other articles:
|
|
 |
|
  K. M. Boini, D. Graf, A. M. Hennige, S. Koka, D. S. Kempe, K. Wang, T. F. Ackermann, M. Foller, V. Vallon, K. Pfeifer, et al. Enhanced insulin sensitivity of gene-targeted mice lacking functional KCNQ1 Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2009; 296(6): R1695 - R1701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. K. Hoffmann, I. H. Lambert, and S. F. Pedersen Physiology of Cell Volume Regulation in Vertebrates Physiol Rev, January 1, 2009; 89(1): 193 - 277. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
, control current; 
, current under hyposmotic conditions; 
, current in the presence of 50 µM 293B). C: exposure of BHK cells pretreated with 50 µM 293B to hyposmotic solution did not cause a significant increase in current amplitude (