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(The FASEB Journal. 2007;21:35-44.)
© 2007 FASEB

Voltage-gated K+ channels support proliferation of colonic carcinoma cells

Melanie Spitzner, Jiraporn Ousingsawat, Kerstin Scheidt, Karl Kunzelmann1,2 and Rainer Schreiber1

Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, Regensburg, Germany

2Correspondence: Institut für Physiologie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany. E-mail: uqkkunze{at}mailbox.uq.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasma membrane potassium (K+) channels are required for cell proliferation. Evidence is growing that K+ channels play a central role in the development and growth of human cancer. Here we examine the contribution and the mechanism by which K+ channels control proliferation of T84 human colonic carcinoma cells. Numerous K+ channels are expressed in T84 cells, but only voltage-gated K+ (Kv) channels influenced proliferation. A number of Kv channel inhibitors reduced DNA synthesis and cell number, without exerting apoptotic or toxic effects. Expression of several Kv channels, such as EagI, Kv 3.4 and Kv 1.5, was detected in patch clamp experiments and in fluorescence-based assays using a voltage sensitive dye. The contribution of EagI channels to proliferation was confirmed by siRNA, which abolished EagI activity and inhibited cell growth. Inhibition of Kv channels did not interfere with the ability of T84 cells to regulate their cell vol, but it restricted intracellular pH regulation. In addition, inhibitors of Kv channels, as well as siRNA for EagI, attenuated intracellular Ca2+ signaling. The data suggest that Kv channels control proliferation of colonic cancer cells by affecting intracellular pH and Ca2+ signaling.—Spitzner, M., Ousingsawat, J., Scheidt, K., Kunzelmann, K., Schreiber, R. Voltage-gated K+ channels support proliferation of colonic carcinoma cells.


Key Words: Eag • T84 • intracellular Ca2+ • growth • Ussing chamber • patch clamp • intracellular pH • ion channels • cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
APART FROM THEIR SPECIFIC epithelial transport function, membrane ion channels are essential for maintaining cellular homeostasis and signaling. Thus, they contribute to the control of essential parameters such as cell vol, intracellular pH, and intracellular Ca2+ concentration. K+ selective ion channels form the largest ion channel protein family, which may be subdivided into voltage gated, Ca2+ dependent, 2-pore domain, and inward rectifier K+ channels. It has been suggested recently that in addition to the physiological parameters controlled by K+ channels, they also affect mitotic cell cycling, proliferation, and development of cancer. A role of K+ channels for proliferation and tumor cell growth has been demonstrated for a number of carcinoma, like those of prostate, colon, lung, breast, and others (41) . K+ channels of different subfamilies have been correlated with tumor proliferation, including Ca2+-activated K+ channels, Shaker-type voltage-gated K+ channels, the ether-a-go-go (EagI) family of voltage-gated K+ channels, and the 2P-domain K+ channels (27 , 28 , 41 , 42) . It is not known currently why different types of K+ channels induce proliferation in the different cancer models. Most of the previous studies describe expression of a particular type of K+ channel in a cancer cell line and correlate expression and functional activity of the channel with proliferation. However, mammalian cells always express a whole array of various types of K+ channels, and it is not clear to what degree these individual K+ channels contribute to proliferation, or whether a specific association exists between particular K+ channel subtypes and proliferation. Moreover, the mechanisms by which these K+ channels facilitate cell proliferation remain obscure.

Colonic carcinomas belong to the most frequent cancers in human. A previous study found a correlation between the activity of voltage-gated K+ channels and proliferation and metastasis of a colonic cancer cell line and native colonic cancers, respectively (2 , 24) . Interestingly Ca2+ influx into colonic cancer cells was affected by voltage-gated K+ channels in one study (2) . Identification of K+ channels, which are relevant for the growth of colonic carcinomas, may provide novel therapeutic targets (9 , 36) . Moreover, K+ channels abnormally expressed in colonic cancer cells have the potential to serve as useful as markers for malign transformation. In the present study, we screened T84 colonic carcinoma cells for expression of a broad spectrum of K+ channels. We then tried to identify the K+ channels that have an impact on proliferation of these cells. Using a variety of K+ channel blockers and siRNA, we found that voltage-gated K+ channels take part in controlling cell proliferation, while other K+ channel subtypes do not. A further aim of this study was to uncover how these channels affect proliferation. The data suggest that voltage-gated K+ channels control basic cellular properties like intracellular Ca2+, elicited by agonist stimulation of the cells, and regulation of the intracellular pH. Both effects are likely to be due to the hyperpolarizing effect of voltage-gated channels on the membrane voltage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and proliferation studies
Human colorectal carcinoma epithelial T84 cells (American Type Culture Collection, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle medium (DMEM)/Ham’s F-12 medium (1:1), supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Pen/Strep) (GIBCO, Karlsruhe, Germany) in a 5% CO2 atmosphere at 37°C. Cells were seeded on bovine plasma fibronectin (Invitrogen, Karlsruhe, Germany) and bovine dermal collagen-coated (Cellon, Bereldange, Luxembourg) plastic dishes or glass coverslips. For proliferation assays, T84 cells were plated at a density of 2000 cells/0.35 cm2 on coated 96-well plates (Sarstedt, Nürnberg, Germany). After 2 d, cells were incubated with inhibitors of potassium channels dissolved in serum-reduced Opti-MEM 1 medium (GIBCO). After another 2 d, cell proliferation was assessed by 5-bromo-2'-deoxyuridine (bromodeoxyuridine) incorporation and cell counting. Bromodeoxyuridine (BrdU) incorporation was determined using an immunoassay cell proliferation ELISA kit (Roche, Mannheim, Germany), according to the manufacturer’s protocol. For cell counting, cells were fixed with 3.7% formaldehyde and 0.5% Triton X-100 in ringer solution [mmol/l: NaCl 145; KH2PO4 0.4; K2HPO4 1.6; Glucose (Glc) 5; MgCl2 1; Ca2+-Gluconat 1.3, pH 7.4] for 30 min and stained with Mayers Hemalaun (Merck, Darmstadt, Germany). Digital images were obtained, and nuclei were counted using imaging software. Toxicity of the blockers was assessed by counting nonstained dead cells in the supernatants (trypan blue). Each experiment was performed in triplicate.

Expression of potassium channels in T84 cells and real-time polymerase chain reaction (PCR) analysis
Total RNA was isolated from T84 cells using NucleoSpin RNA II columns (Macherey-Nagel, Düren, Germany). Total RNA (1 µg) was reverse-transcribed for 1 h at 37°C using random primer and RT (M-MLV Reverse Transcriptase, Promega, Mannheim, Germany). Reverse-transcriptase PCR (RT-PCR) was used to detect expression of the mRNAs for potassium channels. The oligonucleotide primers were designed for the mRNA of each gene product (name, gene, accession number: sense and antisense primer, size of PCR product): Kv1.3, KCNA3, NM 002232: 5'-GCCATCCTCTACTACTATCAG-3', 5'-CCACAATGTCGATCAGGTTC-3', 532 bp; Kv1.5, KCNA5, NM 002234: 5'-CTACTTCGACCCCCTGAG-3', 5'-GCTCGAAGGTGAACCAGATG-3', 555 bp; KvLQT, KCNQ1, NM000218: 5'-CCACGGGGACTCTCTTCTG-3', 5'-GGCACCTTGTCCCCATAG-3', 504 bp; Kv3.3/Kv3.4, KCNC3/KCNC4, NM 004977: 5'-GACGTGCTGGGCTTCCTG-3', 5'-GCTTCTGCTTGGCCATGG-3', 442 bp; Kv4.1, KCND1, NM004979: 5'- GGAAGAATACGCTGGACCG-3', 5'-GCGGCATGGGATGGTCTC-3', 472 bp; Kv5.1, KCNF1, NM 002236: 5'-GAGTCGTCGTGCCCGGC-3', 5'-GTCTCTGGATGGCTCTGCTC-3', 530 bp; Kv9.3, KCNS3, NM 002252: 5'-GTGCAGCCTGATCTTCCTC-3', 5'-GTGTCAAACTTCTCCAGC hpTC-3', 540 bp; EagI, KCNH1, NM 002238: 5'-GCATGAACTACCTGAAGACG-3', 5'-CTTCTCAATGTCTGTGGATGG-3', 488 bp; ERG1, KCNH2, NM 000238: 5'-CAATGCCAACGAGGAGGTG-3', 5'-GGAGAGACGTTGCCGAAG-3, 468 bp; ELK1, KCNH8, NM 144633: 5'-GCCCGAACTGAAGTCATGC-3', 5'-CAAAATAAGCCAGTCCCAGC-3', 522 bp; Kir6.1, KCNJ8, NM 004982: 5'-GTTTGGAGTCCACTGTGTGTG-3', 5'-CAGAATAACTATGACCTCCAAG-3', 548 bp; TWIK1, KCNK1, NM 002245: 5'-GCTTCGGCTTCCTGGTGC-3', 5'-GACAAACCCAAGGAGCACG-3', 505 bp; TASK2, KCNK5, NM009740: 5'-GGCCATCACAGGGAACCAG-3', 5'-GCCAGGCCAGCCCCAAG-3', 496 bp; TWIK2, KCNK6, NM 004823: 5'-GATCTTTGCCCACCTCGAG-3', 5'-GATGCTGGGAAACAAAGGAG-3', 520 bp; BKCa channel ß-subunit, KCNMA1, NM 002247: 5'-GTGGATGAAAAAGAGGAGGC-3', 5'-CAAATGGATGAACCCGGCTG-3', 528 bp; BKCa channel ß-subunit, KCNMB3, NM 014407: 5'- CGACCTGCACTGCCATCC-3', 5'-GGGCACCACCTAGCAGAG-3', 398 bp; BKCa channel ß-subunit, KCNMB4, NM 014505: 5'- CCAGGTCTACGTGAACAAC-3', 5'-CTGTTGCCACTGAGGGATG-3', 520 bp; SK4, KCNN4, NM 002250: 5'- GATTTAGGGGCGCCGCTGAC-3', 5'-CTTGCCCCACATGGTGCCC-3', 520 bp. The PCR reactions were performed at 94°C for 2 min, 35 cycles at 94°C for 30 s, annealing temperature 60°C for 30 s, and 72°C for 1 min. PCR products were visualized by loading on agarose gels and were verified by sequencing.

Real-time PCR was performed in a Light Cycler (Roche), using the Quanti Tect SYBR Green PCR Kit (Qiagen, Hilden, Germany). Each reaction contained 2 µl Master Mix (including Taq polymerase, DNTPs, SYBR green buffer), 1 pM of each primer (EagI: 5'-GGAGTTCCAGACGGTGCAC-3', 5'-CCTCATCATCTTGGATCACC-3', 116 bp; ß-actin, ACTB, NM 001101: 5'-CAACGGCTCCGGCATGTG-3', 5'-CTTGCTCTGGGCCTCGTC-3', 151 bp), 2.5 mM MgCl2, and 2 µl cDNA. After 10 min at 94°C for activation of Taq polymerase, cDNA was amplified by 15 s at 94°C, 10 sat 60°C, and 20 s at 72°C, for 50 cycles. The amplification was followed by a melting curve analysis to control of the PCR products. As negative controls, water instead of cDNA was run with every PCR experiment. To verify accuracy of the amplification, PCR products were further analyzed on ethidium bromide-stained 2% agarose gels. Analysis of the data was performed using Light Cycler software 3.5.3. Standard curves for EagI channel mRNA and ß-actin mRNA were produced by using cDNA of 16HBE cells at different dilutions. The ratio of the amount of EagI to ß-actin mRNA was calculated for each sample.

Down-regulation of EagI expression by small interfering RNA
Duplexes of 21-nucleotide siRNA with 3'-overhanging TT were designed according to Ahn et al. 2003 (4) and synthesized by IBA (Göttingen, Germany). The sense strand of the siRNA used to silence the EagI gene was 5'-GACACGAUUGAA AAAGUGCTT-3', corresponding to positions 268–286 of the EagI mRNA, relative to the start codon. The sense strand of the siRNA used to silence the potassium channel sk4 gene was 5'-GCGCUU GCUGGAGCAGGAGTT-3', corresponding to positions 48–66 of the sk4 mRNA relative to the start codon. A control siRNA oligonucleotide, Lmo2, designed to silence the LIM domain only 2 T cell oncogene (Mus musculus), has no target gene in T84 cells and was used as a negative control for transfection. It had the sequence 5'-GCC AUC GAC CAG UAC UGG CTT-3' (positions 133–151 relative to the start codon) of the Lmo2 gene. Transfection of T84 cells was performed in Opti-MEM 1, 1 day after seeding using Oligofectamine (Invitrogen, Karlsruhe, Germany). After a 48 h rest, T84 cells were used for proliferation assays, patch clamp, real-time PCR, and Western blot.

Detection of EagI protein by Western blot
Lysates of T84 cells were resolved by 7% SDS-PAGE, transferred to Hybond-P (Amersham Biosiences, Munich, Germany), and incubated with mouse anti-Kv10.1 (EagI) antibodies (Alomone Labs, Jerusalem, Israel). Proteins were visualized using a goat anti-mouse IgG conjugated to horseradish peroxidase (Acris Antibodies, Herford, Germany) and enhanced chemiluminescence (ECL) Advance Detection Kit (Amersham Biosiences).

Measurement of the intracellular Ca2+ concentration, pH, cell vol, and membrane potential
T84 cells were loaded with 2 µM Fura-2 acetoxymethyl ester (AM) (Molecular Probes, Eugene, OR, USA) in Opti-MEM 1 medium with 2.5 mM probenecid (Sigma, Deisenhofen, Germany) for 1 h at room temperature. For fluorescence measurements of the intracellular Ca2+ concentration [Ca2+]i, cells were perfused with ringer solution containing 2.5 mM probenecide (Sigma) at 37°C. Fluorescence was measured continuously using an inverted microscope IMT-2 (Olympus, Nürnberg, Germany) and a high-speed polychromator system (VisiChrome, Visitron Systems, Puchheim, Germany). Fura-2 was excited at 340/380 nm, and emission were recorded between 470 and 550 nm using a CCD camera (CoolSnap HQ, Visitron Systems). [Ca2+]i was calculated from the ratio of 340/380 nm fluorescence values (after subtraction of background fluorescence). The formula used for calculation of [Ca2+]i was [Ca2+]i = Kd x (R-Rmin)(Rmax–R) x (Sf2/Sb2), where R is the observed fluorescence ratio. The values Rmax, Rmin (maximum and minimum ratios), and the constant Sf2/Sb2 (fluorescence of free and Ca2+-bound Fura-2 at 380 nm) were calculated using 2 µmol/l ionomycin (free acid; Calbiochem, Merck Biosciences, Bad Soden, Germany), 5 µmol/l nigericin (Sigma), 10 µmol/l monensin (Sigma) and 5 mmol/l EGTA to equilibrate intracellular and extracellular Ca2+ in intact Fura-2-loaded cells. The dissociation constant for the Fura-2-Ca2+ complex was taken as 224 nmol/l.

For assessment of intracellular pH, cells were incubated in standard bath solution (mM: 145 NaCl, 0.4 KH2PO4, 1.6 K2HPO4, 5 Glc, 1 MgCl2, 1.3 Ca-gluconate, pH 7.4) containing 8 µM BCECF-AM and 0.05% Pluronic for 30–60 min at room temperature (RT). For cell vol measurements, M1 cells were loaded with 2.5 µM calcein-AM and 0.025% Pluronic in standard bath solution for 30–60 min at RT. Excitation wavelengths of 488/440 and 500 nm were used for BCECF and calcein-loaded cells, respectively. Emission wavelengths were >520 nm for BCECF fluorescence and 520–550 nm for calcein fluorescence. We screened for the effect of K+ channel blockers on the membrane voltage using the voltage-sensitive dye FMP (6) . Cells were incubated with FMP (FLIPR Membrane Potenial Assay Kit; Molecular Devices, Ismaning, Germany) in ringer solution at room temperature. FLIPR was exited with 485 nm and emission was detected between 515 and 560 nm. All experiments were controlled and analyzed using the software package Meta-Fluor (Universal Imaging, Afton, NY, USA). All optical filters and dichroic mirrors were from AHF (Tübingen, Germany).

Patch clamp
Cell culture dishes were mounted on the stage of an inverted microscope (IM35, München, Zeiss) and kept at 37°C. The bath was perfused continuously with Ringer solution at ~10 ml/min. Patch clamp experiments were performed in the fast whole-cell configuration. Patch pipettes had an input resistance of 2–4 M{Omega} when filled with a solution containing (mM) KCl 30, K-gluconate 95, NaH2PO4 1.2, Na2HPO4 4.8, EGTA 1, Ca-gluconate 0.758, MgCl2 1.034, D-glucose 5, ATP 3. pH was adjusted to 7.2, the Ca2+ activity was 0.1 µM. The access resistance was measured continuously during the recordings and was 8.3–14.2 M{Omega}. Currents (voltage clamp) and voltages (current clamp) were recorded using a patch clamp amplifier (EPC 7, List Medical Electronics, Darnstadt, Germany), the LH1600 interface and PULSE (HEKA, Lambrecht/Pfalz, Germany) and Chart (AD-Instruments, Spechbach, Germany) software. In regular intervals, membrane voltages (Vc) were clamped in steps of 10 mV from –50 to +50 mV relative to resting potentials. The membrane conductance Gm was calculated from the measured current (I) and Vc values, according to Ohm’s law.

Materials and statistical analysis
All compounds used were of highest available grade of purity. 4-aminopyridine (4-AP), TPeA, astemizole, calciseptine, EIPA, carbachol, quinidine, and terfenadine were all from Sigma. BDS-I, charybdotoxin, and iberiotoxin were from Alamone Labs. Clotrimazole was from Calbiochem (Merck Biosciences). Scyllatoxin was from Latoxan (Valence, France). TRAM-34 was a generous gift by Dr. H. Wulff (Department of Medical Pharmacology and Toxicology, University of California Davis, San Francisco, USA). 293B, AVE0118, and AVE1231 were gifts from Aventis Pharma (Frankfurt, Germany). All other chemicals were obtained from Merck. The acetomethyl ester [2',7')-bis(carboxyethyl)-5 (6)-carboxyfluorescein (BCECF-AM)], calcein (calcein-AM), Fura-2-AM, and Pluronic were all from Molecular Probes. Student’s t test (for paired or unpaired samples as appropriate) and ANOVA was used for statistical analysis. P < 0.05 was accepted as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of cancer-related K+ channels in T84 colonic carcinoma cells
We broadly screened T84 cells for expression of K+ channels based on previous reports, which indicated a role of K+ channels for proliferation of cancer cells [database SAGEmap of the Cancer Genome Anatomy Project, http://cgap.nci.nih.gov, (22) and recent publications]. RT-PCR detected transcripts of a number of voltage-gated channels (Fig. 1 A). Moreover, the ATP-sensitive K+ channels KIR6.1 and the two-pore (2P)-domain K+ channels TWIK1, TWIK2, and TASK2 are expressed in T84 cells (Fig. 1B ). Although, transcripts of the {alpha}-subunit of the large conductance Ca2+-sensitive (BK) channel could not be detected, the two ß-subunits ß3 and ß4 are expressed in these cells, along with intermediate conductance Ca2+-activated SK4 K+ channels (Fig. 1C ). Taken together, several K+ channels are expressed in T84 colonic carcinoma cells, which could potentially support cell proliferation.


Figure 1
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  		Figure 1. Expression of K+ channels in T84 cells.

Expression of (A) voltage-gated (Kv3.3/3.4, Kv4.1, Kv5.1, Elk1, KvLQT1, EagI, Kv9.3), (B) Inward rectifier (Kir6.1), two-pore domain (TWIK1, TASK2, TWIK2), and (C) large (BKCaß 3, BKCaß 4) and small (SK4) conductance Ca2+-activated K+ channels, after reverse transcription (RT).

Inhibition of T84 cell proliferation by blockage of Kv channels
In order to identify the K+ channels that affect cell proliferation, we used a variety of inhibitors (Table 1 , Figs. 2 , 3 ). Cell proliferation was assessed independently by quantifying DNA replication (bromodeoxyuridine incorporation) and by cell counting. Potential toxicity of the K+ channel blockers was assessed by trypan blue staining. At the concentrations used in this study, the channel inhibitors caused no toxic effects on T84 cells. Concentration-dependent inhibition of cell proliferation was found for the nonselective Kv channel blockers TPeA and Quinidine (17 , 31) ; for the toxin inhibitor BDS-1, which blocks Kv3.4 channels (11) ; and for the EagI blocker terfenadine (16) (Fig. 2A-D ). Other Kv inhibitors, such as 4-AP (5 µM–2 mM), AVE0118 or AVE1231 (0.5 nM–50 µM), and the KvLQT blocker 293B (1 nM–10 µM) also inhibited cell proliferation, albeit at higher concentrations (Table 1) . Inhibitors of Ca2+-activated K+ channels, such as charybdotoxin, clotrimazole, iberiotoxin, scyllatoxin, and TRAM-34 did not affect proliferation (Table 1) . Thus, Ca2+-activated K+ channels do not seem to support proliferation. Surprisingly, activation of SK4 channels by riluzole (7) enhanced proliferation (Fig. 2E ). These experiments suggest that only endogenous Kv channel activity supports proliferation of T84 colonic carcinoma cells; however, when up-regulated other K+ channels such as intermediate conductance SK4 channels may also enhance proliferation. Endogenous Kv channels not only support proliferation of T84 cells but also those of other colonic cell lines, like HT29. Proliferation of HT29 cells was inhibited between 60–80% (n=4) by 4-AP protein and astemizole. Moreover, Kv channels have been identified recently in human and murine colonic cancers (unpublished observations).


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  		Table 1. Inhibition of T84 cell proliferation by K+ channel blocker


Figure 2
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  		Figure 2. Inhibition of proliferation of T84 cells by Kv channel blockers. Cell proliferation of T84 cells was assessed by BrdU incorporation and cell counting. The K+ channel blockers (A) TPeA, (B) terfenadine, (C) BDS-I, and (D) quinidine caused dose-dependent inhibition of proliferation. E) Additional activation of SK4 channels by riluzole further enhanced proliferation. Asterisks indicate significant differences when compared with control (ANOVA). Experiments were carried out in triplicate (number of experiments).


Figure 3
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  		Figure 3. Effects of Kv channel blockers on membrane voltage whole cell conductance. A) Uptake of the voltage-sensitive fluorescence dye FMP. Loading of T84 cells with FMP induced fluorescence. 4-AP (50 µM) further increased fluorescence intensity due to depolarization of Vm. BaCl2 (5 mM) completely inhibited K+ channels, thereby inducing maximal depolarization and further fluorescence increase. The right panel shows the summary of the effects of the K+ channel blockers TPeA (50 µM), quinidine (10 µM), terfenadine (10 µM), astemizole (5 µM), 4-AP (50 µM), 293B (10 µM), TRAM-34 (100 nM), and clotrimazole (100 nM) on FMP fluorescence, relative to the effects of BaCl2. B) Effect of TPeA and terfenadine on whole cell currents obtained in T84 cells and summary i/v curves in the absence or presence of the inhibitor. C) Summary of the effects of the K+ channel blockers on whole cell conductance and membrane voltage at concentrations as indicated above. Concentrations for the other inhibitors were BDS-I (10 nM), charybdotoxin (100 nM), iberiotoxin (10 nM), AVE0118 (10 µM), and AVE1231 (10 µM). IC50 values were determined experimentally (in bold) or were obtained from literature. Asterisks indicate significant differences when compared to control (paired Student’s t test) (number of experiments).

Kv channels are active in colonic carcinoma cells
We screened for the effects of K+ channel blockers in a fluorescence-based assay, using the voltage-sensitive dye FMP. After loading of the cells with FMP, the Kv channel blocker 4-AP (50 µM) enhanced fluorescence intensity due to depolarization of the membrane voltage. The effects of individual blockers were analyzed as a fraction of the effect of BaCl2 (5 mM), which induced maximal depolarization. The largest changes were observed for the Kv channel blockers 4-AP (50 µM), quinidine (10 µM), terfenadine (10 µM), and astemizole (5 µM). This finding demonstrates a contribution of Kv channels to the overall K+ conductance in T84 cells (Fig. 3A , right panel). K+ channel blockers were also applied in whole cell patch clamp experiments. The blockers were used at lower concentration range in order to achieve best specificity. As shown in Fig. 3B, C , K+ channel blockers had small but significant effects on whole cell conductance and membrane voltage (Vm). Blockers were typically applied in the presence of Ringer solution; however, similar effects were also observed when blockers were examined in the presence of culture medium (Opti-MEM), i.e., under proliferation assay conditions (data not shown). Taken together, Kv channels contribute only ~10–15% to the total membrane conductance, which is sufficient to support proliferation of T84 cells. Similar has been observed in previous studies (21) .

Down-regulation of EagI inhibits proliferation
Proliferation of T84 cells may be supported by a number of different Kv channels. Although patch clamping and expression studies suggested a dominant role of Eag channels, we used siRNA-EagI to specifically down-regulate expression of EagI protein. Quantitative analysis of gene expression using real-time PCR indicated a significant loss of EagI transcripts after treatment with siRNA-EagI, and reduced EagI expression was verified by Western blotting (Fig. 4 A). We found that down-regulation of EagI expression inhibited proliferation, while mock transfection, unrelated siRNA (for Lmo2 oncogene), or siRNA inhibition of the Ca2+-activated K+ channel SK4 did not reduce cell proliferation (Fig. 4B ). In the FMP fluorescence assay we found a reduced effect of astemizole (5 µM) on membrane voltage, i.e., FMP uptake ({Delta} FMP) (Fig. 4C ). Along this line, suppression of astemizole-sensitive EagI conductance was detected in whole cell patch clamp experiments. In contrast TRAM-34 sensitive SK4 conductance was eliminated by siRNA-SK4, which had no impact on proliferation of T84 cells (Fig. 4D ). Taken together, endogenous EagI currents control proliferation of T84 colonic carcinoma cells, while endogenous SK4 currents are not relevant for cell growth.


Figure 4
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  		Figure 4. Down-regulation of EagI inhibits cell proliferation and astemizole sensitive currents. A) Suppression of EagI-mRNA and protein in T84 cells by siRNA-EagI but not by unrelated siRNA (Lmo2; SK4), or in mock-transfected cells. B) Inhibition of cell proliferation by siRNA-EagI but not by unrelated control siRNA. C) Reduced astemizole-sensitive FMP fluorescence ({Delta} FMP) in siRNA-EagI transfected cells but not in control cells. D) siRNA-EagI abolished astemizole-sensitive Gm (left panel). In contrast, siRNA-SK4 abolished TRAM-34 sensitive Gm (right panel), which, however, did not affect proliferation of T84 cells (shown in B). Asterisks indicate significant difference when compared to control (ANOVA, Student’s t test). Proliferation assays were carried out in triplicates (number of experiments).

Kv channels control regulation of intracellular pH and Ca2+ signaling but not cell volume
Kv channels contribute to the negative membrane voltage, which may be crucial for vol and pH regulation, as well as proper Ca2+ signaling particularly during cell cycling (18 , 21 , 39 , 42) . We therefore asked whether Kv channels enhance proliferation by affecting cell vol or pH regulation, or by supporting intracellular Ca2+ signaling. T84 cells were swollen by removing 120 mmol/l mannitol (Hypo) or were shrunken by adding 120 mmol/l mannitol (Hyper) to an isotonic Ringer solution. Swelling and shrinkage were assessed by changes in calcein fluorescence (Fig. 5 A). Repetitive exposure to hypotonic bath solution produced identical changes in cell vol (n=41, data not shown). Cell swelling did not differ in the presence or absence of Kv channel blockers (Fig. 5B, C ). We further examined whether pH recovery from an acid (NH4Cl; 20 mM) load and thus regulation (recovery) of intracellular pH is affected by inhibitors of Kv channels. To that end, cells were acid loaded using a NH4Cl pulse. The slope for initially recovery from cellular acidification was determined in the absence or presence of Kv channel blockers. Repetitive acidification under control condition showed similar recovery of the intracellular pH (n=62, data not shown). Recordings of the intracellular pH and the summary for the recovery rates demonstrate impaired pH regulation in T84 cells when exposed to the Kv channel blockers 4-AP or astemizole (Fig. 5D, E ). These results indicate that Kv channels are important for pH regulation in colonic carcinoma cells. pH recovery is primarily due to the function of the Na+/H+ exchanger NHE (21 , 37) . In fact, blockage of NHE by 5-(N-ethyl-N-Isopropyl) amiloride (EIPA) inhibited proliferation of T84 cells (Fig. 5F ).


Figure 5
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  		Figure 5. Kv channels affect pH regulation but not cell swelling. A) T84 cells were swollen (removal of 120 mmol/l mannitol; Hypo) or shrunken (adding 120 mmol/l mannitol; Hyper), as assessed by changes in calcein fluorescence. Repetitive exposure to hypotonic bath solution produced identical changes in cell vol (n=41, data not shown). B) Kv channel inhibitors 4-AP or TPeA did not affect cell swelling. C) Summary of the effects of 4-AP, astemizole and TPeA on cell swelling, as measured by calcein fluorescence. D) Original recording of intracellular pH and effect of astemizole on pH recovery from acid (NH4Cl)load. E) Summary of the effects of 4-AP protein and astemizole on pH recovery after acid load. F) Effect of the NHE1- inhibitor EIPA on cell proliferation. Asterisks indicate significant difference when compared to control (Student’s t test). (number of experiments).

We further examined potential effects of Kv channels on intracellular Ca2+ signaling. As shown in Fig. 6 , baseline intracellular Ca2+ in resting cells was not affected by Kv channel inhibitors. We stimulated T84 cells with the muscarinic secretagogue carbachol (CCH, 100 µM). This induced a release of Ca2+ from intracellular stores (transient peak response) and a subsequent influx through store-operated Ca2+ channels (Ca2+ plateau) (original traces in Fig. 6 ). Both Ca2+ peak and plateau were significantly reduced in the presence of the nonselective Kv blockers 4-AP (50 µM) and TPeA (5 µM) (Fig. 6A ). Similar effects on [Ca2+]i were observed with the EagI inhibitor astemizole (0.5 µM) and the KV3.4 blocker BDS-I (10 nM) (Fig. 6) . Moreover, inhibition of EagI expression by siRNA also attenuated Ca2+ signaling, while siRNA suppression of SK4 had no effects on the carbachol induced Ca2+ signaling (Fig. 6B, C ). The contribution of K+ channels to Ca2+ signaling was further confirmed by the nonselective K+ channel inhibitor Ba2+, which also attenuated Ca2+ signaling T84 cells (data not shown). Taken together, Kv channels affect intracellular Ca2+ signaling, probably by their hyperpolarizing effects, which provide a driving force for Ca2+ release and influx through store-operated Ca2+ channels (SOCs) (15 , 19) . Although up-regulation of voltage-gated Ca2+ channels (VOCC) in T84 cells has been reported previously (40) , it is probably of limited importance for proliferation since we did observe any impact of the VOCC inhibitor calciseptine had no effect on proliferation (Fig. 7 A). Moreover, agonist (carbachol)-induced Ca2+ signaling was not affect by calciseptine in either T84 or HT29 cells (Fig. 7B, C ).


Figure 6
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  		Figure 6. Kv channels affect carbachol induced Ca2+ signaling. A) Original recordings of [Ca2+]i in Fura-2 loaded T84 cells and effects of carbachol (CCH; 100 µM) and 4-AP on transient (store release; peak) and persistent (influx; plateau) Ca2+ increase. Right panel: Summary of the effects of 4-AP (50 µM) and TPeA (5 µM) on Ca2+ peak and plateau. B) Original recordings of the effects of carbachol and astemizole. Right panel: Summary of the effects of BDS-I (10 nM) and astemizole (0.5 µM) on Ca2+ peak and plateau. C) Original recordings of the effects of carbachol on control cells and cells treated with siRNA-EagI or siRNA-SK4. Right panel: Summary of the effects of siRNA-EagI or siRNA-SK4 on Ca2+ peak and plateau. Asterisks indicate significant difference when compared to control (ANOVA, Student’s t test). (number of experiments).


Figure 7
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  		Figure 7. Voltage-gated Ca2+ channels do not affect proliferation or Ca2+ signaling in colonic cancer cells. A) Cell proliferation of T84 cells was not affected by the inhibitor of L-type Ca2+ channels, calciseptine. Carbachol (CCH, 100 µM) induced Ca2+ increase in T84 (B) or HT29 (C) cells was not affected by calciseptine (number of experiments).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
K+ channels and cancer
Several types of K+ channels have been detected in human cancers (21) . Ca2+-activated K+ channels were found in prostate cancer (1) , uterine cancer (38) , human gliomas (32) , gastric cancer (13) , pituitary adenomas (10) , and colorectal cancer (1 , 24 , 43) . In other tumors proliferation is supported by ATP-sensitive KIR channels or two-pore (2P)-domain channels (20 , 25 , 30) . Nevertheless, in the majority of cancer cells, Kv channels were correlated to proliferation (2 , 3 , 8 , 14 , 27 , 28 , 29) . Other data from human biopsies and carcinogen-treated mice suggest high levels of EagI protein in colonic cancers, which is not observed in the native colon (unpublished observations). Also in the present study we found a role of Kv channels for proliferation of cultured colonic carcinoma cells. Thus, the results obtained in T84 cells may be representative for the changes that occur in the native colon during carcinogenesis.

Kv but not other K+ channels control proliferation in T84 cells
Kv channels operate at rather depolarized membrane voltages (Vm), which are typically not found in terminally differentiated epithelial cells (27 , 28) . The depolarized Vm of carcinoma cells provides the basis for activation of Kv channels (5) . When studied under proliferative conditions, serum containing culture medium depolarizes Vm (34) . In vivo, cells are exposed to 20–30 g/l protein present in the interstitial fluid, which may depolarize Vm and allow for activation of Kv channels in carcinoma cells in situ (35) . Remarkably, the contribution of Kv channels to the overall conductance in T84 cells was only 10–15%. However, this is in line with previous reports, which indicate that proproliferative K+ currents are often of smaller amplitude, while large K+ currents are detected during apoptosis (21 , 23) .

Because of the limited specificity of most K+ channel inhibitors (12) , we also used siRNA to identify the K+ channels in charge of cell proliferation. Only Kv channels appear to affect cell proliferation, although other K+ channels are expressed and functional in T84 colonic carcinoma cells, such as SK4 channels. Obviously EAG1 and other Kv channels operate well in proliferating T84 cells, while SK4 channels only support proliferation after additional activation by riluzole. Surprisingly, even the K+ ionophore valinomycin increased proliferation of T84 cells when applied at very low (100 fM) concentrations (unpublished data from the author’s laboratory). We suggest that the oncogenic potential of Kv channels is linked to the K+ channel function. This has been demonstrated recently for TASK3 channels (33) . In principle, any type of K+ channel might support proliferation, depending on additional cell specific properties that determine K+ channel activity.

How do Kv channels control cell proliferation?
The present study was performed to determine the mechanism by which K+ channels affect cell growth. Progression through the cell cycle is dependent on K+ channels, and blocking these channels causes inhibition of proliferation (42) . This clearly suggests a cell cycle specific function of K+ channels. Apart from this specific role, Kv channels may also have homeostatic functions in T84 cells. The present data demonstrate an impact of Kv channels on intracellular Ca2+ signaling and pH regulation. The importance of pHi for cell proliferation and cancer development is well established (39) . pHi varies during the cell cycle and needs to be tightly controlled, probably by the Na+/H+ exchanger NHE1 (37) . The experiments with the NHE blocker EIPA presented here clearly show the importance of NHE for cell proliferation. Na+/H+ exchange requires low intracellular Na+ concentrations, and Vm need to stay hyperpolarized, Both are facilitated by the Na+/K+-ATPase in parallel with K+ channels. Kv channels may provide a K+ recycling pathway and hyperpolarize the membrane voltage and may thus contribute indirectly to pH regulation.

Equally important is the control of intracellular Ca2+ in proliferating cells, especially during the mitotic cell cycle (37) . We found that agonist-induced Ca2+ release and influx of Ca2+ through SOCs, dependents on the function of Kv channels. Thus Ca2+-dependent activation of ion currents was reduced significantly by 3.2 ± 0.5 nA (100 µM 4-AP, n=4) and 2.8 ± 0.4 nA (5 µM astemizole, n=5). It has been demonstrated by others (15) that depolarization of the membrane voltage reduces Ca2+ signaling. Since inhibitors of Kv channels depolarize Vm, this may explain why Ca2+ signaling is affected. Furthermore, a previous study demonstrated that activation of store-operated Ca2+ channels (SOCs) in T84 cells induces proliferation (19) . Moreover, capacitative Ca2+ influx through SOCs is reduced at acidic pH (26) . Since Kv channels also affect intracellular pH, this may provide an independent mechanism by which Kv channels affect intracellular Ca2+ signaling. VOCC are probably not related to the effects of Kv channel inhibitors, although L-type Ca2+ channels are expressed in T84 colonic carcinoma cells (40) . This is further substantiated by the fact that the L-type Ca2+ channel inhibitor calciseptine did not reduce proliferation or Ca2+ signaling (Fig. 7) . Taken together hyperpolarization of Vm by Kv channels may be necessary to maintain pH regulation and Ca2+ signaling, which allows progression through the G1 phase of the cell cycle (18 , 42) .


   ACKNOWLEDGMENTS
 
Supported by DFG SCHR 752/2–1 und Wilhelm Sander-Stiftung 2005.063.1. We gratefully acknowledge the supply of TRAM-34 (Dr. H. Wulff, Department of Medical Pharmacology and Toxicology, University of California, Davis, USA) and 293B, AVE0118 and AVE1231 by Aventis (Frankfurt, Germany). We acknowledge the expert technical assistance by Ms. E. Tartler and Ms. A. Paech.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication April 12, 2006. Accepted for publication August 21, 2006.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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M. Spitzner, J. R. Martins, R. B. Soria, J. Ousingsawat, K. Scheidt, R. Schreiber, and K. Kunzelmann
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