Pflugers Arch - Eur J Physiol (2008) 457:149–159 DOI 10.1007/s00424-008-0516-3

ION CHANNELS, RECEPTORS AND TRANSPORTERS

Functional coupling of TRPV4 cationic channel and large conductance, calcium-dependent in human bronchial epithelial cell lines

José M. Fernández-Fernández & Yaniré N. Andrade & Maite Arniges & Jacqueline Fernandes & Cristina Plata & Francisca Rubio-Moscardo & Esther Vázquez & Miguel A. Valverde

Received: 28 January 2008 /Accepted: 11 April 2008 / Published online: 6 May 2008 # Springer-Verlag 2008

Abstract Calcium-dependent potassium channels are im- solutions (related to mechanical stress) revealed the activa- 2+ plicated in electrolyte transport, cell volume regulation and tion of BKCa channels subsequent to extracellular Ca mechanical responses in epithelia, although the pathways influx via TRPV4, an effect lost upon antisense-mediated for calcium entry and their coupling to the activation of knock-down of TRPV4. Further analysis of BKCa modula- potassium channels are not fully understood. We now show tion after TRPV4 activation showed that the Ca2+ signal can molecular evidence for the presence of TRPV4, a calcium be generated away from the BKCa location at the plasma permeable channel sensitive to osmotic and mechanical stress, membrane, and it is not mediated by intracellular Ca2+ and its functional coupling to the large conductance calcium- release via ryanodine receptors. Finally, we have shown that, dependent potassium channel (BKCa) in a human bronchial unlike the reported disengagement of TRPV4 and BKCa in epithelial cell line (HBE). Reverse transcriptase polymerase response to hypotonic solutions, cystic fibrosis bronchial chain reaction, intracellular calcium imaging and whole-cell epithelial cells (CFBE) preserve the functional coupling of patch–clamp experiments using HBE cells demonstrated the TRPV4 and BKCa in response to high-viscous solutions. presence of TRPV4 messenger and Ca2+ entry, and outwardly rectifying cationic currents elicited by the TRPV4 Keywords TRP. Channel . Ca2+ . BK . Airways . specific activator 4α-phorbol 12,13-didecanoate (4αPDD). Mechanical . Viscosity. Osmotic . Cystic fibrosis Cell-attached and whole-cell patch–clamp of HBE cells exposed to 4αPDD, and hypotonic and high-viscosity Introduction José M. Fernández-Fernández and Yaniré N. Andrade contributed equally to this work, and both should be considered first authors. Potassium channels are required for effective electrolyte : : : J. M. Fernández-Fernández: : Y. N. Andrade: M. Arniges : transport and cell volume regulation in the epithelia [5, 24, J. Fernandes C. Plata F. Rubio-Moscardo E. Vázquez 31, 47]. Recycling of K+ through basolateral K+ channels M. A. Valverde serves two important functions [46]: First, K+ efflux Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, hyperpolarize cell membrane potential, thereby maintaining − Universitat Pompeu Fabra, the driving force for Cl exit and Ca2+ entry via conductive PRBB, C/ Dr. Aiguader 88, pathways; second, prevents accumulation of K+ that could Barcelona 08003, Spain cause cell swelling. Intimately related to this later function + M. A. Valverde (*) is the role of K channels in cell-volume regulation. Laboratory of Molecular Physiology and Channelopathies, Although signaling pathways may differ depending on the Departament de Ciències Experimentals i de la Salut, stimuli [22], hypotonic and isotonic epithelial cell swelling Universitat Pompeu Fabra, after nutrient absorption trigger a regulatory mechanism Parc de Recerca Biomedica de Barcelona, C/ Dr. Aiguader 88, Barcelona 08003, Spain (regulatory volume decrease, RVD) that involves the loss of e-mail: [email protected] osmolytes and osmotically obliged water via the activation 150 Pflugers Arch - Eur J Physiol (2008) 457:149–159 of Cl− [12, 41]andK+ channels [11, 21] (for a review, see TRPV4-mediated activation of Ca2+-dependent K+ channel [15]). The molecular identity of the swelling-activated Cl− activation in control and CF airways. channel is still controversial [29, 40], while different K+ We now report on the functional coupling of TRPV4 and channels have been identified in epithelial cells, many of BKCa channels in response to the synthetic activator of them sensitive to cell volume changes [7, 13, 18, 21, 46]. TRPV4, 4α-phorbol 12,13-didecanoate (4αPDD), and Among the swelling-sensitive K+ channels, Ca2+-dependent physiological osmotic and mechanical (high-viscous load) K+ channels are particularly relevant, as epithelial RVD is stimuli in HBE cells. A Ca2+ signal generated by TRPV4 2+ normally triggered by changes in intracellular Ca concen- channels, not necessarily in close proximity to BKCa, tration [24], although exceptions exist. Large (KCNMA1, also triggers the activity of the potassium channel. We have known as BKCa or Maxi-K) [11], intermediate (KCNN4, also demonstrated that mechanically induced (high-viscous also known as IK) [44], and small conductance (KCNN2, also loads) functional coupling of TRPV4 and BKCa channels, known as SK2) [9]K+ channels activated by osmotic cell unlike the osmotically induced coupling [3], is preserved in swelling have been reported. Our previous study with the a human CF bronchial epithelial cell line (CFBE). human bronchial epithelial cell line 16HBE14o- indicated the presence of the BKCa channel under resting conditions and its modulation by cell swelling [11]. Materials and methods Classically, the source of Ca2+ has been attributed to its influx via non-selective cation channels activated either Cells, antisense oligonucleotides, and reverse transcriptase directly in response to the membrane stretch or after polymerase chain reaction swelling-activated intracellular signaling [15, 16, 26], although intracelullar Ca2+ stores may also participate [35, Human bronchial epithelial cell line 16HBE14o- [6] and the 51]. However, only recently, the molecular nature of cystic fibrosis bronchial epithelial cell line CFBE41o- different swelling-sensitive Ca2+ influx pathway has been (ΔF508/ΔF508) [13] (both a gift from Dr. D. C. Gruenert, unveiled [4, 14, 20]. In the case of a human tracheal San Francisco, CA, USA) were grown in modified Eagle’s epithelial cell line, we have previously shown that the medium with Earle’s salts (Gibco, Life Technologies), 10% TRPV4 channel provides the main pathway for Ca2+ influx fetal bovine serum (Gibco, Life Technologies), and 1% and the subsequent RVD [3]. TRPV4 is a member of the gentamicin (Sigma). Cells were grown on flasks coated vanilloid subgroup of the transient receptor potential (TRP) with a solution containing 1 mg human fibronectin family of channels [23] that responds to osmotic and (Stratech), 0.33 mg Vitrogen (Imperial Laboratories), and mechanical stimuli after the activation of phospholipase A2 10 mg bovine serum albumin (ICN Flow) per 100 ml of 2 (PLA2)[1, 45]. Different TRPV4 splice variants have been modified Eagle’s medium with Earle’s salts. The 25 cm identified in human airway epithelial cells, although not all plastic flasks were coated overnight (37°C) with the fibronec- of them form functional channels [2]. TRPV4 channel tin-based solution. Any excess fibronectin was aspirated mediates swelling-dependent Ca2+ influx in a human before cell seeding. For antisense experiments, HBE cells tracheal epithelia cell line, resulting in the activation of were seeded in 12-mm wells at 80% confluency and exposed Ca2+-dependent, intermediate-conductance K+ channels and to 1 μM TRPV4 antisense (5′-GCCTTCGCTGGAATCCGC subsequent RVD [3]. Besides, TRPV4 appears to be CAT-3′) or ß-globin (negative control) antisense morpholino involved in the response of ciliary beat frequency (CBF) oligonucleotides (5′-CCTCTTACCTCAgTTACAATTTATA- to changes in the viscosity load in the hamster oviduct 3′, Tools, Corvallis, OR, USA), plus 100 ng of pEGFP [1, 10]. CBF is a critical factor in mucociliary transport and, plasmid (ClonTech Laboratories) diluted into 25 μlserum- hence, clearance of the airways [27]. Typically, changes in free medium. Cells were transfected by a Lipofectamine mucus viscosity occurs in cystic fibrosis (CF) airways [17], Plus (Invitrogen) procedure following the manufacturer’s therefore reducing the mucociliary clearance due to the instructions as described previously [3]. Functional experi- increased stickiness of the mucus, apparently without ments were carried out in cells seeded onto plastic dishes or significant changes in CBF [25, 52]. Functional coupling glass coverslips and used within 2 days. RNA extraction between swelling-activated TRPV4 and Ca2+-dependent K+ (Nucleospin RNA II kit, Macherey-Nagel) was carried out channels is lost in the cystic fibrosis human tracheal and 48 h after transfection and reverse transcriptase polymerase bronchial epithelial cell lines (CFT1 and CFBE, respec- chain reaction (RT-PCR; One Step kit, Qiagen) was performed tively) [3]. Consistently, CFT1 and CFBE cells show as described previously [3, 11]. Real-time RT-PCR was impaired RVD [3, 44], a process also described for jejunal performed on an ABI Prism 7900HT (Applied Biosystems, crypts of null and ΔF508 CF mice [42, 43]. However, it is Foster City, CA, USA) with SYBR-Green (Sybr-Green not known whether high-viscous loads also triggered Power PCR Master Mix, Applied Biosystems) and TRPV4 Pflugers Arch - Eur J Physiol (2008) 457:149–159 151 primers (forward 5′-AGAGGTGGAGGAAGAAGATC-3′ also used in several experiments. Single-channel currents and reverse 5′-AGTTAATGAACTCCCGCATG-3′). were obtained by clamping the cells at different potentials (−20, −40, −60, −80, and −100 mV) for 30 s. Currents were Electrophysiology low-pass filtered at 1 kHz and sampled at 10 kHz. Single- channel current levels were measured by all-points histo- Ionic currents were measured using the whole-cell or cell- grams, and open probability (NPo) was calculated from attached recording mode of the patch–clamp technique [11]. 30-s recordings at the indicated potential. All chemicals pClamp8 software (Axon Instruments, Foster City, CA, were purchased from the Sigma-Aldrich Company. USA) was used for pulse generation, data acquisition through 2+ an Axon Digidata A/D interface, and subsequent analysis. [Ca ]i Measurements Cells were plated in 35-mm plastic dishes and mounted on the stage of an Inverted Olympus IX70. Whole-cell cationic Intracellular Ca2+ measurements were carried out as de- currents were recorded using borosilicate glass electrodes scribed previously [3, 11]. Cells were incubated in isotonic (2–4MΩ) filled with a solution containing (in mM) 120 CsCl, solution containing 2 μM fura-2 AM for 30 minutes at room

1 MgCl2, 1 ethylene glycol bis(2-aminoethyl ether)-N,N,N′N′- temperature. Cells were then washed thoroughly with tetraacetic acid (EGTA), 30 4-2-hydroxyethyl-1-piperazinee- isotonic solution. Video microscopic measurements of Ca2+ thanesulfonic acid (HEPES), 4 adenosine triphosphate (ATP), were obtained using an Olympus IX70 inverted microscope and 0.1 guanosine triphosphate (GTP; 290 mosmoles/l, with a ×40 oil-immersion objective (Hamburg, Germany). pH 7.25). Bath solution contained (in mM) 125 NaCl, 1.5 The excitation light (340 and 380 nm) was supplied by a

MgCl2, 1 EGTA, 10 HEPES, pH 7.36, and 305 mosmoles/l Polychrome IV monochromator (Till Photonics, Martinsried, (adjusted with D-mannitol). Cells were held at 0 mV and Germany) and directed towards the cells under study by a ramps from −140 mV to +100 mV with a duration of 200 ms 505DR dichromatic mirror (Omega Optical, Brattleboro, VT, were applied at a frequency of 0.2 Hz. Ramp data were USA). Fluorescence images were collected by a digital CCD acquired at 10 KHz and low-pass filtered at 1 kHz. camera (Hamamatsu Photonics, Japan) after passing through Whole-cell K+ currents were measured using pipette a 535DF emission filter (Omega Optical) using the solution containing (in mM) 140 KCl, 2 MgCl2, 0.15 AquaCosmos software program (Hamamatsu Photonics, CaCl2, 0.5 EGTA, 4 ATP, 0.1 GTP, and 10 HEPES Japan). A 340/380-nm ratio images were computed every 5 s. (298 mosmoles/l, pH 7.25; the intracellular-free Ca2+ concentration was 50 nM as calculated using EqCal from Statistics Biosoft (Cambridge, UK)). The isotonic bathing solution contained (in mM) 100 NaCl, 5 KCl, 1.2 CaCl2, 0.5 MgCl2, Data are expressed as the mean ± SEM. Student’s t test or 5 glucose, 10 HEPES, and 90 D-mannitol (305 mosmoles/l, analysis of variance (ANOVA) were performed with the pH 7.35). The hypotonic bathing solution (osmolality, SigmaPlot 5, SPSS, and Statistica 6.0 programs. Bonferroni’s 215 mosmoles/l) was prepared by omitting D-mannitol test was used for post hoc comparison of means. The criterion from the isotonic solution. Ca2+-free bath solutions, con- for a significant difference was a final value of P<0.05. 2+ taining 0 Ca , 1.5 mM MgCl2, and 1 mM EGTA, were also used in several experiments. Cells were clamped at −80 mV and pulsed for 400 ms from −100 mV to +100 mV Results and discussion in 20 mV steps. High-viscous isotonic solutions of 73 cP were obtained by adding 20% dextran T-500 (500,000 TRPV4 activity in human bronchial epithelial cells (HBE) Daltons; Pharmacia, Uppsala, Sweden) to isotonic bathing solutions (1 cP). The activity of the TRPV4 channel was evaluated using a Cell-attached single-channel recordings were carried out synthetic activator, 4αPDD, specific for the TRPV4 using electrodes (4–6MΩ) filled with a solution containing channel [48]. Application of 1 μM4αPDD-induced typical

(in mM) 100 NaCl, 5 KCl, 1.2 CaCl2, 0.5 MgCl2,5 TRPV4 cationic currents that rectified outwardly when glucose, and 10 HEPES at pH 7.25 (osmolality, adjusted pipettes were loaded with permeant cation-containing 2+ with D-mannitol, 300±5 mOsm; n=15), and the isotonic solutions in the absence of extracellular Ca to prevent bathing solution contained (in mM) 105 KCl, 1.2 CaCl2, the reduction of the current amplitude and its decay [49] 0.5 MgCl2, 5 glucose, 10 HEPES, 80 D-mannitol at pH 7.25 (Fig. 1a; see inset for the time course of TRPV4 activation). (osmolality, 305±4 mOsm; n=10). Hypotonic bathing used The intracellular Ca2+ signal induced by 1 μM4αPDD has already been described above. Ca2+-free solutions, (Fig. 1b) was abolished in the presence of 100 μMGd3+ 2+ containing 0 Ca , 1.5 mM MgCl2 and 1 mM EGTA, were (Fig. 1C), a blocker of different cationic channels, among 152 Pflugers Arch - Eur J Physiol (2008) 457:149–159

Fig. 1 TRPV4 channel expres- sion and activity in HBE cells. a Current/voltage relationships of non-selective cationic cur- rents recorded with a CsCl- containing pipette solution in HBE cells under control (white circle, n=6), 1 μM4αPDD (black circle, n=6) and after washing out the drug (gray circle, n=3). Inset shows the time course of TRPV4 activa- tion measured at −100 and +100 mV. b Increase in cyto- solic Ca2+ after incubation with 1 μM4αPDD in HBE cells (n=7). c Effect of 100 μM Gd3+ (n=7) and d extracellular Ca2+ (n=8) on the cytosolic calcium response induced by 4α-PDD. Solutions were added at the time marked by the bar

them TRPV4 [38], that does not block BKCa [11], or in the induce an increase in BKCa activity despite the presence of absence of extracellular Ca2+ (Fig. 1d). Altogether, the Ca2+ in the extracellular pipette solution. Figure 3e shows results demonstrated the presence of functional TRPV4 mean NPo for all four conditions. channels in HBE cells. Functional coupling between TRPV4 and BKCa has been demonstrated in vascular smooth muscle cells in response to

Functional coupling of TRPV4 and BKCa channels in HBE vasodilatory factors, although in this case, a third player also cells (I): modulation by 4αPDD and hypotonicity participates in the process, the (RyR). Ca2+ entry through TRPV4, either directly or through increased Cell-attached recordings in HBE cells exposed to 4αPDD Ca2+ loading into the sarcoplasmic reticulum, activates RyR 2+ (1 μM) revealed a pronounced activation of BKCa channels and generates local Ca sparks that augment BKCa activity (Fig. 2a), an effect lost upon removal of extracellular Ca2+, [8]. Other laboratories have previously presented evidence despite the presence of Ca2+ in the pipette solution (Fig. 2b) that HBE cells lack functional RyR receptors [36]. However, or addition of 100 μMGd3+ (Fig. 2c). Figure 2d shows we did some additional experiments to exclude the involve- mean NPo for all three conditions. Single-channel conduc- ment of RyR in the coupling of TRPV4 and BKCa in HBE tance of the BKCa was 125±9 pS (n=27) in the absence and cells. Cells pretreated for 25 min with the RyR inhibitor 127±10 pS (n=16) in the presence of 4αPDD, similar to dantrolene (10 μM) showed the same level of BKCa channel the BKCa conductance previously reported in these [11] and activation by 4αPDD (NPo=0.011±0.001 and 0.5±0.16 other epithelial cells [33, 34]. The coupling of BKCa and before and after 4αPDD; n=4) than the untreated cells (see TRPV4 and its dependence on extracellular Ca2+ was Fig. 2; P>0.05 untreated vs dandrolene-treated cells). further studied in response to a hypotonic solution. Cell- The Ca2+ signal generated in condition C (Fig. 3) most attached recordings of BKCa channels in HBE cells exposed likely results from the activation of TRPV4 channels to hypotonic solutions were carried out in the presence or situated outside the membrane patch and travels to reach 2+ absence of Ca ions in the pipette and bathing solutions the BKCa channels located within the membrane patch. (Fig. 3). Interestingly, only in those conditions where Ca2+ Previous reports showed that Ca2+ signals produced by the was present in the extracellular bathing solution, was the activation of stretch-activated cation channels in endothelial 2+ activity of BKCa increased (Fig. 3a and c). In Ca -free cells [16] or produced by RyR secondary to the activation extracellular bathing solutions, hypotonic stimulus did not of TRPV4 in vascular smooth muscle [8] are generated in Pflugers Arch - Eur J Physiol (2008) 457:149–159 153

Fig. 2 Activation of BKCa channel by 4αPDD. Represen- tative cell-attached single- channel recordings from HBE cells before (top) and after (bottom) the addition of 1 μM 4αPDD to isotonic bathing solutions containing 1.2 mM Ca2+ (a), 0 Ca2+ (b), and 100 μMGd3+ + 1.2 mM Ca2+ (c). Mean NPo responses under the conditions indicated above. 4αPDD, n=12; 0 Ca2+, n=5; and Gd3+, n=5. Dotted line represents zero current level

2+ 2+ the proximity of BKCa. However, in HBE cells, the Ca mechanically activated Ca signaling is also a fundamental signal can be produced away from the BKCa location mechanism for CBF regulation [28]. Transporting cilia (outside the membrane patch within the pipette tip), and it appear to present adaptations that allow them to beat under is not mediated by Ca2+-induced Ca2+ release via RyR, as conditions of varying viscosity of the mucus, thereby the current response is maintained in the presence of the preventing the collapse of mucociliary transport, a process RyR inhibitor. In conditions B (Fig. 2)andD(Fig.3), with known as autoregulation of CBF [1, 19]. Moreover, it has Ca2+ ions present only in the pipette (extracellular) solution, been previously suggested that mucus itself or changes in no increased BKCa activity was observed in response to its viscosity may be a trigger for mechanical stimulation in either 4αPDD or hypotonic solutions. Considering that ciliated epithelia [37]. According to our most recent studies, TRPV4 channels overexpressed in HEK-293 cells can be a TRPV4-mediated Ca2+ signal links the response of the activated in cell-attached patches under hypotonic conditions CBF to the viscosity of the external medium in hamster [38], the most likely explanation for the lack of activation of oviductal ciliated cells [1]. We have also demonstrated the

BKCa in such conditions is that the number of endogenous activation of TRPV4 currents by high-viscous loads in TRPV4 channels within the membrane patch of HBE cells is native ciliated cells and TRPV4-expressing HeLa cells [1]. not enough to generate an effective Ca2+ signal. Therefore, the functional coupling between TRPV4 and

BKCa channels was also studied in response to high Functional coupling of TRPV4 and BKCa channels in HBE viscosity conditions (presumably, exerting a mechanical cells (II): modulation by mechanical stress (high-viscous stimulation related to shear stress, viscous resistance to loading) ciliary beat or cell membrane fluctuations [39, 50]). The whole-cell recording mode, instead of single-channel

Airway-ciliated epithelia carry out the task of transporting recording mode, was mainly used to test BKCa channel mucus and trapped particles, the efficiency of which activity. The use of high-viscous solutions complicated the strongly depends on the CBF [30]. In this process, acquisition of long-lasting single-channel recordings. 154 Pflugers Arch - Eur J Physiol (2008) 457:149–159

Fig. 3 Effect of extracellular 2+ Ca concentration on BKCa channel activation by hypotonic- ity. Representative cell-attached single-channel recordings from HBE cells under isotonic (top) and hypotonic conditions (bottom) with 1.2 mM Ca2+ in both pipette and bathing solu- tions (a), 0 Ca2+ in both pipette and bathing solutions (b), 1.2 mM Ca2+ in the bathing solution and 0 Ca2+ in the pipette (c), and 0 Ca2+ in the bathing solution and 1.2 mM Ca2+ in the pipette (d). e Mean NPo under conditions mentioned above; n=13, 6, 9, and 5, respectively. *P<0.05 compared with corresponding isotonic condi- tions (paired t test)

Whole-cell BKCa currents were recorded in HBE cells containing 20% dextran. Cell-attached single-channel exposed to high-viscous loading (73 cP) using isotonic recordings from a HBE cell exposed to 20% dextran solutions (305 mosmoles/l) containing 20% dextran. High solutions show single-channel current with characteristics viscosity induced a substantial increase in typical BKCa similar to BKCa (Fig. 5b). currents (Fig. 4a). The increase in BKCa currents in Activation of TRPV4 channel by both hypotonic and response to 20% dextran solutions was prevented in the high-viscosity solutions depends on previous activation of 2+ 3+ absence of extracellular Ca or in the presence of Gd PLA2 [1, 45]. Accordingly, the presence of the PLA2 (100 μM) in the extracellular dextran-containing solution. inhibitor arachidonyl trifluoromethyl ketone (AACOCF3, Figure 4b shows the BKCa current/voltage relationships and 50 μM) in the bathing solutions prevented whole-cell BKCa Fig. 4c the mean maximal BKCa current density recorded at activation in response to 20% dextran-containing solutions +100 mV under different conditions. To further confirm the [ratio BKCa(dextran)/BKCa(control) is 2.9±0.3 (n=6) in the nature of the high viscosity-induced potassium current, we absence and 1.06±0.16 (n =3) in the presence of used iberiotoxin, an inhibitory toxin specific for BKCa AACOCF3; P<0.05] and hypotonic solutions [BKCa(hypo)/ (Fig. 5a). In the presence of iberiotoxin (100 nM), no BKCa(control) is 3.85±0.5 (n=4) in the absence and 0.98± activation of BKCa was observed in response to solutions 0.02 (n=3) in the presence of AACOCF3]. Pflugers Arch - Eur J Physiol (2008) 457:149–159 155

Fig. 4 Response of BKCa cur- rents to 20% dextran solutions in HBE cells. a Whole-cell BKCa currents recorded under control and 5 min after bathing the cells with a isotonic bathing solutions containing 1.2 mM Ca2+ + 20% dextran, Ca2+-free 20% dextran, or 100 μMGd3+ + 1.2 mM Ca2+ + 20% dextran. Mean I/V curves (b) and maxi- mal BKCa current densities obtained at +100 mV (c) under control (n=13), 20% dextran (n=6), Ca2+-free 20% dextran (n=5), and Gd3+ + 20% dextran (n=6). *P<0.05 com- pared with the control, untreated cells (one-way ANOVA and Bonferroni post hoc)

TRPV4 antisense oligonucleotides prevent BKCa BKCa currents, TRPV4 expression was suppressed using modulation by high-viscosity, osmotic and 4α-PDD stimuli antisense oligonucleotides as previously described [3].

Figure 6ashowsBKCa whole-cell currents obtained from Previous data obtained from human tracheal epithelial cells HBE cells treated with control β-globin antisense (left) and treated with TRPV4 antisense oligonucleotides showed TRPV4 antisense (right), and exposed to 20% dextran diminished TRPV4 expression, reduced activation solutions. High-viscosity-induced currents were preserved of intermediate conductance Ca2+-dependent K+ channels in HBE cells treated with control β-globin antisense but lost by hypotonic stimulation, and impaired RVD, pointing to in TRPV4 antisense-treated cells. Semiquantitative RT-PCR TRPV4 as the main Ca2+ entry pathway activated by was used to show that TRPV4 messenger RNA levels were hypotonic stress [3]. Similarly, the activation of TRPV4 by diminished (without affecting the pore-forming α-subunit of high-viscous solutions was prevented in hamster oviductal the BKCa) in TRPV4 antisense- but not in β-globin ciliated cells loaded with a specific TRPV4 antibody [1]. antisense-treated cells (inset), as previously shown [8]. These To further confirm the relationship between high-viscosity- findings, were confirmed using real-time RT-PCR to deter- induced, TRPV4-mediated Ca2+ influx and modulation of mine the relative abundance (normalized to β-actin) of

Fig. 5 Pharmacology and single-channel conductance of the high-viscosity-induced BKCa current. a Whole-cell BKCa currents recorded under control and 5 min after bathing the cells with a isotonic bathing solutions containing 1.2 mM Ca2+ + 20% dextran + 100 nM iberiotoxin. b Representative cell-attached single-channel recordings from a HBE cell before (left) and after (right) exposure to a bathing solution containing 1.2 mM Ca2+ + 20% dextran 156 Pflugers Arch - Eur J Physiol (2008) 457:149–159

Fig. 6 Effect of antisense-induced knockdown of TRPV4 on BKCa dextran for untreated (n=6), TRPV4 antisense (n=11), and ß-globin channel activity. a Whole-cell BKCa currents recorded under control antisense (n=12) treated HBE cells. *P<0.05 compared with the and 5 min after bathing ß-globin antisense (left) and TRPV4 antisense control, untreated cells (one-way ANOVA and Bonferroni post hoc). (right) treated HBE cells with a 20% dextran solution. Inset c Mean BKCa NPo from untreated (n=12), TRPV4 antisense (n=7), Semiquantitative RT-PCR demonstrating the effects of antisense and ß-globin antisense-treated cells (n=5) exposed to hypotonic or treatment on TRPV4 and the pore-forming α-subunit of the BKCa 4αPDD stimuli. *P<0.05 compared with corresponding isotonic expression in HBE cells (V4 TRPV4, ßG ß-globin, AS antisense, NT conditions (paired t test) no template control). b Mean ratio of BKCa currents activated by 20%

TRPV4 transcript in β-globin and TRPV4 antisense-treated latter presenting a significantly reduced response to 20% cells, 0.99±0.03 and 0.39±0.1, respectively (triplicate dextran-containing solutions. The response of the BKCa measurements; P<0.001, Student’s t test). Figure 6bshows channel to hypotonic and 4αPDD stimuli was also checked mean increased ratios of BKCa currents under control, in antisense-treated cells (Fig. 6c). Mean NPo response of β-globin antisense, and TRPV4 antisense-treated cells, the BKCa channel to hypotonic and 4αPDD stimuli was also

Fig. 7 Response of BKCa cur- rents to 20% dextran solutions in CFBE cells. a Whole-cell BKCa currents recorded under control and 5 min after bathing the CFBE cells with a isotonic bathing solutions containing 1.2 mM Ca2+ + 20% dextran, Ca2+-free 20% dextran, or 100 μMGd3+ + 1.2 mM Ca2+ + 20% dextran. Mean I/V curves (b) and maximal BKCa current densities obtained at +100 mV (c) under control (n=13), 20% dextran (n=6), Ca2+-free 20% dextran (n=7), and Gd3+ + 20% dextran (n=6) solutions. *P<0.05 compared with the control, untreated cells (one-way ANOVA and Bonferroni post hoc) Pflugers Arch - Eur J Physiol (2008) 457:149–159 157 significantly reduced in TRPV4 antisense-treated cells. coordination with swelling-activated Cl− channels providing Although, in response to the potent activator of TRPV4, the pathway for KCl exit during RVD. Thus, Ca2+ entry 2+ + 4αPDD, BKCa channel activity in TRPV4 antisense-treated pathways and Ca -dependent K channels form a functional cells is above control conditions, statistically significant unit in different cell types. We have now shown that, in a increases in BKCa activity were only reached in untreated or human bronchial epithelial cell line, such functional coupling β-globin antisense treated cells (Fig. 6c). While other TRP exists between the calcium-permeable TRPV4 cationic channels have been implicated in Ca2+ influx after mechan- channel and the large conductance Ca2+-dependent K+ ical or osmotic stimulation of different cell types [4, 14, 20], channel (BKCa). This functional coupling occurs in response based on the antisense experiments, it appears that TRPV4 to 4αPDD (a synthetic activator of TRPV4), and osmotic and plays a key role in the activation of BKCa by 4αPDD, and mechanical (high-viscous load) stimulation, and unlike the osmotic and high-viscous stimuli in the HBE cell line. TRPV4-BKCa coupling describe in vascular smooth muscle [8], does not involve the ryanodine receptor. Our study also

Human cystic fibrosis bronchial cells show normal BKCa reveals that, unlike the hypotonicity-induced TRPV4-BKCa modulation by mechanical stress functional coupling, the high-viscosity-induced TRPV4-

BKCa coupling is maintained in CFBE cells, suggesting that The cystic fibrosis transmembrane conductance regulator CFTR requirement for TRPV4 activation in airway epithelia (CFTR) protein, the product of the CF gene, is both a Cl− is stimulus-dependent. In this sense, we have recently channel and a channel regulator [32]. CFTR, through a still demonstrated that TRPV4 activation by 20% dextran or elusive mechanism, is linked to the activation of TRPV4 30% hypotonic solutions employs slightly different intracel- under hypotonic conditions in human tracheal epithelial lular signaling [10]. While TRPV4 activation by either cells [3]. The absence of CFTR or the presence of the stimuli requires activation of PLA2, high-viscosity-mediated mutant ΔF508 protein diminishes TRPV4 activation by activation is more dependent on phosphopipase-C and osmotic cell swelling and the subsequent enhanced activity inositol trisphosphate signaling than osmotic activation of of Ca2+-dependent K+ channels, resulting in a defective the channel [10]. Whether this difference is related to the RVD. Human CF bronchial epithelial cells (CFBE) preservation of viscosity-induced but not hypotonicity- obtained from a patient homozygous for ΔF508 express induced TRPV4-BKCa coupling in CF cells is unknown at TRPV4 but show no hypotonicity-mediated Ca2+ entry, no present, offering a hypothesis to test in future studies. activation of BKCa, and no RVD in response to hypotonic solutions [3]. Functional TRPV4 channels in CFBE cells Acknowledgment We thank D. C. Gruenert (San Francisco, USA) 2+ for the gift of the cell lines. Work funded by the Spanish Ministry of were demonstrated by measuring intracellular Ca concen- Education and Science (SAF2006-04973 to M.A.V. and SAF2006- trations in response to 4αPDD, which also allowed RVD 13893-C02-02 to J.M.F.-F.), Ministry of Health (Fondo de Investigación recovery due to direct activation of TRPV4 channels [3]. Sanitaria, red HERACLES RD06/0009 to M.A.V.), Generalitat de Together, the results suggested that CFBE, like CF human Catalunya (SGR05-266 to M.A.V.), and Fundació la Marató de TV3 (061331 to J.M.F.-F.). J.M.F.-F. is a Ramón y Cajal Fellow. The authors tracheal epithelial cells [3], lack an effective RVD due to a declare that they have no competing financial interests. deficient Ca2+ influx via TRPV4. Therefore, we set out to test whether BKCa activation by high-viscous solutions was also altered in CFBE cells. Figure 7a shows whole-cell References BKCa current activation in response to 20% dextran- containing solutions (73 cP), which was similar in terms 1. Andrade YN, Fernandes J, Vazquez E, Fernandez-Fernandez JM, of the I/V curve and magnitude (Fig. 7b,c) to that recorded Arniges M, Sanchez TM, Villalon M, Valverde MA (2005) from HBE cells. BKCa current activation was also pre- TRPV4 channel is involved in the coupling of fluid viscosity vented in the absence of extracellular Ca2+ and in the changes to epithelial ciliary activity. J Cell Biol 168:869–874 3+ 2. Arniges M, Fernandez-Fernandez JM, Albrecht N, Schaefer M, presence of Gd (Fig. 7c). Valverde MA (2006) Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. J Biol Chem 281:1580–1586 Conclusions 3. Arniges M, Vazquez E, Fernandez-Fernandez JM, Valverde MA (2004) Swelling-activated Ca2+ entry via TRPV4 channel is 2+ defective in cystic fibrosis airway epithelia. J Biol Chem Ca signaling has been shown to induce the activation of 279:54062–54068 Ca2+-dependent K+ channels, thereby hyperpolarizing the 4. Christensen AP, Corey DP (2007) TRP channels in mechanosen- membrane potential and adjusting different cellular functions sation: direct or indirect activation? Nat Rev Neurosci 8:510–521 5. Cotton CU (2000) Basolateral potassium channels and epithelial depending on the cell type: reducing cell excitability in ion transport. Am J Respir Cell Mol Biol 23(3):270–272 neurons and muscle cells, and increasing the electrochemical 6. Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, gradient for Ca2+ entry in non-excitable cells or in Finkbeiner WE, Widdicombe JH, Gruenert DC (1994) CFTR 158 Pflugers Arch - Eur J Physiol (2008) 457:149–159

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