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J. Biol. Chem., Vol. 276, Issue 45, 42268-42275, November 9, 2001

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The Small Conductance K⁺ Channel, KCNQ1

EXPRESSION, FUNCTION, AND SUBUNIT COMPOSITION IN MURINE TRACHEA^*

Florian Grahammer^§, Richard Warth^§¶, Jacques Barhanin^¶, Markus Bleich^**, and Martin J. Hug^§§

From the  Institute of Physiology, Albert-Ludwigs-Universität, Hermann-Herder-Strae 7, D-79104 Freiburg, Germany, ^¶ Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France, ^** Aventis Pharma Deutschland GmbH, D-65926 Frankfurt am Main, Germany, and  Institute of Physiology, Westfälische Wilhelms Universität, Robert-Koch-Strae 27a, D-48149 Münster, Germany

Received for publication, May 31, 2001, and in revised form, August 21, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The gene KCNQ1 encodes a K⁺ channel -subunit important for cardiac repolarization, formerly known as K_vLQT1. In large and small intestine a channel complex consisting of KCNQ1 and the -subunit KCNE3 (MiRP2) is known to mediate the cAMP-activated basolateral K⁺ current, which is essential for luminal Cl secretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However, previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Cl secretion by these cells is not reduced by the knock-out of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K⁺ channel in murine tracheal epithelial cells. As described for colonic epithelium, the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B-sensitive current was present after stimulation with forskolin and agonists that increase Ca²⁺ as well as after administration of the pharmacological K⁺ channel activator, 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K⁺ channel not only for Cl secretion but also for Na⁺ reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K⁺ conductance in tracheal epithelial cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The consistency and the electrolyte content of the airway surface liquid are tightly controlled by numerous transport processes. Disturbances in the underlying secretion and absorption mechanisms lead to malfunction of the ciliary clearance, which is needed to transport mucous and foreign particles from the bronchial system toward the pharynx (1, 2). One of the diseases in which these transport properties are disturbed is the autosomal recessively inherited condition, cystic fibrosis (CF)¹ (3, 4). Mutations in the CFTR gene lead to viscous mucous due to Na⁺ hyperabsorption as well as reduced Cl secretion and predispose individuals to bacterial infections (5). To enhance Cl secretion in CF patients, interest has focused over the last years on activating basolateral K⁺ channels (6, 7). This approach is based on the finding that Cl secretion requires the concomitant activation of basolateral K⁺ channels (8, 9). There are two differently regulated subsets of K⁺ channels in airway epithelia, one group regulated by Ca²⁺ and the other group via cAMP. IK1 (KCNN4) is thought to be one of the Ca²⁺-activated channels (10). The benzimidazolone 1-EBIO became known as the first substance that pharmacologically enhances the Ca²⁺-activated K⁺ current and has been suggested as an approach to ameliorate electrolyte imbalances in CF (6, 7). The molecular basis of the cAMP-regulated channels has been a controversial issue (6).

KCNQ1-KCNE3 channel complexes have been shown to mediate cAMP-activated Cl secretion in small and large intestine (11, 12). In addition to their expression in the gut they are also expressed in the kidney, stomach, and lung (13). In the heart and inner ear, KCNQ1 is known to co-assemble with KCNE1, another member of the expanding KCNE family, to form the native channel complex (14-16). In the airway epithelium it had been a matter of debate whether KCNQ1 channel complexes play a functional role at all (17). Pharmacological evidence with the KCNQ1-specific inhibitor, the chromanol 293B, supports a role of KCNQ1 in human nasal epithelium (18, 19). Previous reports have suggested a functional role for KCNE1 in rat and mouse airway epithelium (20, 21). Here we provide evidence that as in the gut KCNQ1 co-assembles with KCNE3 in murine tracheal epithelial cells to form a basolateral K⁺ channel complex, which functions as the main driving force for epithelial anion secretion and base-line Na⁺ absorption.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- KCNE1 knock-out and wild-type mice were generated as described previously and were back-crossed with C57Bl6 mice (Charles River, Sulzfeld, Germany) (15). 6-12-week-old animals derived from heterozygous mating were used and genotyped by PCR prior to experiments. Other than for breeding, C57Bl6 mice were used directly for all other functional experiments not concerning the KCNE1 gene knock-out. All animals had free access to standard chow and tap water and were kept according to the German law for the care and use of laboratory animals. The experimental protocols were approved by the local council on animal care.

Mice were anesthetized with a mixture of ketamine/xylazine (ketamine: 100 mg/kg of body weight, intraperitoneal, Sigma; xylazine: 4 mg/kg of body weight, intraperitoneal, Bayer, Leverkusen, Germany) and killed by dissection of the abdominal aorta. With a parasternal incision the thoracic cavity and the neck were opened, and the trachea was exposed from adjacent structures. After the main bronchi were cut off with a pair of scissors, a scalpel blade was used to separate the trachea from the esophagus. The trachea was then cut from the larynx and freed from surrounding connective tissue under a dissection microscope.

RT-PCR-- mRNA expression of KCNQ1, KCNE1, and KCNE3 was assessed by RT-PCR. Mouse tracheae were removed as described and placed for 15 min at 37 °C in a Ca²⁺-free solution containing (in mM): 127 NaCl, 5 KCl, 5 D-glucose, 1 MgCl₂, 5 Na⁺-pyruvate, 10 HEPES, 5 EDTA, pH 7.4. Under a dissection microscope (Stemi 2000, Zeiss) the epithelial layer was carefully removed, and the epithelial cells were placed immediately thereafter in lysis buffer (QuickPrep Micro mRNA Purification Kit, Amersham Pharmacia Biotech, Little Chalfont, UK). mRNA purification and cDNA preparation were performed according to the manufacturer's instructions (SensiScript RT Kit, Qiagen, Hilden, Germany). 0.1-0.2 µg of mRNA were used to create a cDNA library. For the respective PCR reactions (1 unit of Taq polymerase, 30 nM MgCl₂, 4 nM dNTPs, 2 µl of 10× PCR buffer, ddH₂O to a reaction volume of 20 µl (all from Life Technologies, Inc.); KCNQ1, 45 s at 95 °C, 30 s at 55 °C, 45 s at 72 °C; KCNE1, 45 s at 95 °C, 30 s at 57 °C, 30 s at 72 °C; KCNE3, 30 s at 95 °C, 30 s at 55 °C, 30 s at 72 °C; for 35 cycles each), the following mouse-specific primers (10 pM) were used: KCNQ1 (product length, 421 bp), sense 5'-CCC TCT TCT GGA TGG AGA T-3', antisense 5'-ATC TGC GTA GCT GCC AAA C-3', generated according to GenBank^TM accession number NM008434; KCNE1 (297 bp), sense 5'-GCT CGT AAG TCT CAG CTC CG-3', antisense 5'-CGA CAA TGG CTT CAG TTC AGG-3', generated according to GenBank^TM accession number NM008424; KCNE3 (302 bp), sense 5'-AAC GGG ACT GAG ACC TGG TA-3', antisense 5'-CAT CAG ATC ATA GAC ACA CGG-3', generated according to GenBank^TM accession number NM020574.

RNA Quantification and Subsequent Southern Probing-- cDNA was synthesized using mouse total RNA from tracheal epithelial cells, colon, and kidney. A second set of mouse-specific primers was used for these experiments: mKCNQ1 (sense, bp 756-775; antisense, bp 1387-1406; from GenBank^TM accession number MMU70068, Tm 62 °C); mKCNE1 (sense, bp 98-117; antisense, bp 528-547; from GenBank^TM accession number NM008424, Tm 62 °C); mKCNE3 (sense, bp 211-230; antisense, 664-683; from GenBank^TM accession number NM020574, Tm 58 °C). They were amplified using the following PCR conditions: 3 min at 94 °C; 15 s at 94 °C, 25 s at Tm, 20 s at 72 °C for 32 cycles. After product quantification with the intensity of the glyceraldehyde-3-phosphate dehydrogenase signal, specific PCR products were detected by Southern probing using internal oligonucleotides. Radioactivity was measured after 10 min with a phosphorimaging device (Fuji bio-imaging analyzer).

Immunofluorescence-- 2.5 µm, thin, paraffin-embedded tracheal sections of KCNE1 / and +/+ mice were used. After removal of paraffin and re-hydration, the sections were boiled for 3 min in citrate buffer (10 mM, pH 6.0). The affinity-purified polyclonal KCNQ1 antibody (Davids Biotechnologie, Regensburg, Germany) was directed against the peptide CPADLGPRPRVSLDPRVSIY of the cytosolic N terminus (22). The polyclonal KCNE1 antibody was directed against the whole polypeptide (23). The antibodies were dissolved in antibody dilution solution (1:500 and 1:1000, respectively; Dako, Carpinteria, CA). Alexa488 goat anti-rabbit (Molecular Probes, Leiden, The Netherlands) was used as secondary antibody and HOE 333342 (2 µM, Molecular Probes) for staining of the nuclei. Control experiments were performed by omitting either primary of secondary antibody respectively. The sections were examined with a confocal microscope (LSM 510, Zeiss, Jena, Germany; Alexa488, excitation at 488 nm, emission at 505-550 nm).

Ussing Chamber Experiments-- The trachea was split along the anterior side, and the pars membranacea of the trachea was then mounted into an Ussing chamber (area 0.64 mm²) with the aid of a dissection microscope. The chamber (2 ml) was maintained at 37 °C and continuously perfused on both sides at a rate of 10-15 ml/min with a solution containing (in mM): 145 NaCl, 0.4 KH₂PO₄, 1.6 K₂HPO₄, 5 D-glucose, 1 MgCl₂, 1.3 calcium gluconate, and 1 µM Indomethacin (Sigma) to inhibit endogenous cAMP formation; pH was adjusted to 7.4. To prevent muscular contraction, 10 nM tetrodotoxin (Molecular Probes) was added to the basolateral solution. The tissue was given at least 40 min of equilibration time prior to experiments. Trans-epithelial resistance (R_te) was determined from the voltage deflection, V_te, caused by the injection of short current pulses (0.2 Hz, 0.9 s duration, 0.5 µA); the resistance of the empty chamber was subtracted. Equivalent short circuit current (I_sc) was calculated from trans-epithelial voltage (V_te), and R_te was calculated according to Ohm's law. The sign of the I_sc and V_te refer to the lumen. Amiloride (50 µM, Sigma), azosemide (500 µM, Sanofi Winthrop, München, Germany), cyclopiazonic acid (50 µM, Calbiochem, Bad Soden, Germany), 1-EBIO (1 mM, Sigma), and 293B (10 nM-10 µM, Aventis, Frankfurt, Germany) were dissolved in Me₂SO; forskolin (5 µM, Aventis) was dissolved in methanol.

Statistics-- Data are shown as the mean values ± S.E. from n observations. Paired as well as unpaired Student's t tests were used as appropriate. A p value of 0.05 was accepted to indicate statistical significance, which is marked in the figures with the  symbol.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RT-PCR showed an mRNA expression of KCNQ1 and KCNE3 in harvested tracheal epithelial cells of wild-type and KCNE1 knock-out mice. mRNA expression of KCNE1 in the cells of wild-type mice could not be detected with this sensitive method, albeit parallel experiments with the same amount of kidney mRNA gave a clearly visible band at the expected length (Fig. 1A). To verify our results we performed a second RT-PCR with a different set of primers, quantified the amount of cDNA according to the glyceraldehyde-3-phosphate dehydrogenase signal of the different tissues, and used a Southern blot specifically to probe and quantify the reaction products (Fig. 1B). In these experiments in addition to cDNA from tracheal epithelial cells we used colon and kidney cDNA as a reference for KCNQ1/KCNE3 and KCNE1, respectively. KCNQ1 and KCNE3 were both expressed in colon and trachea, whereas KCNE1 could not be detected in these tissues, which was in striking contrast to kidney. We therefore conclude that there is no relevant mRNA expression of the KCNE1 gene in murine tracheal epithelial cells. Immunofluorescence with KCNQ1- and KCNE1-specific antibodies showed a cytoplasmatic as well as cell membrane expression pattern of KCNQ1 in tracheal sections of both genotypes, whereas a KCNE1-specific signal could not have been established in KCNE1 wild-type mice (Fig. 2). Controls, omitting either primary or secondary antibody, were both negative (data not shown). In paraffin-embedded KCNE1 wild-type kidney sections, the KCNE1 protein could easily be detected in the brush-border membrane of proximal tubules, which was already shown for the rat (24). As expected, this signal was absent in kidney sections of KCNE1 knock-out mice (data not shown). Thus, these findings suggest the presence of KCNQ1 and KCNE3 in murine tracheal epithelial cells and a lack of KCNE1 expression in this tissue.

View larger version (56K): [in this window] [in a new window]   Fig. 1.   A, RT-PCR results with KCNQ1-, KCNE1-, and KCNE3-specific primers on tracheal epithelial cell cDNA of KCNE1 knock-out (/) and wild-type (+/+) mice. Both genotypes produce a band at the expected length for KCNQ1 (421 bp) and KCNE3 (302 bp), but KCNE1 (+/+) mice do not show a band for KCNE1 (297 bp), albeit cDNA from kidney gives a clearly visible band at the respective length. RT-controls were performed without Superscript RT to check for DNA contamination. B, Southern probing was used in a second set of experiments to detect even minute amounts of PCR products. KCNQ1 and KCNE3 were both present in trachea and colon, whereas KCNE1 was absent in these tissues but strongly expressed in kidney. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used to estimate total mRNA amount.

View larger version (55K): [in this window] [in a new window]   Fig. 2.   In contrast to the KCNE1 antibody (left panel), a KCNQ1-specific antibody (right panel) gives a green signal in the cytoplasm and the cell membrane of tracheal epithelial cells when examined with a laser scanning microscope, corresponding well with the earlier findings in RT-PCR experiments. Nuclei are stained with Hoe 333342 (blue color); the gray color represents the underlying differential interference contrast image.

To establish whether the expression of these two genes' products correlates with a detectable functional role, we performed in vitro Ussing chamber experiments with freshly excised murine tracheas. After the I_sc had reached a stable plateau (~40 min), the KCNQ1-specific inhibitor 293B (0.1-10 µM) was added to the basolateral side under three different conditions: 1) luminal control solution, 2) luminal amiloride (50 µM), and 3) both luminal amiloride and basolateral forskolin (5 µM) (Fig. 3, Table I). In the presence of luminal control solution, basolateral 293B (10 µM) reduced the I_sc by 39.1 ± 5.5 µA/cm² (n = 7). When amiloride was added to the luminal solution, the effect of 293B was reduced to 19.8 ± 2.2 µA/cm² (n = 6). Stimulation by forskolin in the presence of luminal amiloride induced a rise in I_sc of 49.1 ± 6 µA/cm² (n = 7). The current under this condition was inhibited by 293B by 45.7 ± 4.6 µA/cm² (n = 7; Figs. 3 and 4A, Table I). Compared with the current in the presence of luminal amiloride the 293B-sensitive current was doubled by the administration of forskolin, which underscores the importance of KCNQ1 complexes for cAMP activated secretion (Fig. 5). When added after the administration of amiloride and 293B, forskolin still induced a rise in I_sc of 26.4 ± 1.6 µA/cm² (n = 6, Table I). This current was completely inhibited by the selective inhibitor of the basolateral Na⁺2ClK⁺ cotransporter, azosemide (500 µM), indicating a 293B-insensitive component of cAMP-stimulated Cl secretion.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Original recording of an Ussing chamber experiment. 293B (10 µM) decreases Vte reversibly under bilateral control solution as well as in the presence of basolateral forskolin (5 µM). Voltage deflections are caused by short current pulses (500 nA) to estimate the trans-epithelial resistance and to calculate Isc according to Ohm's law after the experiment.

View larger version (62K): [in this window] [in a new window]   Fig. 4.   A, original recording showing the concentration-response relationship for luminally applied 293B in the presence of luminal amiloride (50 µmol/liter) and basolateral forskolin (5 µmol/liter). B, fitted concentration-response relationship for luminal (0.01-10 µM; , n = 8) and basolateral (0.1-10 µM; , n = 7) administration of the compound 293B.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of the chromanol 293B (10 µM) under bilateral control solution, luminal amiloride (50 µM), and also luminal amiloride and basolateral forskolin (5 µM). 293B reduced Isc under all conditions, suggesting a marked functional role for KCNQ1 complexes in tracheal epithelial transport.

The IC₅₀ of 293B given basolaterally after stimulation of Cl secretion by forskolin was 1.6 µM, which is in good agreement with previously published results in other tissues (25). The basolateral connective and muscular tissue in our experimental set-up, which could not be removed completely, constitutes a pharmacokinetic hindrance. Therefore we also established the IC₅₀ of 293B after luminal administration of the compound. Under these conditions the IC₅₀ was 0.4 µM, which is very close to the recently published value of 0.3 µM in human nasal epithelium (Fig. 4B) (19).

In addition to the molecular approach, we compared functional parameters in tracheal tissue from wild-type (KCNE1 +/+) and knock-out mice (KCNE1 /). Basal I_sc was increased in KCNE1 / to 228.4 ± 19.4 µA/cm² (n = 8) as compared with 165.5 ± 12.2 µA/cm² (n = 7) in KCNE1 +/+. KCNE1 / also showed an enhanced effect of forskolin (5 µM) and 293B (10 µM) compared with KCNE1 +/+. The forskolin-induced increase in I_sc was 83.7 ± 7.8 µA/cm² (n = 8) in KCNE1 / and 49.1 ± 6 µA/cm² (n = 7) in KCNE1 +/+, respectively. 293B decreased I_sc by 70.9 ± 10 µA/cm² (n = 8) in KCNE1 / compared with 45.7 ± 4.6 µA/cm² (n = 7) in wild-type mice (Fig. 6). The effect of 293B under control conditions was 30.2 ± 3.5 µA/cm² (n = 8) in KCNE1 /, which was not different from its effect in KCNE1 +/+ mice (39.1 ± 5.5 µA/cm², n = 7).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   KCNE1 knock-out mice (white bars, n = 8) had a stronger effect on forskolin and 293B thereafter compared with KCNE1 wild-type mice (black bars, n = 7). The 293B effect under bilateral control solution was not different between genotypes, and neither was the effect of amiloride.

To establish the contribution of KCNQ1 complexes to Ca²⁺-activated secretion, we used either luminal ATP (100 µM) or cyclopiazonic acid (CPA, 50 µM). Both agents are known to increase intracellular Ca²⁺ in the presence and absence of 293B. To rule out receptor desensitization or incomplete washout of 293B, these experiments were performed in a permutated manner. In the presence of luminal amiloride (50 µM) and basolateral forskolin (5 µM), 293B (10 µM) reduced the ATP- and CPA-induced peak rise in I_sc by about 80% (Table I). The effect of ATP without 293B was 461.9 ± 36.2 µA/cm² (n = 16), and the effect of CPA was 277.8 ± 40.3 µA/cm² (n = 6), respectively. After administration of 293B the short circuit current was reduced: 104.6 ± 8.1 µA/cm² (n = 16) for ATP and 49.8 ± 17 µA/cm² (n = 6) for CPA (Figs. 7 and 8), respectively. The rise in I_sc during the plateau phase after administration of ATP was not affected by 293B; it was 31.2 ± 5.4 µA/cm² (n = 16) without 293B and 32.1 ± 4.1 µA/cm² (n = 16) with 293B (10 µM). This result indicates the activation of channels other than KCNQ1 complexes to ensure the necessary basolateral K⁺ conductance during the plateau phase of ATP induced secretion.

View larger version (55K): [in this window] [in a new window]   Fig. 7.   Original recording demonstrating the effect of CPA (50 µM) in the presence and absence of 293B (10 µM). When CPA was administered under 293B, Vte increased only modestly. However, a strong increase in Vte was observed after the washout of 293B, paralleled by a decrease in Vte that corresponded to a great rise in Isc.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Summary of the effects of 293B (10 µM) on ATP (100 µM, n = 16)- and CPA (50 µM, n = 6)-induced Isc peak increase. Peak Isc was reduced by about 80% in the presence of 293B.

To establish the role of KCNQ1 complexes during pharmacological stimulation of secretion independent of changes in cytosolic cAMP or Ca²⁺, we compared the effect of 293B (10 µM) before and after the administration of 1-ethylbenzimidazolone (1-EBIO, 1 mM). In this series 293B reduced the basal I_sc by 13.3 ± 2.3 µA/cm² (n = 8). As expected, 1-EBIO after washout of 293B increased the I_sc from 29.4 ± 3.2 µA/cm² (n = 8) to a stable plateau of 66.1 ± 4.2 µA/cm² (n = 8). Surprisingly, the effect of 293B during administration of 1-EBIO was more than doubled as compared with control conditions (Fig. 9, Table I). 293B reduced the I_sc by 29.7 ± 5.2 µA/cm² (n = 8). Hence, 50% of the 1-EBIO effect was sensitive to inhibition by the chromanol 293B.

View larger version (64K): [in this window] [in a new window]   Fig. 9.   Original recording illustrating the effect of luminal 293B (10 µM) under luminal amiloride (50 µM). After washout of 293B, 1-EBIO (1 mmol) is administered luminally, leading to a rise in Vte, which subsequently stabilizes in a plateau. The latter is decreased largely by luminal application of 293B. The withdrawal of 1-EBIO results in a further decrease of Vte.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The role of KCNQ1 channel complexes in airway epithelial cells has not been studied thoroughly, which is in contrast to studies in small and large intestine (11, 26-28). So far there has even been doubt as to whether KCNQ1 plays a functional role in airway epithelium at all (17). In contrast to the -subunit KCNQ1, the -subunit KCNE1 had been associated in several studies with a functional role in airway epithelial cells (20, 21). We confirmed the expression of the -subunit KCNQ1 by RT-PCR alone, RT-PCR and subsequent Southern probing, immunofluorescence, and by demonstrating a 293B-sensitive current. KCNQ1 is expressed in the cytoplasmatic and the basolateral membranes of murine tracheal epithelial cells. In contrast to other groups, we could neither establish a relevant mRNA expression of KCNE1 with sensitive RT-PCR methods nor could we detect a protein expression with the help of a specific antibody in murine tracheal epithelial cells. As the molecular evidence for an expression of KCNE1 in trachea previously relied on RT-PCR in primary cultured rat tracheal epithelial cells, the apparent discrepancy of these results to our study might be explained by: (a) a species difference between rat and mouse, (b) an altered gene expression due to the 10-day exposure to culture medium (29), or (c) fundamental differences in the experimental protocols used (20). Instead of KCNE1 we found the related -subunit KCNE3 to be expressed in murine tracheal epithelial cells.

In KCNE1 knock-out mice, we did not observe a loss or reduction of function as one would expect if KCNE1 participates in Cl secretion. However, the I_sc was increased under control conditions and after stimulation with forskolin in KCNE1 knock-out mice. The latter finding is probably because of changes in systemic hormone levels of these animals that in turn might influence expression of genes important for fluid transport (30). In fact it has recently been shown that KCNE1 / mice do have an altered water and electrolyte balance, which is due in part to a renal Na⁺ loss (31) The latter finding might also explain the apparent conflict between our data and a recent publication demonstrating marked differences in volume regulation of airway epithelial cells of wild-type and KCNE1 / mice, respectively (21). However, the precise hormonal status and its possible impact on ion transport in airways need further investigation. Taking the molecular and functional experiments together, it seems prudent to conclude that KCNE1 is not detected in tracheal epithelial cells and that the KCNQ1 channel predominantly resembles a complex of KCNQ1 and KCNE3 as had been shown previously for the gut (11).

In the trachea, KCNQ1-KCNE3 channel complexes are already active under control conditions, which could be shown by the reduction of the basal I_sc by the chromanol 293B. This finding is in contrast to the colon where there is only little if any 293B-sensitive current without previous stimulation with agonists increasing cAMP (28). As the ion transport under control conditions predominately resemble Na⁺ reabsorption via the epithelial sodium channel (ENaC) and only slight anion secretion, it was interesting to address the question of whether KCNQ1 complexes also form part of the K⁺ conductance, which is needed for Na⁺ reabsorption. Indeed, the 293B-sensitive current was reduced by 50% after the administration of amiloride, indicating a role for these channel complexes not only in secretion but also in reabsorption, a function that has not yet been established in colon.

On the other hand, KCNQ1-KCNE3 complexes seem to mediate the basolateral K⁺ current that is necessary for base-line anion secretion. Previous reports have suggested that this basal anion secretion is due to relatively high intracellular Ca²⁺ levels at rest and is in part due to Cl secretion (32). In our experiments azosemide was not able to reduce the I_sc further than 293B, indicating that the chromanol had already blocked the base-line Cl secretion completely by inhibiting the relevant basolateral K⁺ conductance. Interestingly, even in the presence of 500 µM azosemide and 10 µM 293B a remaining I_sc of 22.8 ± 2.1 µA/cm² could be detected, which might be due in part to Na⁺-coupled uptake of glucose (33, 34).

Forskolin itself doubled the 293B-sensitive component of I_sc. In addition, a part of the forskolin-induced rise in trans-epithelial short circuit current was not inhibited by 293B, suggesting that K⁺ channels other than the KCNQ1 complexes were activated as well. In contrast to the colon it had been shown in freshly harvested tracheal epithelial cells that forskolin not only augments intracellular cAMP levels but also induces a rise in intracellular Ca²⁺ (17, 32, 35). It had been suggested that a considerable amount of the forskolin-activated secretion is due to this rise in intracellular Ca²⁺ levels (29). We tested the effect of 293B on the ATP- and CPA-induced rise in I_sc. ATP increases intracellular Ca²⁺ levels and activates the protein kinase C via generation of diacylglycerol (36, 37). The fungus toxin cyclopiazonic acid inhibits the sarcolemmal Ca²⁺ ATPase and therefore solely leads to an increase of intracellular Ca²⁺ levels without concomitantly activating the protein kinase C via diacylglycerol. Under both circumstances 293B reduced the peak rise in I_sc by about 80% without affecting the following plateau. As Ca²⁺ levels are highest during the peak rise, it might be that KCNQ1 channel complexes have a lower Ca²⁺ sensitivity than other known Ca²⁺-activated K⁺ channels (such as mIK1). The lower affinity of KCNQ1 complexes might explain why the channel is only activated in the Ca²⁺ peak but not during the following plateau phase. A positive regulation of KCNQ1 complexes by both Ca²⁺ and cAMP would therefore seem reasonable in this epithelium.

This hypothesis is supported further by the fact that mice in contrast to humans have a considerable amount of Ca²⁺-activated Cl secretion, which is due to the expression of mCLCA1, a Ca²⁺-activated Cl channel (38, 39). The abundance of mCLCA1 might also explain the lack of a lung phenotype in murine CF models (40, 41). This notion is supported by the finding in a recent mRNA study of little CFTR expression in murine trachea (42). In fact, the nearly 10-fold increase of I_sc observed after administration of ATP compared with forskolin points in this direction. Bearing this in mind it seems justified to conclude that KCNQ1-KCNE3 complexes in murine tracheal epithelial cells are regulated via both Ca²⁺ and cAMP. The cAMP component might be underestimated in murine trachea due to a somewhat scanty expression of CFTR in this tissue (42). This is in contrast to the colon, where cAMP is known to decrease intracellular Ca²⁺ levels, and hence such a regulation of KCNQ1 channel complexes would not be favorable (43). As the channel subunit composition in mouse trachea seems to be the same as in the colon, one could speculate that a yet unidentified third player confers these characteristics to the native KCNQ1 channel complex.

1-EBIO doubled the base-line I_sc in our in vitro trachea preparation and thereby confirmed data gathered from experiments with primary cultured human and murine airway epithelial cells (7, 44). Surprisingly, part of this 1-EBIO-induced rise of I_sc was 293B-sensitive, which stands in clear contrast to the literature, where this current is predominantly attributed to mIK1 channels. There are at least three different explanations for our findings. 1) 1-EBIO not only activates mIK1 but also KCNQ1 channel complexes and hence leads to an increase in KCNQ1 mediated conductance. 2) 1-EBIO activates luminal Cl channels, which in consequence increases the basal anion secretion. Administration of 293B then leads to an apparently increased 293B-sensitive I_sc without an activation of KCNQ1 complexes (6). 3) 1-EBIO, as already described for mesenteric arteries, increases nitric oxide formation in tracheal epithelial cells, which leads to an activation of KCNQ1 channel complexes via a protein kinase G II-mediated process (45). From our experiments we cannot judge which possibility is the appropriate one. Further studies are required to elucidate the precise mechanism by which 1-EBIO activates trans-epithelial ion secretion.

In summary, we conclude that the native KCNQ1 channel complex in mouse tracheal epithelial cells is formed by KCNQ1-KCNE3 and constitutes the dominating basolateral K⁺ conductance and is activated by both cAMP as well as Ca²⁺. It is not only a prerequisite for Cl secretion but also supports Na⁺ reabsorption via the epithelial sodium channel. To our surprise it is in one way or the other also involved in the secretion mechanisms induced by 1-EBIO. Our findings suggest that 1-EBIO enhances not only the IK1 but also a KCNQ1 channel complex.

	ACKNOWLEDGEMENT

We thank Prof. Dr. R. J. Bridges and Prof. Dr. H. Oberleithner for critical reading of the manuscript. We acknowledge the expert technical assistance of M. Grimm and W. Rohm.

	FOOTNOTES

^* This work was supported by the Forschungsförderung des Landes Baden-Württemberg.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.

This work is dedicated to our mentor and colleague, Prof. Dr. Rainer Greger, Institute of Physiology, Albert-Ludwigs-Universität, Frieburg, Germany.

^§ These authors contributed equally to this work.

Supported by a fellowship from the European Molecular Biology Organization (EMBO).

^§§ To whom correspondence should be addressed: Physiologisches Institut Abteilung Vegetative Physiologie, Westfälische Wilhelms Universität Münster, Robert-Koch-Str. 27a, D-48149 Münster, Germany. Tel.: ++49 251-83-55327; Fax: ++49 251-83-5331; E-mail: hugma@uni-muenster.de.

Published, JBC Papers in Press, August 29, 2001, DOI 10.1074/jbc.M105014200

	ABBREVIATIONS

The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; bp, base pair(s); m, mouse; CPA, cyclopiazonic acid; 1-EBIO, 1-ethylbenzimidazolone; Tm, melting temperature.

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