The cardiac K+ channel KCNQ1 is essential for gastric acid secretion

GASTROENTEROLOGY 2001;120:1363–1371

The Cardiac Kⴙ Channel KCNQ1 Is Essential for Gastric Acid Secretion FLORIAN GRAHAMMER,* ANDREAS W. HERLING,‡ HANS J. LANG,‡ ANNETTE SCHMITT–GRA¨FF,§ OLIVER H. WITTEKINDT,* ROLAND NITSCHKE,* MARKUS BLEICH,‡ JACQUES BARHANIN,储 and RICHARD WARTH 储 Institutes of *Physiology and §Pathology, Albert-Ludwigs-University, Freiburg, Germany; ‡Aventis Pharma GmbH, Frankfurt am Main, Germany; and 储IPMC, CNRS, Valbonne Sophia Antipolis, France

Background & Aims: Gastric Hⴙ secretion via the Hⴙ/ Kⴙ–adenosine triphosphatase is coupled to the uptake of Kⴙ. However, the molecular identity of luminal Kⴙ channels enabling Kⴙ recycling in parietal cells is unknown. This study was aimed to investigate these luminal Kⴙ channels. Methods: Acid secretion was measured in vivo and in vitro; KCNQ1 protein localization was assessed by immunofluorescence, and acid-sensitivity of KCNQ1 by patch-clamp. Results: We identified KCNQ1, which is mutated in cardiac long QT syndrome, as a Kⴙ channel located in tubulovesicles and apical membrane of parietal cells, where it colocalized with Hⴙ/Kⴙ–adenosine triphosphatase. Blockade of KCNQ1 current by 293B led to complete inhibition of acid secretion. The putative KCNQ1 subunits, KCNE2 and KCNE3, were abundant in human stomach; KCNE1, however, was absent. Coexpression of KCNE3/KCNQ1 in COS cells led to an acid-insensitive current; KCNE2/KCNQ1 was activated by low extracellular pH. Conclusions: We identified KCNQ1 as the missing luminal Kⴙ channel in parietal cells and characterized its crucial role in acid secretion. Because KCNE3 and KCNE2 are expressed in human stomach, one or both are candidates to coassemble with KCNQ1 in parietal cells. Thus, stomach- and subunit-specific inhibitors of KCNQ1 might offer new therapeutical perspectives for peptic ulcer disease.

he ionic mechanisms underlying gastric acid secretion have been a matter of debate for decades. Purification and functional characterization of the gastric H⫹/K⫹–adenosine triphosphatase (ATPase)1,2 and cloning of the ␣- and ␤-subunit of the enzyme3,4 led to an understanding of proton secretion on the molecular level. The H⫹/K⫹-ATPase couples H⫹ exit to K⫹ uptake. Therefore, luminal K⫹ recycling and Cl⫺ exit are essential for HCl secretion.5–9 A splice variant of the ubiquitous CLC-2 Cl⫺ channel10 and parchorin,11 a CLC-related protein, were identified as luminal Cl⫺ channels. Whole cell experiments revealed a Ca2⫹-activated K⫹

T

conductance in a human gastric epithelial cell line.12 K⫹ channels in the basolateral membrane of oxyntic cells have been characterized by electrophysiological methods.13,14 There is also good evidence for K⫹ channels in the luminal membrane5,15; however, the molecular identity of the luminal K⫹ channels remained unknown. The K⫹-channel gene KCNQ1 (also called KvLQT1) was found to be mutated in the hereditary Long QT syndrome type 1.16 In the heart and inner ear, KCNQ1 coassembles with KCNE1 (IsK,17 minK) to form the native K⫹ channel.18,19 Besides its expression in the heart and inner ear, KCNQ1 is abundant in a variety of epithelia, i.e., kidney, pancreas, small and large intestine, and stomach.20 –22 Previously, we have shown that KCNQ1 is a prerequisite for adenosine 3⬘,5⬘-cyclic monophosphate (cAMP)-activated Cl⫺ secretion in colonic mucosa,23,24 where it coassembles with KCNE3 (MiRP2) to form the native channel.25 Very recently, KCNE2 (MiRP1), which underlies cardiac IKr current when associated with HERG,26 was also shown to coassemble with KCNQ1.27 We examined the distribution of KCNQ1 in gastric mucosa and characterized its function during acid secretion. We found the putative KCNQ1 subunits KCNE2 and KCNE3 abundant in human stomach and examined the acid sensitivity of KCNE2/KCNQ1 and KCNE3/ KCNQ1 channels in COS cells. In contrast to rodent stomach, KCNE1 was absent in human tissue. To investigate the role of KCNE1 in mouse stomach, we measured acid secretion in wild-type and KNCE1-knockout mice.28 Abbreviations used in this paper: CCH, carbachol; CHO, Chinese hamster ovary; dbcAMP, dibutyryladenosine 3ⴕ,5ⴕ-cyclic monophosphate; PCR, polymerase chain reaction. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.24053

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Materials and Methods Animals The experimental protocols were approved by the local councils for animal care and were conducted according to the German law for the care and use of laboratory animals.

Immunofluorescence Paraffin-embedded sections of human oxyntic gastric biopsy specimens (archive material) and mouse stomach were used. After removal of paraffin, the sections were boiled for 3 minutes in citrate buffer (10 mmol/L, pH 6.0). The KCNQ1 antibodies were obtained by immunizing rabbits with a peptide (CPADLGPRPRVSLDPRVSIY) of the cytosolic N-terminus (Davids Biotechnologie, Regensburg, Germany). The KCNQ1 antibodies were affinity purified before use. A monoclonal antibody was used for H⫹/K⫹-ATPase staining (Dianova, Hamburg, Germany29). The antibodies (1:500 and 1:1000) were dissolved in antibody dilution solution (Dako, Carpinteria, CA). Alexa488 goat anti-rabbit (Molecular Probes, Leiden, The Netherlands) and cy3 goat anti-mouse (Dianova) were used as secondary antibodies, and HOE 33342 (2 ␮mol/L; Molecular Probes) was used for staining of the nuclei. The sections were examined with a confocal microscope using sequential excitation to avoid emission cross talk (LSM 510; Zeiss, Jena, Germany; Alexa488: excitation 488 nm, emission 505–550 nm; Cy3: excitation 543 nm, emission ⬎560 nm).

Western Blots Human KCNQ1-transfected and nontransfected Chinese hamster ovary (CHO) cells were washed with phosphatebuffered saline (PBS) and resuspended in 70 ␮L lysis buffer (10 mmol/L 3-(N-morpholino)propanesulfonic acid, 1 mmol/L EDTA, 100 mmol/L dithiothreitol, 2% sodium dodecyl sulfate, and 1 mmol/L phenylmethylsulfonyl fluoride, pH 7.2). After swelling on ice for 30 minutes, the cell suspension was sonicated and incubated on a shaker at room temperature for 15 minutes. The proteins were separated in a Mini-Protean II Gel chamber (Bio-Rad, Hercules, CA) by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto nitrocellulose. The membrane was blocked with PBS/0.1% Tween 20 containing 5% blocking agent (Amersham Pharmacia, Freiburg, Germany) for 1 hour at room temperature and incubated with affinity-purified, polyclonal KCNQ1 antibodies (1:1000) overnight at 4°C. Primary antibodies were labeled with peroxidase-conjugated anti-rabbit antibodies (1:5000; Amersham Pharmacia) for 1 hour at room temperature. Chemiluminescence was detected by exposing a chemiluminescence film (ECL kit and Hyperfilm ECL; Amersham Pharmacia) for 20 seconds to the membrane. 14C-Aminopyrine

Accumulation in Isolated

Gastric Glands Rabbits (2–3 kg body wt) were killed by cervical dislocation during anesthesia. Gastric glands were isolated as described elsewhere30 and transferred into a medium contain-

ing (in mmol/L) 100 NaCl, 5 KCl, 0.5 NaH2PO4 , 1 Na2HPO4 , 1 CaCl2 , 1.5 MgCl2 , 20 NaHCO3 , 20 HEPES, 1.1 glucose, and 1 g/L rabbit albumin. Finally, the glands were diluted to a concentration of 2– 4 g dry wt/L. Samples of 1 mL gland suspension were equilibrated for 20 minutes at 37°C in 1 mL medium containing 0.1 mCi/L 14C-aminopyrine and 293B (Aventis Pharma GmbH, Frankfurt, Germany) or vehicle, respectively. Next, either carbachol (CCH, 100 ␮mol/L; Sigma, Deisenhofen, Germany) or dibutyryladenosine 3⬘,5⬘cyclic monophosphate (dbcAMP) (300 ␮mol/L; Merck, Darmstadt, Germany) was added for 30 and 45 minutes, respectively. Then the glands were separated from the medium by brief centrifugation. Radioactivity of aliquots of both the digested gland pellet and the supernatant was determined in triplicate in a liquid scintillation counter. The 14C-aminopyrine accumulation as a measure of acid secretion was calculated as the ratio between 14C-aminopyrine in the intraglandular water and in the incubation medium.31

Stomach-Lumen–Perfused Mice KCNE1-knockout mice were fasted 2– 4 hours before the experiment with free access to 5% glucose solution. Mice were anesthetized with ketamine (100 mg/kg body wt intraperitoneally [IP]; Sigma)/xylazine (4 mg/kg body wt IP; Bayer, Leverkusen, Germany) and trachea and femoral vein were cannulated. A modified saline solution (0.9% NaCl, 5% glucose, and 0.1% bovine serum albumin, pH 7.4) was infused at a rate of 0.15 ␮L 䡠 min⫺1 䡠 g⫺1. The abdominal cavity was opened and pylorus was ligated. After preparation of the vagus nerve, the esophagus was ligated also. A double-lumen perfusion cannula was fixed in the forestomach.32 The stomach was perfused continuously with saline (37°C; pH 7.0; 200 ␮L/ min). After an equilibration period of 30 minutes, the perfusate was collected every 10 minutes and retitrated to pH 7.0 to measure acid output. Thirty minutes under control conditions was followed by a stimulation phase (histamine ⫹ CCH ⫹ pentagastrin intravenously [IV]). After another 30 minutes of stimulation, 293B was administered IP.

Stomach-Lumen–Perfused Rats Male Sprague–Dawley rats (300 –350 g) were anesthetized with 30% (wt/vol) urethane (5 mL/kg intramuscularly) and tracheotomized. Abdominal surgical procedures for rats were similar to those already described for mice. The stomach was perfused at a rate of 1 mL/min, and the perfusate was collected every 15 minutes. The corresponding acid output was measured by retitration to pH 7.

Heidenhain Pouch in Dogs Male Beagle dogs were prepared with Heidenhain pouches.33,34 After surgery, they were allowed to recover for at least 6 weeks and were trained to stay in a Pavlov stand. Food was withdrawn 18 hours before the experiment, but tap water was given ad libitum. During the experiments, gastric juice was collected from the pouches every 30 minutes and acid output was measured by retitration to pH 7.

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Patch-Clamp Experiments on Transfected Cells Human KCNE2 and KCNE3 were cloned in the pIRES-CD8 mammalian expression vector.35 COS cells were transiently cotransfected with pCI-hKCNQ1 and either KCNE2 or KCNE3 (each 0.5 ␮g per dish) using the FuGENE transfection reagent (Roche, Mannheim, Germany). Standard whole cell recordings were performed on transfected cells, as visualized by CD8 expression. The patch pipette solution was (in mmol/L): 110 K-gluconate, 30 KCl, 4.8 Na2HPO4 , 1.2 NaH2PO4 , 5 glucose, 2.38 MgCl2 , 0.73 CaCl2 , 1 ethylene glycol-bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid, and 3 adenosine triphosphate, pH 7.2. The extracellular solution was (in mmol/L): 150 NaCl, 5 KCl, 2 MgCl2 , 1 CaCl2 , and 5 HEPES, pH 7.4 or 5.5.

Polymerase Chain Reaction Experiments Complementary DNA was synthesized using human stomach total RNA (Cat. No. #64090-1; Clontech, Palo Alto, CA). Human KCNQ1 (base pairs [bp] 2078 –2645 from GenBank AF000571), KCNE1 (bp 53– 425 from GenBank NM000219), KCNE2 (bp 136 –513 from GenBank AF302095), and KCNE3 (bp 298 – 615 from GenBank AF302494) were amplified using 18-bp primers. The polymerase chain reaction (PCR) conditions were: 3 minutes at 94°C denaturation; 30 seconds at 94°C, 25 seconds at 55°C, and 20 seconds at 72°C for 31 cycles. Specific PCR products were detected by Southern probing using internal oligonucleotides.

Determination of the 293B Concentration in Serum 293B was extracted from mice serum samples via an EXTRELUT column (Merck) and analyzed by high-performance liquid chromatography at Aventis Pharma GmbH.

BCECF pH Measurements Intracellular pH was measured with a confocal laser scanning microscope (LSM 510 UV; Zeiss, Jena, Germany) using the ratiometric pH dye BCECF (2⬘,7⬘-bis-[2-carboxyethyl]-5-[and-6]-carboxy-fluorescein). BCECF was excited at 457 nm and 488 nm using the bidirectional scan mode; emission signal was collected at ⬎505 nm. Images were taken with a 40⫻ objective (C-APO 40/1.2 water; Zeiss) at a pixel time of 2.24 microseconds. HeLa cells grown on glass cover slips (30 mm A) were loaded with BCECF-AM (10 ␮mol/L; Molecular Probes) for 30 minutes at 20°C. Mean fluorescence ratios were calculated for single cells on a pixel by pixel basis using the Metafluor software (Universal Imaging, West Chester, PA).

Chemicals Standard chemicals were purchased from Sigma and Aldrich (Deisenhofen, Germany), and desglugastrin, 293B, 377B, and 389B were synthesized by Aventis Pharma GmbH. 293B, 377B, and 389B were dissolved in dimethyl sulfoxide

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(0.1 mol/L) and diluted to their final concentration in the experimental solutions.

Statistics Data are shown as mean values ⫾ SEM from n observations. The paired and unpaired Student t tests were used as appropriate. A P value of ⱕ0.05 was accepted to indicate statistical significance.

Results Localization of KCNQ1 in Gastric Mucosa Previously, Northern hybridization revealed a strong KCNQ1 expression in human stomach.21 However, the cellular and subcellular localization and the functional role of KCNQ1 in this tissue remained unclear. To examine the distribution of KCNQ1 in gastric mucosa, we performed immunofluorescence measurements on paraffin-embedded sections from murine (data not shown) and human oxyntic gastric mucosa (from biopsy specimens). CHO cells transfected with human KCNQ1 were used to confirm specificity of the affinitypurified antibodies, and nontransfected CHO cells served as a control (Figure 1A and B). In Western blots, a 75-kilodalton band was detected by the antibodies using proteins from transfected cells (Figure 1C). In human and murine gastric mucosa, KCNQ1 staining was prominent in a cell type, which could be identified by differential interference contrast optics as parietal cells. The staining pattern in parietal cells suggested a localization of KCNQ1 in the tubulovesicles and the canaliculi, which belong to the luminal membrane compartment. This hypothesis was confirmed by costaining of gastric mucosa with KCNQ1- and H⫹/ K⫹-ATPase–specific antibodies (Figure 2A–F ). In fact, the localization of the H⫹/K⫹-ATPase staining in parietal cells was similar to that for KCNQ1. Besides the abundance in parietal cells, a weaker KCNQ1 staining was observed at the basolateral membrane of gastric foveolar cells (Figure 2B). Role of KCNQ1 During Acid Secretion In Vivo We addressed the question whether KCNQ1 plays a role during gastric acid secretion. For this purpose, we examined the effect of the KCNQ1 inhibitor 293B (trans-6-cyano-4-[N-ethylsulfonyl-N-methylamino]3-hydroxy-2,2-dimethyl-chromane36) on gastric acid secretion in stomach-perfused rats. Under control conditions, acid secretion was 0.77 ⫾ 0.12 ␮mol H⫹/min (n ⫽ 14). Next we stimulated secretion with CCH, desglugastrin, or histamine. 293B (5 mg/kg bolus IV)

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Figure 1. (A ) No KCNQ1-specific ( green) staining in CHO control cells; blue HOE33342 staining of the nuclei. (B) Specific fluorescence ( green) in CHO cells transfected with human KCNQ1. Transfection efficiency was in the range of 50%. (C ) Western blot of CHO control cells and hKCNQ1-transfected CHO cells. A 75-kilodalton band was detected by the antibodies. Higher molecular weight bands might be caused by aggregation of several subunits.

Figure 2. KCNQ1 in human oxyntic gastric mucosa from a biopsy. (A ) H⫹/K⫹-ATPase–specific staining was observed in parietal cells. (B) KCNQ1-specific antibodies show a distribution pattern in parietal cells similar to that observed for the monoclonal H⫹/K⫹-ATPase antibody. Besides the strong staining of parietal cells, the basolateral membrane of foveolar cells (upper part of the picture) was stained, suggesting that KCNQ1 also plays a role for secretion in this cell type. The inserts show control experiments without secondary (upper insert) and without primary (lower insert) antibodies. (C ) Overlay of KCNQ1 ( green), H⫹/K⫹-ATPase (red ), cell nuclei staining (HOE33342; blue), and differential interference contrast picture. Colocalization of H⫹/K⫹-ATPase and KCNQ1 led to the yellow color. (D) H⫹/K⫹-ATPase staining in human parietal cells at a higher magnification. (E ) KCNQ1-specific fluorescence in the same area as in D. (F ) Overlay of KCNQ1 ( green), H⫹/K⫹-ATPase (red ), HOE33342 nuclei staining (blue), and differential interference contrast picture; colocalization in yellow.

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reduced H⫹ secretion from the plateau value in the presence of CCH (30 ␮g 䡠 kg⫺1 䡠 h⫺1) from 5.74 ⫾ 0.52 to 0.54 ⫾ 0.09 ␮mol H⫹/min (n ⫽ 4), during desglugastrin (100 ␮g 䡠 kg⫺1 䡠 h⫺1) stimulation from 5.71 ⫾ 0.93 to 0.64 ⫾ 0.09 ␮mol H⫹/min (n ⫽ 5), and during histamine (10 mg 䡠 kg⫺1 䡠 h⫺1) stimulation from 5.94 ⫾ 0.85 to 1.00 ⫾ 0.29 ␮mol H⫹/min (n ⫽ 5; Figure 3A). The effect of 293B was maximal after 30 minutes and partially reversible within 90 minutes. The 293B-related substances 377B (trans-6-cyano-4-[N-(dimethylamino)sulfonyl-N-methylamino]-3-hydroxy-2,2-dimethylchromane)and389B(trans-4-[N-ethylsulfonyl-N-methylamino]-3-hydroxy-6-tetramethylene-chromane)23 also inhibited acid secretion at 5 mg/kg IV by 85% and 30%, respectively (each n ⫽ 6). To exclude species-specific effects of 293B, we examined gastric acid secretion in Heidenhain pouch dogs. Dogs received histamine (50 ␮g 䡠 kg⫺1 䡠 h⫺1 IV) or CCH (10 ␮g 䡠 kg⫺1 䡠 h⫺1 IV) for stimulation of H⫹ secretion. 293B (3 mg/kg bolus IV) reversibly diminished histamine-induced acid output from 107 ⫾ 29 to 18 ⫾ 2

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␮mol H⫹/min (n ⫽ 3) and CCH-induced acid output from 145 ⫾ 31 to 22 ⫾ 18 ␮mol H⫹/min (n ⫽ 3, Figure 3B). In the rodent stomach, KCNE1,17 KCNE2, and KCNE3 are expressed (data not shown). We investigated the role of KCNE1 in mouse parietal cells using the KCNE1-knockout mouse. Acid secretion of stomachperfused 8-week-old KCNE1–wild-type (KCNE1 ⫹/⫹) and -knockout mice (KCNE1⫺/⫺) was measured under control and after maximal gastric stimulation (pentagastrin 44 ␮g 䡠 kg⫺1 䡠 h⫺1 ⫹ carbachol 3 ␮g 䡠 kg⫺1 䡠 h⫺1 ⫹ histamine 128 ␮g 䡠 kg⫺1 䡠 h⫺1, IV). This stimulation induced a peak secretion followed by a stable plateau phase (Figure 3C). In KCNE1 ⫹/⫹, this stimulation increased H⫹ output from 33 ⫾ 1 to 570 ⫾ 100 nmol H⫹/min (n ⫽ 8), and even more in KCNE1⫺/⫺ from 41 ⫾ 8 to 920 ⫾ 130 nmol H⫹/min (n ⫽ 7). 293B (5 mg/kg IP) almost completely inhibited gastric acid secretion in the plateau phase from 430 ⫾ 80 to 100 ⫾ 30 nmol H⫹/min in KCNE1 ⫹/⫹ (n ⫽ 8) and from 550 ⫾ 190 to 110 ⫾ 40 nmol H⫹/min in KCNE1 ⫺/⫺ (n ⫽ 7). The dose of

Figure 3. (A ) Acid secretion in rat lumen perfused stomach. H⫹ secretion was stimulated by either histamine (10 mg 䡠 kg⫺1 䡠 h⫺1 IV, n ⫽ 5), desglugastrin (gastrin, 100 ␮g 䡠 kg⫺1 䡠 h⫺1 IV, n ⫽ 5), or carbachol (CCH, 30 ␮g 䡠 kg⫺1 䡠 h⫺1 IV, n ⫽ 4). 293B (5 mg/kg bolus IV) strongly reduced secretion. (B) Heidenhain pouch experiments in dogs. After activation of H⫹ secretion by histamine (50 ␮g 䡠 kg⫺1 䡠 h⫺1), 293B (3 mg/kg bolus IV) reversibly inhibited secretion (n ⫽ 3). (C ) Effect of acid stimulation (pentagastrin 44 ␮g 䡠 kg⫺1 䡠 h⫺1 IV, carbachol 3 ␮g 䡠 kg⫺1 䡠 h⫺1 IV, histamine 128 ␮g 䡠 kg⫺1 䡠 h⫺1 IV) and inhibition by 293B (5 mg/kg bolus IP) in KCNE1 ⫹/⫹ (open bars, n ⫽ 8) and KCNE1 ⫺/⫺ (filled bars, n ⫽ 7) mice. Basal secretion was not different; after stimulation, the peak value was even higher in KCNE1 ⫺/⫺ (indicated by §), indicating that the KCNE1 gene knockout does not diminish murine gastric H⫹ secretion. (D) Concentration-response curve of 293B obtained from 14C-aminopyrine accumulation assays in isolated rabbit gastric glands in the presence of carbachol (100 ␮mol/L, n ⫽ 6).

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5 mg/kg 293B IP corresponded to a serum concentration of 7.55 ⫾ 0.54 ␮mol/L 293B, indicating a high 293B sensitivity of the gastric KCNQ1 channel complex as reported for KCNE2/KCNQ127 and KCNE3/KCNQ1.23,25 Effect of 239B in Isolated Rabbit Gastric Glands To rule out systemic side effects of 293B as the way of action for the inhibition of acid secretion, we examined the effect of 293B on H⫹ secretion in isolated rabbit gastric glands using the 14C-aminopyrine accumulation method. The ratio of 14C-aminopyrine in glandular water and medium was determined as a measure of acid production. Under control conditions, the 14Caminopyrine ratio was 14.7 ⫾ 1.0 (n ⫽ 12). Histamine (100 ␮mol/L) induced an increase to 42.6 ⫾ 4.1 (n ⫽ 7), dbcAMP (300 ␮mol/L) to 49.7 ⫾ 4.3 (n ⫽ 10), and CCH (100 ␮mol/L) to 61.5 ⫾ 8.5 (n ⫽ 6). 293B inhibited the accumulation of 14C-aminopyrine in a concentration-dependent manner with half maximal inhibition in the range of 3–20 ␮mol/L (Figure 3D). These IC50 values are in good agreement with values obtained from other tissues where KCNQ1 is expressed.37 These data indicate that the inhibition of gastric acid secretion by 293B was not due to “extragastric” side effects. Acid Sensitivity of Possible Gastric KCNQ1 Channels KCNQ1 is known to coassemble with proteins of the KCNE family. We examined expression of KCNE1-3 in human gastric mucosa using PCR techniques. KCNE1, KCNE2, and KCNE3 were abundant in mouse gastric mucosa. In human stomach KCNE2 and KCNE3 were strongly expressed; however, KCNE1 was absent (Figure 4A). Next we examined the effect of acidic pH on COS cells coexpressing human KCNQ1 and either human KCNE2 or human KCNE3. Whole cell current was determined under control conditions (pH 7.4) and in acidic bath solution (pH 5.5) using clamp voltages from ⫺95 to ⫹5 mV (2-second pulse duration, 20-mV steps). In KNCE2/ KCNQ1-expressing cells the reduction of extracellular pH led to a strong increase in 293B-sensitive K⫹ current, paralleled by a hyperpolarization. In contrast, KCNE3/KCNQ1 current was not affected by a reduction of external pH (Figure 5A and B). Does 293B Act as a Protonophore? In the past several compounds suggested to block acid secretion via inhibition of distinct target proteins turned out to act as protonophores.7 Although the chemical structure of 293B suggested that a direct protono-

Figure 4. Expression of KCNQ1, KCNE1, KCNE2, and KCNE3 in the human stomach. Southern probing identified specific PCR products. KCNQ1 (567 bp), KCNE2 (377 bp), and KCNE3 (317 bp) were abundant. No PCR product was obtained with primers for KCNE1 (372 bp), which gave rise for specific product in human heart (not shown).

phoretic action is unlikely, we performed BCECF fluorescence measurements of HeLa cells to exclude an increase in H⫹ permeability because of unspecific effects of 293B. Acidification of the external solution from pH 7.4 to 6.0 led to an acidification of the cells. Addition of 293B (100 ␮mol/L) did not further increase the rate of acidification (Figure 5C shows 1 of 8 similar experiments), nor did it change cytosolic pH under control conditions (not shown). In contrast, acetate and NH4⫹ induced the expected pH changes due to nonionic diffusion of acetic acid and NH3 and following deprotonization and protonization, respectively.

Discussion Previous studies elucidated the key role of the H⫹/K⫹-ATPase for gastric acid secretion in parietal cells.1,38 The H⫹/K⫹-ATPase takes up K⫹ whenever H⫹ is pumped out of the cell. Therefore, a K⫹ recycling pathway is required. Vesicle studies revealed conductive pathways for Cl⫺ and K⫹ in oxyntic cells.6,39 It was suggested that the activation of the luminal K⫹ conductance might regulate H⫹ output since the H⫹/K⫹ATPase itself seems to be constitutively active during stimulation of acid secretion.7 Up to now, the luminal K⫹ channel of mammalian parietal cells has not been characterized on a single channel level and its molecular identity has been unknown. In the present study we show an immunostaining of KCNQ1-specific antibodies in human parietal cells, which is similar to the distribution observed for H⫹/K⫹ATPase–specific monoclonal antibody,29 namely a location in the tubulo-vesicles, the canaliculi system, and the luminal membrane. It clearly differs from the basolateral

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Figure 5. (A ) Effect of extracellular pH on KCNE2/KCNQ1-coexpressing COS cells. Total whole cell current (normalized to the capacitance of the cells, left-hand side) and 293B-sensitive current (293B, 10 ␮mol/L, right-hand side) are depicted. Reduction of external pH from 7.4 to 5.5 increased both total current and 293B-sensitive current (n ⫽ 7, paired experiments). (B) Effect of acidic pH on KCNE3/KCNQ1 (same protocol as in A ). Whole cell current and 293B-sensitive current were not affected by acidic pH (n ⫽ 5, paired experiments). (C ) Effect of 293B on BCECF fluorescence ratio (488 nm/458 nm) as a measure of cytosolic pH. In the presence of acidic extracellular solution (pH 6.0), 293B (100 ␮mol/L) did not further increase the rate of acidification. In contrast, acetate and NH4⫹ elicited the expected changes in cytosolic pH (original traces of 7 cells).

staining shown for aquaporin-4 in parietal cells.40 However, H⫹/K⫹-ATPase and KCNQ1 were not completely colocalized. Most likely, the expression of KCNQ1 and H⫹/K⫹-ATPase are differentially regulated and might depend on cell cycle or function. The staining pattern suggests a functional coupling of both proteins in the same cell compartment rather than a fixed stoichiometry

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or direct protein-protein interaction. Therefore, KCNQ1 might play a role in K⫹ recycling over the luminal membrane. To examine the physiologic role of KCNQ1 in parietal cells we performed measurements of gastric acid secretion in rats and dogs in vivo. The chromanols 293B, 377B, and 389B, which are known KCNQ1 inhibitors,23,36,41 were used as pharmacologic tools to examine the function of KCNQ1 during secretion. Irrespective of the hormone used for acid secretion stimulation (CCH, histamine, desglugastrin), 293B reversibly blocked acid secretion. 377B exerted a similar effect; 389B was less potent. At concentrations higher than 20 ␮mol/L 293B also blocks Kv4.3, Kv1.5,41 and ITo channels.42 However, gastric H⫹ secretion was much more sensitive for inhibition by 293B: a 293B serum concentration of approximately 8 ␮mol/L was sufficient to inhibit acid secretion in mice by some 85%. These data speak in favor of a 293B inhibition of KCNQ1 associated to a subunit that increased 293B sensitivity as described for KCNE2/ KCNQ127 and KCNE3/KCNQ1.25 However, unspecific effects of 293B via inhibition of other ion channels cannot be completely ruled out, although such effects are not likely at the concentrations used in this study. Moreover, 293B did not inhibit H⫹ secretion via a protonophoretic action. KCNQ1 is expressed in a variety of epithelial tissues, inner ear, and heart muscle. To exclude an indirect inhibition of acid secretion, we performed in vitro experiments with isolated rabbit gastric glands. Using the 14C-aminopyrine accumulation method as a measure of acid secretion we observed activation of secretion in the presence of CCH, histamine, and dbcAMP. 293B blocked H⫹ secretion irrespective of the hormone by which secretion was activated indicating a direct action on parietal cells. Moreover, the inhibitory effect on dbcAMP-induced secretion ruled out indirect inhibition of H⫹ secretion via blockade of hormone receptors or cAMP production. In heart and inner ear KCNQ1 coassembles with KNCE1 (IsK or minK) to form the native channel complex.18,19 We failed to detect KCNE1 mRNA in human stomach, but there is good evidence for expression in rodent stomach.17 Thus, we examined the role of KCNE1 for gastric acid secretion in rodents using the KCNE1knockout mouse. Interestingly, there was no defect in maximal acid secretion and normal 293B sensitivity in KCNE1-knockout mice, indicating that KCNE1 is not crucially involved in acid secretion in mice. KCNE2 and KCNE3 are abundant in both rodent and human stomach. KCNE3/KCNQ1 was shown to be ac-

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tivated by cAMP25 and, therefore, KCNE3 could be a candidate for the small 18-kilodalton protein which was described to induce a PKA-dependent K⫹ conductance in isolated gastric microsomes.43 KCNQ1 and KCNE1/KCNQ1 were shown to be inhibited by acidic extracellular pH.44 In contrast, our whole cell data of KCNE2/KCNQ1-expressing COS cells suggest that this channel complex is activated by external pH 5.5 (more acidic solutions could not be applied to COS cells). Clearly, further studies are required to examine the regulation of KCNE2/KCNQ1, since changes in cytosolic pH and/or Ca2⫹ cannot be excluded when the external pH is changed to 5.5. KCNE3/KCNQ1 channels were not affected by pH 5.5 on the extracellular side. We conclude from our data that KCNE2/KCNQ1 and KCNE3/KCNQ1 channels conduct a substantial K⫹ current in the presence of acidic external pH. In conclusion, our data suggest that KCNQ1 is the missing luminal K⫹ channel in gastric parietal cells of several species including humans. KCNQ1 may be required for acid secretion, perhaps playing a role in K⫹ recycling. The KCNQ1 inhibitor 293B blocked gastric secretion with a half-maximal inhibitory concentration in the micromolar range. Most likely, KCNE2, KCNE3, or another yet unknown protein coassembles with KCNQ1 in human parietal cells. The precise subunit composition and its regulation during secretion remain to be elucidated. The tissue-specific subunit composition of KCNQ1 and the peculiar pharmacokinetics of the stomach could offer new therapeutic perspectives for the treatment of peptic ulcer disease via inhibition of the KCNQ1 channel complex.

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Received September 14, 2000. Accepted January 10, 2001. Address requests for reprints to: Dr. Richard Warth, Institut de Pharmacologie du CNRS, c/o Dr. J. Barhanin, 660 route des Lucioles, 06560 VALBONNE SOPHIA-ANTIPOLIS, FRANCE. e-mail: warth@ipmc. cnrs.fr; fax: (33) 493-95-77-04. Supported by the Forschungsfo ¨rderung des Landes Baden-Wu ¨rttemberg. R.W. was supported by an EMBO Fellowship. This work is dedicated to our mentor and colleague Prof. Dr. Rainer F. Greger. The authors gratefully acknowledge the expert assistance by S. Haxelmanns, M. M. Larroque, B. Weinhold, and W. Rohm. Drs. Grahammer and Herling contributed equally to this work.