Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603, Japan
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ABSTRACT |
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Contribution of K+ channels derived from the expression of ERG, KCNQ, and KCNE subtypes, which are responsible for rapidly and slowly activating delayed rectifier K+ currents (IKr and IKs, respectively) in cardiac myocytes, to membrane currents was examined in stomach circular smooth muscle cells (SMCs). The region-qualified multicell RT-PCR showed that ERG1/KCNE2 transcripts were expressed in rat stomach fundus and antrum SMCs and that KCNQ1/KCNE1 transcripts were expressed in antrum but not fundus. Western blotting and immunocytochemical analyses indicate that ERG1 proteins were substantially expressed in both regions, whereas KCNE1 proteins were faintly expressed in antrum and not in fundus. Both IKr- and IKs-like currents susceptible to E-4031 and indapamide, respectively, were identified in circular SMCs of antrum but only IKr-like current was identified in fundus. It is strongly suggested that IKr- and IKs-like currents functionally identified in rat stomach SMCs are attributable to the expression of ERG1/KCNE2 and KCNQ1/KCNE1, respectively. The membrane depolarization by 1 µM E-4031 indicates the contribution of K+ channels encoded by ERG1/KCNE2 to the resting membrane potential in stomach SMCs.
rat stomach; multicell polymerase chain reaction; Western blotting; immunocytochemistry; whole cell voltage clamp
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INTRODUCTION |
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EXCITABLE MEMBRANES DISPLAY a variety of voltage-gated K+ (Kv) channels with different functional properties (6). In smooth muscles (SMs), Kv channels play crucial roles in the regulation of contraction via the control of membrane potential and excitability (1, 15). Electrophysiological properties of K+ currents in gastrointestinal (GI) SMs are recorded as rapidly activating K+ conductance, which may regulate the amplitude of the upstroke depolarization phase of a slow wave (28, 39). The molecular basis for electrical rhythmicity in GI SMs has also been substantially clarified (10). Rapidly activating (IKr) and slowly activating delayed rectifier K+ (IKs) currents are found in many different cell types, including neurons and cardiac muscles. They are major determinants of the threshold firing properties of neurons (42, 46) and of the repolarization of the ventricular action potential (31). IKr displays inward rectification and is selectively blocked by several class III antiarrhythmic drugs such as E-4031. On the other hand, IKs is blocked by several class III antiarrhythmic drugs, the diuretic agent indapamide, and chromanol 293B and is increased in the absence of external K+ (24, 30, 31). In cardiac muscle, the heterogeneity of IKr and IKs density in different areas strongly affects action potential duration and its rate dependence (41). In GI SMs, electrophysiological recordings provide evidence for components of IKr- and IKs-like channels (3, 5).
To date, three ERG (ERG1-3), five KCNQ (KCNQ1-5), and five KCNE (KCNE1-4 and 1L) are identified in mammals. It is well known that the heterologous expression of ERG1/KCNE2 and KCNQ1/KCNE1 results in K+ currents with characteristics similar to those of endogenous cardiac IKr and IKs, respectively (2, 30). Mutations in these genes are responsible for the human genetic disease long QT syndrome (44, 47). Recent studies have confirmed that their subtypes play significant roles in central and peripheral nervous systems. The expression patterns of heteromeric KCNQ2/KCNQ3 corresponds to the neuronal M channel (43), and the inheritance in their mutant forms is associated with benign familial neonatal convulsions (7, 38). Also, KCNQ4 and KCNQ1/KCNE3 are responsible for dominant deafness in human sensory outer hair cells and cystic fibrosis in the intestine crypt cells, respectively (14, 34). The physiological roles of ERG2 and ERG3, which are exclusively expressed in mammalian nervous system in contrast to ERG1, remain uncertain (37). In GI SMs, the relationship between the molecular components of ERG, KCNQ, and KCNE subtypes and the functional expression of IKr- and IKs-like currents remain unclear.
The aim of the present study is to elucidate the molecular and functional characterization of IKr- and IKs-like channels in rat stomach SMCs on the basis of both molecular biological and electrophysiological techniques. To address this issue, RT-PCR and Western blotting analyses were initially performed to identify the molecular components of ERG, KCNQ, and KCNE subtypes expressed in the rat stomach. Subsequently, to exclude the possible contamination of mRNA signal nonmyocytes, the multicell PCR and immunocytochemical analyses were performed using single SM cells (SMCs). Moreover, we investigated whether IKr- and IKs-like currents could be detected in single SMCs of the rat stomach antrum and fundus using whole cell patch-clamp techniques and pharmacological tools, E-4031, and indapamide, which have been shown to block IKr and IKs in cardiac myocytes, respectively (29, 40).
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METHODS |
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RNA extraction and RT-PCR.
Five- to six-week-old male Wistar rats were anesthetized with ether and
killed by bleeding. All experiments were carried out in accordance with
the guiding principles for the care and use of laboratory animals (the
Science and International Affairs Bureau of the Japanese Ministry of
Education, Science, Sports, and Culture) and also with the approval of
the ethics committee in Nagoya City University. Total RNAs were
extracted from homogenates of SM layers or mucosa in selected or whole
areas of stomach by the acid guanidium thiocyanate-phenol method
following digestion with RNase-free DNase, and RT was performed
according to GIBCO BRL protocol as previously described
(25). Oligonucleotide sequences of primers specific for
ERG1-3, KCNQ1-3, KCNE1-2, and
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) of the rat were shown
in Table 1. The thermal cycler program
used for PCR amplification included a 0.5-min denaturation step at
94°C, a 0.5-min annealing step at 55°C, and a 0.5-min primer
extension step at 72°C for 32 or 28 (GAPDH) cycles (GeneAmp 2400, Perkin Elmer ABI). Amplified products were separated on 1.5% agarose
gels in Tris acetate/EDTA buffer, visualized with 1 µg/ml ethidium
bromide, and documented on FluorImager 595 (Molecular Dynamics). After
recovery from gel fragments using GENECLEAN II (BIO 101), the amplified
fragments were ligated into pBluescript II SK(+) (Stratagene). Cloned
cDNAs were sequenced by the chain-termination method with a DSQ-1000L
sequencer (Shimadzu).
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Multicell RT-PCR. Antrum and fundus SMCs of the rat stomach were isolated using a slight modification as previously reported by Muraki et al. (22). Individual myocytes (50-60 cells) were first identified by morphology and then harvested by using negative pressure through a glass pipette (30- to 50-µm tip diameter). The entry of the cells into the glass pipette was monitored visually, with care taken to avoid picking up other types of cells. The content of each pipette was transferred into a sterile tube that contained an RT reaction solution without RTase. Genomic DNA was removed by adding 1 unit RNase-free DNase (Promega) followed by incubation for 30 min at 37°C. After the sample was heated for 5 min at 95°C to inactivate DNase activity, RT-PCR was performed as described above, but the cycle numbers were increased to 45.
Western blotting. Membrane fractions of the rat tissues were prepared using a protocol reported by Barry et al. (4), and protein contents were measured with a protein assay kit (BioRad) with BSA as a standard. Rat membrane proteins (50 µg/lane) were fractionated by SDS-PAGE (8%) and transferred to polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia). After the membranes were incubated into blocking solution (PBS with 1% BSA and 0.1% Tween 20), they were incubated with either the anti-human ERG1 (anti-HERG) (1:200 dilution), the anti-KCNE1 (1:200 dilution), or the anti-large-conductance Ca2+-activated K+ channel (BK; 1:400 dilution) polyclonal antibody (Alomone Laboratory) in PBS with 0.1% Tween 20 (buffer A) at 4°C overnight. The next day, membranes were washed three times with buffer A for 10 min and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (Chemicon) diluted 1:2,000 in buffer A. An enhanced chemiluminescence detection system (Amersham Pharmacia) was used for the detection of the bound antibody. Resulting images were analyzed by an image reader (LAS-1000, Fujifilm), and the digitized signals were quantified with Image Gauge software (version 3.0, Fujifilm).
Immunocytochemistry. Single antrum and fundus SMCs of the rat stomach were seeded onto glass-bottom dishes, respectively. Before staining, isolated myocytes were fixed with 3% paraformaldehyde for 10 min. They were subsequently permeabilized with PBS containing 0.2% Triton X-100. Nonspecific binding sites were blocked with PBS containing 0.2% Triton X-100 and 1% normal goat serum (buffer B) (4). Cells were then exposed to anti-HERG or the anti-KCNE1 polyclonal antibody diluted 1:50 in buffer B for 24 h at 4°C. Excess primary antibody was removed by repeating washing with PBS, and the cells were exposed to biotin-conjugated goat anti-rabbit IgG (H & L) antibody (1:200 dilution, Chemicon). After 1-h incubation at room temperature, excess secondary antibody was removed by repeating washing with PBS. Then the cells were labeled with FITC-conjugated streptavidin (1:50 dilution, Chemicon). After 1-h incubation at room temperature, excess streptavidin was removed by repeated washing with PBS. Digital images were viewed on a scanning confocal microscope (LSM510, Zeiss).
Electrophysiological measurement. Single antrum and fundus SMCs were isolated as described above. Whole cell voltage and current clamps were applied to single cells with patch pipettes using a patch-clamp amplifier (CEZ-2400, Nihon Kohden). For electrical recordings, HEPES-buffered solution having the following composition was used as the external solution (in mM): 137 NaCl, 5.9 KCl, 2.2 CaCl2, 1.2 MgCl2, 14 glucose, 10 HEPES, 0.1 CdCl2, and 0.001 penitrem A (pH 7.4). In some experiments, high K+ solution having the following composition was used (in mM): 140 KCl, 5.9 NaCl, 0 CaCl2, 1.2 MgCl2, 14 glucose, 10 HEPES, and 0.1 CdCl2 (pH 7.4). When IKr- and IKs-like currents were recorded, the pipette-filling solution contained (in mM) 140 KCl, 0.3 EGTA, 4 MgCl2, 5 ATP-Na, and 10 HEPES (pH 7.2). All experiments were done at room temperature (23 ± 1°C). Penitrem A and indapamide were purchased from Sigma.
Data storage, data analysis, and statistics. Membrane potential and current were stored on videotape after they were digitized by PCM-recording system (PCM 501 ES, Sony; modified a frequency response from direct current to 20 kHz). The data on the videotape were replayed later and loaded into a computer (IBM-AT) through an analog-to-digital converter (Data translation; DT 2801A) for analysis by use of data-acquisition and analysis programs as reported by Imaizumi et al. (12). Data analysis was done on a computer using software (Cell-Soft) developed at the University of Calgary. Pooled data were expressed as means ± SE. Statistical significance was tested according to Dunnett's test or paired t-test and is indicated by *P < 0.05 and **P < 0.01.
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RESULTS |
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Molecular identification of ERG, KCNQ, and KCNE subtypes expressed
in rat SMs.
The expression levels of ERG1-3, KCNQ1-3, and KCNE1-2
transcripts in various SM tissues of the rat were examined by use of conventional RT-PCR and compared with those in brain, heart, and kidney. The subtype-specific PCR primers are listed in Table 1. GAPDH
primers were used to confirm the integrity of RNA preparations and to
confirm that there was no contamination with genomic DNA (these primers
were designed to span 2 introns). Intron-containing bands (~650 bp)
were not detected in any tissues examined (not shown). The negative
controls were run by addition of water in place of RT, resulting in no
detectable signal (not shown). The specificity of each PCR product was
confirmed by DNA sequence analysis (not shown). It has been established
that ERG1 coassembly with KCNE2 (ERG1/KCNE2) forms the
IKr channel in heart (2). RT-PCR
revealed expression of ERG1/KCNE2 in heart and that of ERG1/KCNE2,
ERG2, and ERG3 transcripts in brain (Fig.
1A), as has been reported
previously. Among SM tissues, ERG1/KCNE2 transcripts were observed in
stomach and urinary bladder, whereas no or very weak signals were
detected in aorta, colon, ileum, and vas deferens (Fig. 1A).
In stomach, ERG2 transcript was also expressed at relatively high
levels.
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Regional expression of ERG, KCNQ, and KCNE transcripts in stomach.
To examine in more detail the distribution of ERG1/KCNE2, ERG2,
KCNQ1/KCNE1, and KCNQ3 transcripts in stomach SMs, RT-PCR was performed
using cDNAs prepared from the following preparations: SM layers of
antrum and fundus, mucosa of whole stomach, and SM layers of lower
esophagus and duodenum. ERG1/KCNE2 transcripts were expressed at
relatively high levels in fundus and antrum compared with those in
esophagus, mucosa, and duodenum (Fig.
2A). On the other hand,
KCNQ1/KCNE1 transcripts were expressed at relatively high levels in
antrum and mucosa, whereas no detectable signals were observed in
fundus (Fig. 2B). In fundus, faint signals of both KCNQ1 and
KCNE1 were detected when the amplification cycle was increased to 40 (not shown). Similar results were obtained from eight separate
experiments.
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Expression of ERG1 and KCNE1 proteins in stomach.
Wymore et al. (45) have suggested the limitations in
comparing ERG1 transcript levels with IKr
amplitudes in the absence of the measurements of ERG1 protein
expression. The expression levels of both ERG1 and KCNE1 proteins were
therefore verified in rat stomach SMs using Western blotting. As shown
in Fig. 3, typical results were obtained
by use of the antibodies specific for ERG1 (Fig. 3A) and
KCNE1 (Fig. 3B). The anti-ERG1 antibody recognized double
bands at 205 and 165 kDa in heart as reported by Pond et al. (Fig.
3A, lane 1) (27). Bands of the same
sizes were also detected in stomach (lane 2) and urinary
bladder (lane 3) membranes. The densitometric signals in
heart were over twofold more abundant than those in stomach and urinary
bladder. Similar results were obtained in four separate experiments.
The signal in heart was specifically blocked when the anti-ERG1
antibody was preincubated with the excess antigen peptide against which the antibody was generated (lane 4). These results are
consistent with the results about the expression levels of ERG1
transcripts by conventional RT-PCR analyses.
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Expression of ERG, KCNQ, and KCNE in SMCs of the rat stomach.
To avoid the contamination of signals from nonmyocytes, multicell
RT-PCR analyses were performed on freshly isolated antrum and fundus
circular SMCs of the stomach (see METHODS). As shown in
Fig. 4, both ERG1/KCNE2 and KCNQ1/KCNE1
transcripts were expressed in antrum SMCs (top) and
ERG1/KCNE2 were expressed in fundus SMCs (bottom). Very weak
or no detectable signal for KCNQ1 and KCNE1 was detected in fundus
SMCs. The signals of ERG2 and KCNQ3 were not detected under these
conditions but were slightly detected when cycle number was increased
to 50 in antrum alone (not shown). The negative controls were run by
addition of water in place of RT, resulting in no detectable signal
(not shown), and intron-containing bands (~650 bp) were not detected
in any cell preparations examined (not shown). Similar results were
obtained from six separate experiments. These results are mostly
consistent with those from conventional RT-PCR. Interestingly, when
multi cell RT-PCR experiments for the transcripts of ERG1/KCNE2 and
KvLQT1/KCNE1 were performed on freshly isolated longitudinal SMCs of
the antrum and fundus (~20 cells), results similar to those in
circular SMCs were obtained by PCR for 45 cycles (not shown).
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IKr- and IKs-like currents in stomach myocytes. To determine whether corresponding IKr- and IKs-like currents can be recorded in antrum and fundus SMCs of the rat stomach, electrophysiological and pharmacological experiments were carried out. E-4031, an anti-arrhythmic agent, and indapamide, a diuretic agent, are known to cause long QT syndrome and ventricular arrhythmias by the selective blockage of IKr and IKs (29, 40). IKr- and IKs-like currents in antrum and fundus were therefore isolated as the E-4031- and indapamide-sensitive currents, respectively. To inhibit Ca2+ currents and Ca2+-activated K+ currents, 100 µM Cd2+ and 1 µM penitrem A were added to the external solution, respectively.
Cells were depolarized to
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DISCUSSION |
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Relationship between ERG1/KCNE2 expression and IKr in rat stomach SM. ERG1 transcript is widely expressed not only in cardiac muscles but also in other tissues such as brain, adrenal gland, thymus, retina, and skeletal muscle (30). In the present study, results obtained by RT-PCR, multicell PCR, Western blot, and immunocytochemistry indicate that ERG1/KCNE2 transcripts and ERG1 protein were abundantly expressed in rat stomach SMCs at similar levels to those in heart. Particularly, immunocytochemical experiments demonstrated the strong and homologous staining patterns of ERG1 proteins along cell membrane in both antrum and fundus SMCs. The abundant expression of ERG1 proteins in the present study is a rather unexpected finding, because IKr has not been previously reported in stomach SMCs.
Western blot analysis of ERG1 indicated that 165- and 205-kDa bands were detected in rat stomach SM membranes, as has been reported in rat ventricle (27). A lower molecular mass protein corresponding to the expression of the alternatively spliced variants of HERG1 (HERG1b, ~95 kDa) was not detected in rat. These appear to reflect differentially N-linked glycosylation forms of ERG1, which play significant roles in the regulation of cell surface expression of HERG1 (26). Interestingly, in canine cardiac muscles, the ERG1 transcripts are expressed at relatively high levels compared with Kv4.3 transcripts, which are the most abundant transcripts in heart, whereas IKr density is much lower than that of Kv4.3 current. Similarly, in myocytes of rat ventricle and atrium, rabbit atrium, and canine ventricle, IKr densities are rather low: 0.36 ± 0.01 (atRelationship between KCNQ1/KCNE1 expression and functional
IKs in rat stomach SM.
A clear difference in the expression of KCNQ1/KCNE1 was found between
SMCs in antrum and fundus; the transcripts of them and KCNE1 protein
were detected in antrum SMCs. Accordingly, K+ current
sensitive to 100 µM indapamide was recorded only in antrum SMCs.
These results suggest that IKs is functional in
SMCs of antrum but not in those of fundus. Because
IKs is activated at positive potentials with
slow kinetics, the current could be responsible for a repolarizing
phase of slow waves, which are originated in antrum but not propagated
to the fundus region (13). It is notable that the
IK,tail in fundus SMCs was totally resistant to
100 µM indapamide. However, the contribution of
IKs in antrum SMCs to total membrane current on
depolarization was not determined qualitatively. Although the
IK,tail at 50 mV was blocked by indapamide at
the IC50 of 228 µM, IKs component
was not resolved quantitatively from other K+ currents.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. R. Giles (Univ. of Calgary, Canada) for providing data-acquisition and analysis programs.
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FOOTNOTES |
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E-4031 was supplied courtesy of Eisai.
This work was supported by the International Scientific Research Program through joint research grants and by a grant-in-aid for scientific research by the Japanese Society for the Promotion of Science to Y. Imaizumi. Support was also provided by the Research Grant for Cardiovascular Diseases (11C-1) from the Ministry of Health and Welfare to Y. Imaizumi.
Address for reprint requests and other correspondence: Y. Imaizumi, Dept. of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City Univ., 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603, Japan (E-mail: yimaizum{at}phar.nagoya-cu.ac.jp).
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.
10.1152/ajpgi.00200.2001
Received 11 April 2001; accepted in final form 15 October 2001.
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