Molecular and functional characterization of ERG, KCNQ, and KCNE subtypes in rat stomach smooth muscle

Susumu Ohya, Keiichi Asakura, Katsuhiko Muraki, Minoru Watanabe, and Yuji Imaizumi

Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya 467-8603, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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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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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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Table 1.   Oligonucleotide sequence of primers used for RT-PCR

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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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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Fig. 1.   Expression of ERG, KCNQ, and KCNE subtypes in rat smooth muscles (SMs). RT-PCRs were performed with 8 pair primers [ERG1-3 and KCNE2 (A); KCNQ1-3 and KCNE1 (B)] for 32 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used to confirm the integrity of RNA preparations (28 cycles). cDNAs were obtained by RT of the total RNAs extracted from rat aorta, brain, colon, heart, ileum, kidney, stomach, urinary bladder, and vas deferens, respectively. Amplified products were separated on 1.5% agarose gels and analyzed by ethidium bromide staining: 428 bp (ERG1), 417 bp (ERG2), 374 bp (ERG3), 453 bp (KCNQ1), 372 bp (KCNQ2), 424 bp (KCNQ3), 471 bp (KCNE1), 399 bp (KCNE2), and 373 bp (GAPDH).

It has been demonstrated that coassembling KCNQ1 with KCNE1 (KCNQ1/KCNE1) forms IKs channel in heart (30) and that the heteromultimer of KCNQ2 and KCNQ3 (KCNQ2/KCNQ3) contributes to M-like channels in neuronal cells (7). KCNQ1/KCNE1 transcripts were expressed in heart but not in brain, whereas KCNQ2/KCNQ3 were expressed in brain but not in heart (Fig. 1B). In SM tissues, KCNQ1/KCNE1 transcripts were expressed in stomach alone, whereas no or very weak signals were detected in other tissues examined here (Fig. 1B). In stomach, KCNQ3 transcript was also expressed at relatively high levels compared with that in brain. These results provide the first description of the expression of both ERG1/KCNE2 and KCNQ1/KCNE1 in rat stomach. Interestingly, in kidney, ERG1-3 and KCNQ1-3 transcripts were expressed at very low levels, whereas KCNE1 and KCNE2 transcripts were expressed at similar levels to those in brain and heart. Similar results were obtained from six separate experiments.

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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Fig. 2.   Regional expression of ERG, KCNQ, and KCNE subtypes in rat stomach SM. RT-PCRs were performed with 8 pair primers [ERG1-3 and KCNE2 (A); KCNQ1-3 and KCNE1 (B)] for 32 cycles. GAPDH cDNA was used to confirm the integrity of RNA preparations (28 cycles). cDNAs were obtained by RT of the total RNAs extracted from rat esophagus, fundus, antrum, mucosa, and duodenum, respectively. Amplified products were separated on 1.5% agarose gels and analyzed by ethidium bromide staining as shown in Fig. 1.

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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Fig. 3.   Western blotting analysis of ERG1 and KCNE1 proteins in stomach and urinary bladder of the rat. Membrane proteins (50 µg/lane) were transferred to polyvinylidene difluoride membranes after fractionating by SDS-PAGE and blotted with anti-human ERG1 (HERG1) (1:200 dilution; A), anti-KCNE1 (1:200 dilution; B), and anti-large-conductance Ca2+-activated K+ (BK) channel (1:400 dilution; C) antibodies. Lane 1, heart; lane 2, stomach; lane 3, urinary bladder. Lane 4 shows the membrane blotted with each antibody preincubated with the excess antigen. Resultant images were analyzed by an Image Reader (LAS-1000, Fuji Film), and the digitized signals were quantified with an Image Gauge software (Fuji Film). Positive signals were indicated by arrowheads. The migration of size marker is shown on the left.

On the other hand, the anti-KCNE1 antibody recognized a single band at ~15 kDa in heart (Fig. 3B, lane 1). Strong signal was clearly detectable in heart, but unexpectedly, only weak signals were detected in stomach (lane 2) and urinary bladder (lane 3). Densitometric analyses revealed that KCNE1 protein levels in heart were >50-fold more abundant than those in stomach. The signals in heart were also specifically blocked when the anti-KCNE1 antibody was preincubated with the excess fusion protein against which the antibody was generated (lane 4). These findings do not fit with the results of the expression levels of KCNE1 transcript in stomach by conventional RT-PCR analyses. Similar results of Western blotting were obtained from four separate experiments. BK-channel protein was expressed at relatively high levels in stomach and urinary bladder but not in heart (Fig. 3C). Specific bands were not detected with anti-ERG1 and anti-KCNE1 antibodies in membrane fractions from HEK-293 cells transfected with the cDNA of Kv1.2, 1.4, 1.6, 2.1, 4.2, 4.3, or BK channel (not shown).

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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Fig. 4.   Expression of ERG, KCNQ, and KCNE subtypes in antrum and fundus SMCs by multicell RT-PCR. Representative 1.5% agarose gels were loaded with each amplified product, stained with ethidium bromide, and documented on FluorImager 595 (Molecular Dynamics). The migration of size marker (100-bp DNA ladder) is shown on the right.

Furthermore, to confirm that the identified transcripts are translated into channel proteins and expressed on the surface membranes of antrum and fundus SMCs, the subcellular localization of ERG1 and KCNE1 protein was examined by an immunocytochemical approach. Freshly isolated myocytes from circular SM layers in fundus and antrum were stained with anti-ERG1 antibody, and the local distribution of immonoreactivity was visualized by laser-scanning confocal microscopy. The strong staining patterns of ERG1 proteins were localized along cell membrane in both types of myocytes (Fig. 5, A and B). ERG1 signals disappeared after preincubation with the excess antigen (not shown). These results were consistent with those from both Western blotting and multicell RT-PCR analyses. Similar experiments were performed in HEK-293 cells transfected with the cDNA of Kv1.2, Kv2.1, Kv4.3, or BK channel; resultant immunoreactivity to anti-ERG1 antibody was not detected in these transfectants (not shown).


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Fig. 5.   Expression of ERG1 and KCNE1 proteins in single antrum and fundus smooth muscle cells of the rat stomach using immunocytochemical methods. Confocal images of representative freshly isolated smooth muscle cells: antrum (A, C, E, G) and fundus (B, D, F). E and F are magnified regions shown by arrows in C and D, respectively. H and I are confocal images of HEK-293 cells transfected with KCNE1 and mock-transfected ones, respectively. Cells were immunostained with specific antibodies against ERG (A and B), KCNE1 (C-F, H, and I), and BK (G)-channel proteins using the avidin-biotin peroxidase technique. The bars represent 20 (A-D, G-I) or 15 µm (E and F).

On the other hand, the fluorescence staining pattern for anti-KCNE1 antibody was diffuse in both types of cells (Fig. 5, C and D). In antrum alone, however, weak and punctuated signals were identified along cell membrane under higher magnification (Fig. 5E). Immunoreactivity was not detected in both types of SMCs that were treated with control antigen during the incubation with primary antibody (not shown). As a positive control, immunoreactivity was clearly detected uniformly in HEK-293 cells transfected with rat KCNE1 cDNA, whereas it was not detected in mock-transfected HEK-293 cells (Fig. 5, H and I). It was also found that the reactivity to anti-BK-channel antibody was observed in a clustered fashion on the surface in antrum SMCs (Fig. 5G).

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 -30 mV for 200 ms from a holding potential of -90 mV every 20 s, and the tail current (IK,tail) was measured at -50 mV [Fig. 6A, antrum (a), fundus (b)]. Peak current amplitude elicited on depolarization from -90 to -30 mV, which mainly consists of rapidly inactivating and delayed-rectifier K+ currents, was not changed significantly by 1 µM E-4031; in antrum, 28.8 ± 2.9 pA (95.0%, n = 6); in fundus, 28.7 ± 2.4 pA (92.2%, n = 5). Figure 6B displays superimposed IK,tail trace in antrum and fundus SMCs in the absence and presence of 1 µM E-4031. E-4031 rapidly and consistently blocked IK,tail recorded on repolarization to -50 mV. The average decrease in amplitude of IK,tail by 1 µM E-4031 was 40.0 ± 8.0 and 45.3 ± 4.2% in antrum and fundus, respectively (Fig. 6C). In antrum and fundus, the amplitude of the E-4031-sensitive IK,tail [IK,tail(E-4031)] at -50 mV was 3.6 ± 1.0 (n = 6) and 3.1 ± 1.3 pA (n = 5), respectively, and the cell capacitance was 32.0 ± 2.0 (n = 6) and 33.2 ± 1.7 pF (n = 5), respectively. With the use of this clamp paradigm, IK,tail(E-4031) density was 0.12 ± 0.02 and 0.11 ± 0.01 pA/pF, respectively (Fig. 6D).


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Fig. 6.   Functional characterization of IKr-like currents in rat stomach SM cells (SMCs). A: after cells were depolarized to -30 mV for 200 ms from -90 mV every 20 s, they were repolarized to -50 mV to measure the tail current (IK,tail; ). IK,tail was observed in the single SMCs of both antrum (a) and fundus (b). B: IK,tail at -50 mV was inhibited by 1 µM E-4031 in both antrum (a) and fundus (b). Arrows (A) and dotted lines (B) indicate 0 current level. C: bar graph showing 1 µM E-4031-induced block of IK,tail at -50 mV in both antrum and fundus. Columns and bars indicate means ± SE. The number of experiments is given in parenthesis, and the statistical significance of the difference vs. 100% is expressed as P < 0.01. D: bar graph showing E-4031-sensitive IK,tail density [IK,tail(E-4031)] in both antrum and fundus. Columns and bars indicate means ± SE. IK,tail(E-4031) was 0.12 ± 0.02 (antrum, n = 6) and 0.11 ± 0.01 pA/pF (fundus, n = 5), respectively. E: E-4031-sensitive components of membrane currents were recorded in symmetrical 140 mM K+ conditions. Cells were hyperpolarized to -90 mV from a holding potential of -30 mV for 250 ms in the absence and presence of 1 µM E-4031 (left). The arrowheads indicate the inward current in the presence of E-4031. Dotted lines indicate the 0 current level. The summarized results about the density of E-4031-sensitive currents at -90 mV (inward current peak) and -30 mV (holding current) are shown at right. No. of experiments is given in parenthesis.

In addition, E-4031-sensitive currents in antrum and fundus SMCs were recorded also in symmetrical 140 mM K+ conditions with nominally free Ca2+. The membrane hyperpolarization to -90 mV from a holding potential of -30 mV showed inward IK,tail, which were partly blocked by 1 µM E-4031 (Fig. 6E). The peak amplitude of the inward currents at -90 mV in the presence of 1 µM E-4031 was reduced to 78.8 ± 3.1 (n = 8; P < 0.01 vs. control) and 80.9 ± 1.0% (n = 7; P < 0.01 vs. control) of the control in antrum and fundus SMCs, respectively (P > 0.05 between antrum and fundus). The holding current at -30 mV was also slightly shifted outwardly by 1 µM E-4031. The density of E-4031-sensitive currents at -90 and -30 mV was summarized in Fig. 6E. E-4031-sensitive conductance appeared to be saturated at potentials positive to -30 mV, suggesting the rectification of the current.

The membrane potential of single SMCs from circular muscle layers of rat stomach antrum and fundus was measured under current-clamp mode (Fig. 7). The resting membrane potential in antrum SMCs was -36.9 ± 2.9 mV and reduced to -31.9 ± 3.9 mV by addition of 1 µM E-4031 (n = 5, P < 0.05; Fig. 7, Aa and Ba). Spontaneous transient depolarizations such as immature action potentials of 15 mV in amplitude were occasionally observed in the presence of E-4031 but not in the absence. The depolarizing effect of E-4031 was removed by withdrawal (-36.1 ± 3.4 mV). Similarly, the resting membrane potential in fundus SMCs was changed by 1 µM E-4031 from -41.1 ± 4.2 to -35.8 ± 3.7 mV (n = 5, P < 0.05) and was recovered to -41.4 ± 4.0 mV by withdrawal (Fig. 7, Ab and Bb).


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Fig. 7.   Effects of 1 µM E-4031 on membrane potential in rat stomach SMCs. A: resting membrane potential was measured in the single SMCs isolated from antrum (a) and fundus (b), respectively. Membrane potential was measured in the whole cell configuration during current-clamp mode. Each baseline was shown by dotted line. B: summarized data from A in antrum (a) and fundus (b; n = 5 for each), respectively. Columns and bars indicate means ± SE. Statistical significance was determined by paired t-test.

Half-maximal block of IKs in cardiac myocytes by indapamide was obtained at the concentration of ~100 µM (40). To activate IKs-like currents, cells were depolarized to +30 mV for 7.5 s from a holding potential of -90 mV every 20 s and IK,tail was measured at -50 mV (Fig. 8A). Figure 8B displays superimposed IK,tail trace in antrum and fundus SMCs [antrum (a), fundus (b); n = 7] in the absence and presence of 100 µM indapamide. In both antrum and fundus SMCs, the similar IK,tail of -50 mV were observed [antrum, 19.5 ± 2.3 pA (n = 23); fundus, 16.3 ± 1.3 pA (n = 11)] (not shown); however, in antrum alone, indapamide rapidly and consistently blocked the slowly decaying IK,tail elicited on repolarization to -50 mV. The average decrease in amplitude of IK,tail by 100 µM indapamide was 32.4 ± 4.7% (n = 7) in antrum and 1.1 ± 4.7% (n = 7) in fundus (Fig. 8C). The amplitude of 100 µM indapamide-sensitive IK,tail [IK,tail(indap)] at -50 mV was 5.9 ± 1.8 (antrum, n = 7) and 0.64 ± 0.81 pA (fundus, n = 7). With the use of this clamp paradigm, IK,tail(indap) density was ~0.21 ± 0.18 (antrum, n = 7) and 0.02 ± 0.02 pA/pF (fundus, n = 7) at -50 mV (Fig. 8D). The current amplitude at the peak and at the end of depolarization (+30 mV) was not changed significantly by 100 µM indapamide. In the presence of 100 µM indapamide, the amplitude was 94.8 ± 1.7 and 90.9 ± 4.6% of the control at the peak and end, respectively, in antrum and 93.8 ± 5.3 and 98.9 ± 4.8% in fundus (n = 7, P > 0.05 vs. 100% in each group). At high concentrations, indapamide significantly blocked both the peak and end of outward current during depolarization in a nonspecific manner. Correspondingly, application of 0.03-1 mM indapamide reduced IK,tail in a concentration-dependent manner, and after washout of indapamide, IK,tail almost completely recovered (Fig. 8E). Figure 8F summarized the results about effects of 0.03-1 mM indapamide on the amplitude of IK,tail at -50 mV. The half-inhibition concentration value and Hill coefficient were 228 µM and 1.0, respectively. IK,tail(indap) was not blocked by the application of 1 µM E-4031 and increased in the absence of extracellular K+ concentration (~200%; not shown).


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Fig. 8.   Functional expression of IKs-like currents in rat stomach SMCs. A: after cells were depolarized to +30 mV for 7.5 s from -90 mV every 20 s, they were repolarized to -50 mV to measure IK,tail (). IK,tail was observed in the single SMCs of both antrum (a) and fundus (b). B: effects of 100 µM indapamide on IK,tail at -50 mV. IK,tail in antrum alone was inhibited by 100 µM indapamide. Arrows or dotted lines indicate 0 current level. C: bar graph showing 100 µM indapamide-induced block of IK,tail at -50 mV in both antrum and fundus. Columns and bars indicate means ± SE. The number of experiments is given in parenthesis, and the statistical significance of the difference vs. 100% is expressed as **P < 0.01. D: bar graph showing 100 µM indapamide-sensitive IK,tail density [IK,tail(indap)] in both antrum and fundus. Columns and bars indicate means ± SE. IK,tail(indap) was 0.21 ± 0.18 (antrum, n = 7) and 0.02 ± 0.02 pA/pF (fundus, n = 7), respectively. E: time course of IK,tail during cumulative addition of indapamide. F: concentration-response curve of indapamide for the inhibition of IK,tail in antrum. The current amplitude in the presence of indapamide was normalized by that before the application (1.0). The half-inhibition concentration and Hill coefficient were 228 µM and 1.0, respectively. The number of experiments is given in parenthesis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (at -30 mV), 0.62 ± 0.03 (at -30 mV), 0.8 (at -40 mV), and 0.22 (at -40 mV) pA/pF, respectively (23, 27, 45). The N-linked glycosylation may possibly be responsible for the expression and functional availability of ERG1 proteins in rat stomach SMCs, as well as in cardiac myocytes.

The IKr due to the complex of ERG1/KCNE2 is characteristic of strong inward rectification at positive potentials and peaked at around -40 mV in the relationship between activation potentials and current amplitude in cardiac myocytes (2, 23). The outward current activated at -30 mV in stomach SMCs consisted of several voltage-dependent K+ currents, and E-4031-sensitive current could not be clearly resolved from others in the present study. The current was more clearly detected as inward current in symmetrical high-K+ conditions, as has been demonstrated using reconstituted MiRP1/HERG K+ channels (2). On the basis of the IK,tail amplitude (0.1 pA/pF at -50 mV in both types of cells in normal solution), however, the density of E-4031-sensitive current at -30 mV appears not to be high compared with the levels of ERG1 protein expression. Although the KCNE2 protein expression was not determined in this study, high levels of KCNE2 mRNA were observed in both types of myocytes. KCNE2 protein expression must be quantified to draw a conclusion about the relationship between the abundance of ERG1/KCNE2 expression as a coassembly and the functional IKr density.

BK-channel current is the main component of K+ currents in rat stomach SMCs (not shown) as in other types of SMCs (11, 15). In the immunocytochemical experiments, ERG1 signals in stomach SMCs appear to be stronger than BK signals, whereas the density of IKr measured as the macroscopic current was much lower than that of BK-channel current (not shown). The distribution of BK-channel proteins localized in a clustered fashion on the cell surface in stomach SMCs. Channel clustering is rather a common phenomenon in neurons (19, 20). It has been suggested that, in addition to the simple density of channel proteins, the distribution pattern, such as clustering, also plays an essential role in differential posttranslation modification and functional availability of the channels (36).

It is noteworthy that the tail of outward current activated at -30 mV was reduced by 40-50% in the presence of 1 µM E-4031, which is specific to IKr at this concentration (31). This finding clearly indicates that IKr is one of the major components of delayed rectifier K+ current activated by depolarization to -30 mV for 200 ms in SMCs of rat stomach fundus and antrum. Cisapride, a gastrointestinal prokinetic drug, was widely used for the treatment of gastroesophageal reflux disease and has been associated with QT prolongation torsades de pointes by interacting with unique sites of the ERG1 channel (21). Akbarali et al. (3) have shown that in opossum esophagus SMCs, 1 µM cisapride inhibits IKr-like currents and thereby induces membrane depolarization (~10 mV) and phasic contractions. In rat antrum and fundus SMCs, application of 1 µM E-4031 induced membrane depolarization (~5 mV) under current-clamp mode. These results strongly suggest that IKr-like current plays an important role to maintain the resting membrane potential in both antrum and fundus and may possibly contribute to the regulation of electrical activities by slowing the rate of slow waves and accelerating the action potential repolarization. Although not examined in this study, these functions of the current may counteract the increase in muscle tone by depolarization and downregulate the rhythmic contractility in the stomach SM.

Relationship 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.

The major component of voltage-dependent K+ currents on depolarization in stomach SMCs is apparently not IKs but presumably A-type transient outward K+ current encoded by Kv4.3 in the early part of depolarization (25) and Kv1.5 and/or Kv2.1 in the late part (based preliminary on results using RT-PCR; S. Ohya, unpublished observation). It has been established that coexpression of KCNE1 with KCNQ1 modifies the properties of KCNQ1 and also results in the increment of the functional K+ current (31). Therefore, the small IKs in atrum SMCs, despite the abundant KCNQ1 transcript, is attributable to the low protein levels of KCNE1. Alternatively, mismatches among transcript, protein, and functional K+ current levels may possibly occur, as has been shown for voltage-gated Kv1 channels in mouse glial progenitors (33) and rat ventricular myocytes (17, 23).

Folander et al. (9) and Felipe et al. (8) have reported that the drastic changes in KCNE1 transcript levels occur in myometrium during late pregnancy and delivery and in kidney and cardiac tissues during development. Very recently, Lee et al. (16) showed that disruption of the KCNQ1 gene causes gastric hyperplasia in mice. In cardiac muscle, the heterogeneity of IKr and IKs densities strongly influences action potential duration (APD) and its rate dependence. Viswanathan et al. (41) showed that the changes in the density ratio of IKr to IKs result in heterogeneity of the repolarization properties of cardiac myocytes. For example, in the mouse midmyocardial M cells, smaller density of IKs lengthens APD and IKs plays an important role in APD adaptation. Changes in IKs density may cause the abnormality in the slow-wave amplitude and frequency in antrum and result in the gastric hyperplasia in KCNQ1-deficient mice.

Recent studies have shown that IKr- and IKs-like currents possess obligatory roles in cellular functions in many noncardiovascular tissues. For example, the KCNQ2/KCNQ3 contributes to the native M current, one of the most important regulators of neuronal excitability (35) and in NG108-15 (neuroblastoma × glioma hybrid cells) and GH3/B6 (clonal rat pituitary cells), the ERG1/ERG2 contributes to deactivating ERG currents (18, 32). In antrum SMCs, not only the transcripts of ERG1 and KCNQ1, but also those of ERG2 and KCNQ3 were expressed, although not very convincingly in multicell RT-PCR. Shi et al. (37) showed that rat ERG1-3 currents expressed in Xenopus oocytes are blocked by E-4031 at almost similar concentrations (dissociation constant = 100-200 nM). The possibility therefore cannot be ruled out that ERG1/ERG2 and/or KCNQ1/KCNQ3 heterotetramer(s) might also play some functional roles in antrum of the rat stomach.

In conclusion, IKr in circular SMCs of rat stomach antrum and fundus is probably due to coexpression of ERG1/KCNE2 and possesses rather low density but significant functional roles to control resting membrane potential in both cell types. IKs encoded by combination of KCNQ1/KCNE1 may be expressed and functional only in antrum SMCs. Although the detailed functions of IKr- and IKs-like currents were not fully understood, the present findings provide new insights into the regional differences in the regulation of membrane excitability in stomach SMs.


    ACKNOWLEDGEMENTS

We thank Dr. W. R. Giles (Univ. of Calgary, Canada) for providing data-acquisition and analysis programs.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aaronson, PI, and Smirnov SV. Regulation of voltage-gated K+ channels in vascular smooth muscle cells. In: Smooth Muscle Excitation, edited by Bolton TB, and Tamita T.. London: Academic, 1996, p. 63-74.

2.   Abbott, GW, Sesti F, Splawski I, Buck ME, Lehmann MH, Timothy KW, Keating MT, and Goldstein SAN MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. Cell 97: 175-187, 1999[ISI][Medline].

3.   Akbarali, HI, Thatte H, He XD, Giles WR, and Goyal RK. Role of HERG-like K+ currents in opossum esophageal circular smooth muscle. Am J Physiol Cell Physiol 277: C1284-C1290, 1999[Abstract/Free Full Text].

4.   Barry, DM, Trimmer JS, Merlie JP, and Nerbonne JM. Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels? Circ Res 77: 361-369, 1995[Abstract/Free Full Text].

5.   Benham, CD, and Bolton TB. Patch-clamp studies of slow potential-sensitive potassium channels in longitudinal smooth muscle cells of rabbit jejunum. J Physiol (Lond) 340: 469-486, 1983[ISI][Medline].

6.   Chandy, KG, and Gutman GA. Voltage-gated potassium channel genes. In: Handbook of Receptors and Channels: Ligand and Voltage-Gated Channels, edited by North RA.. Boca Raton, FL: CRC, 1995, p. 1-71.

7.   Charlier, C, Singh NA, Ryan SG, Lewis TB, Reus BE, Leach RJ, and Leppert M. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat Genet 18: 53-55, 1998[ISI][Medline].

8.   Felipe, A, Knittle TJ, Doyle KL, Snyders DJ, and Tamkun MM. Differential expression of Isk mRNAs in mouse tissue during development and pregnancy. Am J Physiol Cell Physiol 267: C700-C705, 1994[Abstract/Free Full Text].

9.   Folander, K, Smith JS, Antanavage J, Bennett C, Stein RB, and Swanson R. Cloning and expression of the delayed-rectifier Isk channel from neonatal rat heart and diethylstilbestrol-primed rat uterus. Proc Natl Acad Sci USA 87: 2975-2979, 1990[Abstract].

10.   Horowitz, B, Ward SM, and Sanders KM. Cellular and molecular basis for electrical rhythmicity in gastrointestinal muscles. Annu Rev Physiol 61: 19-43, 1999[ISI][Medline].

11.   Imaizumi, Y, Henmi S, Nagano N, Muraki K, and Watanabe M. Regulation of Ca-dependent K current and action potential shape by intracellular Ca strage sites in some types of smooth muscle cells. In: Smooth Muscle Excitation, edited by Bolton TB, and Tomita T.. London: Academic, 1996, p. 337-354.

12.   Imaizumi, Y, Muraki K, and Watanabe M. Characteristics of transient outward currents in single smooth muscle cells from the ureter of the guinea-pig. J Physiol (Lond) 427: 301-324, 1990[Abstract].

13.   Kelly, KA, Code CF, and Elveback LR. Patterns of canine gastric electrical activity. Am J Physiol 217: 461-470, 1969[ISI][Medline].

14.   Kubisch, C, Schroeder BC, Friedrich T, Luetjohann B, El-Amraoui A, Marlin S, Petit C, and Jentsch TJ. KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness. Cell 96: 437-446, 1999[ISI][Medline].

15.   Kuriyama, H, Kitamura K, Itoh T, and Inoue R. Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol Rev 78: 811-920, 1998[Abstract/Free Full Text].

16.   Lee, MP, Ravenel JD, Hu RJ, Lustig LR, Tomaselli G, Berger RD, Brandenburg SA, Litzi TJ, Bunton TE, Limb C, Francis H, Gorelikow M, Gu H, Washington K, Argani P, Goldenring JR, Coffey RJ, and Feinberg AP. Targeted disruption of the kvlqt1 gene causes deafness and gastric hyperplasia in mice. J Clin Invest 106: 1447-1455, 2000[Abstract/Free Full Text].

17.   London, B, Wang DW, Hill JA, and Bennett PB. The transient outward current in mice lacking the potassium channel gene Kv1.4. J Physiol (Lond) 509: 171-182, 1998[Abstract/Free Full Text].

18.   Meves, H, Schwarz JR, and Wulfsen I. Separation of M-like current and ERG current in NG108-15 cells. Br J Pharmacol 127: 1213-1223, 1999[Abstract/Free Full Text].

19.   Mi, H, Deerinck TJ, Ellisman MH, and Schwarz TL. Differential distribution of closely related potassium channels in rat Schwann cells. J Neurosci 15: 3761-3774, 1995[Abstract].

20.   Mi, H, Harris-Warrick RM, Deerinck TJ, Inman I, Ellisman MH, and Schwarz TL. Identification and localization of Ca2+-activated K+ channels in rat sciatic nerve. Glia 26: 166-175, 1999[ISI][Medline].

21.   Mitcheson, JS, Chen J, Lin M, Culberson C, and Sanguinetti MC. A structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci USA 97: 12329-12333, 2000[Abstract/Free Full Text].

22.   Muraki, K, Imaizumi Y, and Watanabe M. Sodium currents in smooth muscle cells freshly isolated from stomach fundus of the rat and ureter of the guinea-pig. J Physiol (Lond) 442: 351-375, 1991[Abstract].

23.   Muraki, K, Imaizumi Y, Watanabe M, Habuchi Y, and Giles WR. Delayed rectifier K+ current in rabbit atrial myocytes. Am J Physiol Heart Circ Physiol 269: H524-H532, 1995[Abstract/Free Full Text].

24.   Nerbonne, JM. Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol (Lond) 525: 285-298, 2000[Abstract/Free Full Text].

25.   Ohya, S, Tanaka M, Oku T, Asai Y, Watanabe M, Giles WR, and Imaizumi Y. Molecular cloning and tissue distribution of an alternatively spliced variant of an A-type K+ channal alpha -subunit, Kv4.3 in the rat. FEBS Lett 420: 47-53, 1997[ISI][Medline].

26.   Petrecca, K, Atanasiu R, Akhaven A, and Shrier A. N-linked glycosylation sites determine HERG channel surface membrane expression. J Physiol (Lond) 515: 41-48, 1999[Abstract/Free Full Text].

27.   Pond, AL, Scheve BK, Benedict AT, Petrecca K, Van Wagoner DR, Shrier A, and Nerbonne JM. Expression of distinct ERG proteins in rat, mouse, and human heart. J Biol Chem 275: 5997-6006, 2000[Abstract/Free Full Text].

28.   Sanders, KM. Ionic mechanisms of electrical rhythmicity in gastrointestinal smooth muscles. Annu Rev Physiol 54: 439-453, 1992[ISI][Medline].

29.   Sanguinetti, MC. Modulation of potassium channels by antiarrhythmic and antihypertensive drugs. Hypertension 19: 228-236, 1992[Abstract].

30.   Sanguinetti, MC, Curran ME, Zou A, Shen J, Spector PS, Atkinson DL, and Keating MT. Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac IKs potassium channel. Nature 384: 80-83, 1996[ISI][Medline].

31.   Sanguinetti, MC, and Jurkiewicz NK. Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol 96: 195-215, 1990[Abstract].

32.   Schafer, R, Wulfsen I, Behrens S, Weinsberg F, Bauer CK, and Schwarz JR. The erg-like potassium current in rat lactotrophs. J Physiol (Lond) 518: 401-416, 1999[Abstract/Free Full Text].

33.   Schmidt, K, Eulitz D, Veh RW, Kettenmann H, and Kirchhoff F. Heterogeneous expression of voltage-gated potassium channels of the shaker family (Kv1) in oligodendrocyte progenitors. Brain Res 843: 145-160, 1999[ISI][Medline].

34.   Schroeder, BC, Waldegger S, Fehr S, Bleich M, Warth R, Greger R, and Jentsch TJ. A constitutively open potassium channel formed by KCNQ1 and KCNE3. Nature 403: 196-199, 2000[ISI][Medline].

35.   Selyanko, AA, Hadley JK, Wood IC, Abogadie FC, Delmas P, Buckley NJ, London B, and Brown DA. Two types of K+ channel subunit, Erg1 and KCNQ2/3, contribute to the M-like current in a mammalian neuronal cell. J Neurosci 19: 7742-7756, 1999[Abstract/Free Full Text].

36.   Sheng, M, and Kim E. Ion channel associated proteins. Curr Opin Neurobiol 6: 602-608, 1996[ISI][Medline].

37.   Shi, W, Wymore RS, Wang HS, Pan Z, Cohen IS, McKinnon D, and Dixon JE. Identification of two nervous system-specific members of the erg potassium channel gene family. J Neurosci 17: 9423-9432, 1997[Abstract/Free Full Text].

38.   Singh, NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R, Ronen GM, Bjerre I, Quattlebaum T, Murphy JV, McHarg ML, Gagnon D, Rosales TO, Peiffer A, Anderson VE, and Leppert M. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat Genet 18: 25-29, 1998[ISI][Medline].

39.   Tomita, T. Electrical activity (spikes and slow waves) in gastrointestinal smooth muscles. In: Smooth Muscle, edited by Bulbring E, Brading AF, Jones AW, and Tomita T.. London: Arnold, 1981, p. 127-156.

40.   Turgeon, J, Daleau P, Bennett PB, Wiggins SS, Selby L, and Roden DM. Block of IKs, the slow component of the delayed rectifier K+ current, by the diuretic agent indapamide in guinea pig myocytes. Circ Res 75: 879-886, 1994[Abstract].

41.   Viswanathan, PC, Shaw RM, and Rudy Y. Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study. Circulation 99: 2466-2474, 1999[Abstract/Free Full Text].

42.   Wang, HS, and MacKinnon D. Potassium channel expression in prevertebral and paravertebral sympathetic neurons: control of firing properties. J Physiol (Lond) 48: 319-335, 1995.

43.   Wang, HS, Pan Z, Shi W, Brown BS, Wymore RS, Cohen IS, Dixon JE, and McKinnon D. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science 282: 1890-1893, 1998[Abstract/Free Full Text].

44.   Wattanasirichaigoon, D, and Beggs AH. Molecular genetics of long-QT syndrome. Curr Opin Pediatr 10: 628-634, 1998[Medline].

45.   Wymore, RS, Gintant GA, Wymore RT, Dixon JE, McKinnon D, and Cohen IS. Tissue and species distribution of mRNA for the IKr-like K+ channel, erg. Circ Res 80: 261-268, 1997[Abstract/Free Full Text].

46.   Yamada, WM, Koch C, and Adams PR. Multiple channels and calcium dynamics. In: Methods in Neuronal Modeling, edited by Koch C, and Segev I.. Cambridge, 1989, p. 97-133.

47.   Yang, WP, Levesque PC, Little WA, Conder ML, Shalaby FY, and Blanar MA. KvLQT1, a voltage-gated potassium channel responsible for human cardiac arrhythmias. Proc Natl Acad Sci USA 94: 4017-4021, 1997[Abstract/Free Full Text].


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