Functional and molecular identification of ERG channels in murine portal vein myocytes

Susumu Ohya1, Burton Horowitz1, and Iain A. Greenwood2

1 Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046; and 2 Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, SW17 0RE London, United Kingdom


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MATERIALS AND METHODS
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Ion channels encoded by ether-à-go-go-related genes (ERG) have been implicated in repolarization of the cardiac action potential and also as components of the resting membrane conductance in various cells. The aim of the present study was to determine whether ERG channels were expressed in smooth muscle cells isolated from portal vein. RT-PCR demonstrated the expression of murine ERG (mERG), and real-time quantitative PCR showed that the mERG1b isoform predominated over the mERG1a, mERG2, and mERG3 in portal vein. Single myocytes from portal vein displayed membrane staining with an ERG1-specific antibody. Whole cell voltage-clamp experiments were performed to determine whether portal vein myocytes expressed functional ERG channels. Large inward currents with distinctive kinetics were elicited that were inhibited rapidly by E-4031 (mean amplitude of the E-4031-sensitive current at -120 mV was -205 ± 24 pA; n = 14). Deactivation of the E-4031-sensitive current was voltage dependent (mean time constants at -80 and -120 mV were 103 ± 9 and 33 ± 2 ms, respectively; n = 13). Because of the rapid kinetics of mERG currents at more negative potentials, there was a substantial noninactivating "window" current that reached a maximum of -66 ± 10 pA at -70 mV. Complete portal veins exhibited spontaneous contractile activity in isometric tension experiments, and this activity was modified significantly by E-4031. These data show that ERG channels are expressed in murine portal vein myocytes that may contribute to the resting membrane conductance.

vascular smooth muscle; ether-à-go-go-related genes; potassium channel


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SEVERAL POTASSIUM CHANNEL GENE FAMILIES are expressed in vascular smooth muscle, and K+ channels play important roles in control of vascular tone by contributing to the resting potential. In resistance vessels such as cerebral artery and myogenic vessels such as portal vein, it has been suggested that voltage-gated, delayed rectifier channels contribute to the membrane K+ conductance (7, 10). Recently in gastrointestinal smooth muscles, it has been suggested that ion channels encoded by ether-à-go-go (eag)-related gene (ERG) are responsible for the regulation of the resting membrane potential (2, 9, 14). ERG channels have various distinctive characteristics that include inhibition by class III antiarrhythmic agents such as E-4031, augmented activation by raised extracellular K+, and marked inward rectification and distinctive "hooked" kinetics due to rapid inactivation (20, 21, 23, 24). Of three ERG members (ERG1-3), ERG1, which encodes the rapidly activating K+ currents (IKr) in cardiomyocytes (17), is responsible for the repolarization phases of the action potential (13, 16) and is implicated in cardiac long QT syndrome arrhythmia (16). ERG1 transcripts have also been observed in various tissues, and the murine gene is alternatively spliced to produce two splice variants, ERG1a and ERG1b, that differ in their NH2-terminal cytoplasmic domains (12). On the other hand, ERG2 and -3 are exclusively expressed in the nervous system, and ERG1-3 contribute to the control of neuronal excitability (6, 18, 19). In addition, the electrophysiological properties of ERG channels are modified by non-pore-forming KCNE subunits (2, 15).

The purpose of the present study was to determine whether myocytes isolated from murine portal veins expressed ERG channels and to compare the characteristics of an ERG-like current with previous studies. The portal vein was chosen because this preparation is spontaneously active and known to generate action potentials (22). Moreover, because KCNE gene products modulate ERG channel properties, we also determined whether these genes were expressed in murine vascular smooth muscles. Using a combination of RT-PCR, patch clamp, immunocytochemistry, and functional vasomotor measurement, we have shown that murine portal vein myocytes express ERG and exhibit an ERG-like current. We suggest from functional experiments that this conductance may play a role in the stabilization of the resting membrane potential and the regulation of the action potential duration in physiological conditions.


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Preparation of portal vein myocytes. BALB/c mice (>30 days old) were sedated by exposure to isoflurane (Baxter Laboratories) and killed by cervical dislocation in accordance with Institutional Animal Care and Use Committee protocols. After mice were killed, portal veins were isolated, cleaned of fat and connective tissue, and then cut into strips. These were then placed in physiological salt solution (PSS) containing 50 µM Ca2+, collagenase type 2 (3 mg/ml), bovine serum albumin (2 mg/ml), trypsin inhibitor (2 mg/ml), and protease type XIV (0.2 mgml) for 12 min at 37°C. Single myocytes were liberated by gentle agitation through a wide-bore Pasteur pipette and stored at 4°C. Freshly dispersed myocytes were then placed in a recording chamber for electrophysiological experiments or for collection and preparation of RNA. All electrophysiological experiments were performed at room temperature by using the whole cell configuration and an Axopatch 200B amplifier. Voltage-clamp protocols were generated and analyzed by using pCLAMP 8 software (Axon Instruments) and Origin 5 software. For RT-PCR analysis, collection of isolated portal vein smooth muscle cells was performed as reported previously (5).

Total RNA extraction and RT-PCR. Total RNA was extracted from tissues and isolated myocytes (~60 cells) with the use of a TRIzol (Life Technology) procedure and a SNAP total RNA isolation kit (Invitrogen), respectively. Total RNA was also isolated from brain and heart tissues. The Superscript II RNase H- (Life Technology) and 200 µg/ml of random hexamer (for tissues) or 500 µg/ml oligo(dT) primer (for cells) were used to reverse transcribe the RNA sample. The PCR amplication profile was as follows: a 10-s denaturation step at 94°C, a 10-s annealing step at 55°C, and a 30-s primer extension step at 72°C. In the tissue- and cell-based RT-PCR, the amplification was performed for 35 and 45 cycles, respectively. The amplified products were separated by electrophoresis on a 2% agarose-1× TAE (Tris, acetic acid, EDTA) gel, and the DNA bands were visualized by ethidium bromide staining. beta -Actin primers were used to confirm that the products generated were representative of RNA (498 bp) and not contaminated with genomic DNA (intron containing 708-bp band), because these primers were designed to span an intron as well as two exons. This control serves the identical purpose as a cDNA reaction lacking reverse transcriptase; however, it can be performed on the same RNA preparation as the test reactions. This is extremely important for isolated cell RNA preparations in which the low amount of RNA prevents the synthesis of more than one cDNA reaction per cell preparation. Each amplified product was sequenced by the chain termination method with an ABI Prizm (model 310; ABI).

PCR primers. The following PCR primers were used: ERG1 (GenBank accession no. AF012868), sense nt 1818-1840 and antisense nt 1923-1945, amplicon = 128 bp (conserved region for both ERG1a and ERG1b); ERG1a (AF012868), sense nt 42-61 and antisense nt 187-206, amplicon = 165 bp; ERG1b (AF012869), sense nt -79 to -60 and antisense nt 27-46, amplicon = 125 bp; ERG2 (BB656358), sense nt 50-69 and antisense nt 171-190, amplicon = 141 bp; ERG3 (AJ291608) sense nt 2995-3016 and antisense nt 3144-3163, amplicon = 169 bp; KCNE1 (NM 08424): sense nt 7-26 and antisense nt 351-370, amplicon = 364 bp; KCNE1-like (KCNE1L) (NM 021487), sense nt 31-50 and antisense nt 412-431, amplicon = 401 bp; KCNE2 (AK008619), sense nt 12-31 and antisense nt 349-368, amplicon = 357 bp; KCNE3 (NM 020574), sense nt 3-22 and antisense nt 255-274, amplicon = 272 bp; and KCNE4 (NM 021342), sense nt 123-142 and antisense nt 488-507, amplicon = 385 bp. Specific primers were also designed for quantitative PCR to determine relative levels of expression of KCNE, as follows: KCNE1, sense nt 6-26 and antisense nt 148-167, amplicon = 162 bp; KCNE1L, sense nt 1-22 and antisense nt 154-173, amplicon = 173 bp; KCNE2, sense nt 66-89 and antisense nt 179-198, amplicon = 133 bp; KCNE3, sense nt 40-61 and antisense nt 197-218, amplicon = 179 bp; and KCNE4, sense nt 232-251 and antisense nt 384-406, amplicon = 175 bp.

Quantitative PCR. Real-time quantitative PCR was performed with the use of Syber Green chemistry on an ABI 5700 sequence detector (PE Biosystems), as reported previously (5). In the SYBR Green Master Mix (PE Biosystems), there is an internal passive dye, ROX, in addition to the SYBR Green dye. The increase in the fluorescence of SYBR Green against that of ROX is measured at the end of each cycle. A sample is considered positive at the cycle in which the change in the fluorescence of SYBR Green relative to that of ROX (Rn) exceeds an arbitrary threshold value. The threshold value is set at the midpoint of the Rn and the cycle number plot. For all the amplifications described in this paper, the threshold value of the Rn was considered to be 0.2. The PCR cycle at which a statistically significant increase in the Rn is first detected is called the threshold cycle (CT). Target DNA copy number and CT values are inversely related. Regression analysis of the mean values of four multiplex RT-PCRs for the log10 diluted cDNA was used to generate standard curves. Unknown quantities relative to the standard curve for a particular set of primers were calculated, yielding transcriptional quantitation of ERG1 and KCNE gene products relative to the endogenous standard (beta -actin). The reproducibility of the assay was tested by analysis of variance (ANOVA) comparing repeat runs of samples, and mean values generated at individual time points were compared by Student's t-test.

Immunocytochemical experiments. Isolated myocytes of the murine portal vein were seeded onto glass-bottomed dishes, respectively. Before staining, isolated myocytes were fixed with 4% 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. As previously reported by Ohya et al. (14), cells were then exposed to anti-ERG1 polyclonal antibody (1:50 dilution; Alomone Labs) and anti-KCNE3 polyclonal antibody [KCNE3 (N-18), 1:40 dilution; Santa Cruz Biotechnology] for 12-16 h at 4°C. Excess primary antibody was removed by repeated washing with PBS, and the cells were exposed to Alexa Fluor 488 goat antirabbit (for anti-ERG) or Alexa Fluor 488 donkey antigoat (for anti-KCNE3) IgG antibody (1:200 dilution; Molecular Probes). After incubation for 1 h at room temperature, excess secondary antibody was removed by repeating washing with PBS. Digital images were viewed on a scanning confocal microscope (MRC600; Bio-Rad). As negative controls, cells were preincubated with excess antigen before the addition of primary antibody (see Fig. 3, B and D).

Electrophysiological protocols. The aim of these electrophysiological experiments was to determine whether vascular smooth muscle cells isolated from murine portal veins exhibited a current with characteristics similar to ERG currents described previously. In many studies the peculiar channel properties of ERG channels have been exploited to generate inwardly rectifying K+ currents with distinctive hooked appearance. Thus in esophageal smooth muscle (2), microglia (25), and lactotrophs (18) as well as studies on heterologously expressed ERG channels (23), currents were elicited by stepping from the holding potential of 0 mV to test potentials between +40 and -120 mV in cells bathed in 140 mM K+ to augment the amplitude of inward currents. With this protocol, ERG channels become inactivated at the holding potential and then reactivate at negative test potentials as inactivation is removed. Channels then deactivate at the negative test potentials. These properties of the channel result in the hooked appearance. This experimental paradigm (termed protocol 1) was used in the present study. Pulses longer than 300 ms were often contaminated by a slowly activating mixed cation current (termed Ih) that is present in this cell type (8), and therefore brief test steps were applied. The ionic nature of the evoked current was investigated by using a protocol based on that used by Schäfer et al. (18). Cells held at 0 mV were stepped initially to -120 mV for 10 ms to reactivate ERG channels, followed by a 70-ms voltage ramp from -120 to +40 mV in solutions that had different concentrations of K+. The protocol was repeated in the same external solutions but with the addition of 1 µM E-4031. Ramp-evoked currents recorded in the presence of E-4031 were subtracted from the corresponding control currents to give the E-4031-sensitive current. The amplitude of the E-4031-sensitive current at each test voltage was then plotted against the test potential. The effects of E-4031 and Ba2+ on the amplitude of the evoked current were determined by constructing an ensemble of currents at voltages between -120 and +40 mV under control conditions and after 3-min application of the agents. Time dependence of block was determined by stepping from 0 to -120 mV for 300 ms every 15 s in the absence and presence of the drug. The concentration dependence of E-4031 block was determined by stepping to -120 mV from a holding potential of 0 mV for 400 ms in the presence of concentrations of E-4031 between 1 nM and 5 µM. Each concentration of E-4031 was applied for 2 min, and a concentration effect curve was constructed by plotting the percentage of control current amplitude after 2 min application of E-4031 against the E-4031 concentration. E-4031-sensitive currents were determined by subtracting currents evoked by protocol 1 described above in the presence of 1 µM E-4031 from currents recorded in the absence of this agent.

Kinetics and voltage dependence parameters. The voltage dependence of channel reactivation at various test potentials was quantified by measuring the time taken to achieve a maximal inward current from the initiation of the voltage step indicated by the peak of start of the capacitative transient. The kinetics of channel deactivation was determined by fitting the decline of E-4031-sensitive currents at each test potential with a single exponential. The availability of the E-4031-sensitive current was determined by stepping to -120 mV to reactivate maximally ERG channels after prepulses of either 300 ms or 5 s to potentials between +40 and -120 mV. Longer protocols were executed in the presence of 5 mM CsCl to block the hyperpolarization-activated cation current present in portal vein myocytes (8).

Solutions. In electrophysiological experiments, external and internal solutions were used to minimize contamination from other conductances and were based on solutions used by Akbarali et al. (2) and Zhou et al. (25). Cells were initially bathed in an external solution (solution A) of the following composition (in mM): 126 NaCl, 5 KCl, 10 HEPES, 20 glucose, 0.1 CaCl2, 1.0 MgCl2, 10 TEA-Cl, and 5 4-aminopyridine (4-AP), and pH was set to 7.2 with 10 M NaOH. Currents through mERG channels were characterized in an isotonic, raised K+ external solution (solution B; in mM): 140 KCl, 10 HEPES, 20 glucose, 0.1 CaCl2, 1.0 MgCl2, 10 TEA-Cl, and 5 4-AP, and pH was set to 7.2 with 10 M NaOH. The internal solution for these experiments was based on one described by Akbarali et al. (2) and had the following composition (in mM): 100 K-aspartate, 30 KCl, 10 HEPES, 5 EGTA, 5 ATP-Na2, 0.1 GTP, and 1 MgCl2. With this pipette solution, activation of Ca2+-dependent Cl- currents that are common in this cell type (5) were precluded by the inclusion of a high concentration of the Ca2+ chelator EGTA. Recording of ATP-sensitive K+ currents was similarly inhibited by the inclusion of 1 mM ATP in the pipette solution, and the presence of TEA and 4-AP should minimize the recording ofvoltage-dependent (Kv-type) K+ channels. The composition of the Krebs solution used in functional experiments was (in mM) 120 NaCl, 5.9 KCl, 15 NaHCO3, 1.2 NaH2PO4, 1.2 Mg Cl2, 11 glucose, and 2.5 CaCl2. All enzymes and reagents were purchased from Sigma except E-4031, which was bought from Wako Industries (Japan).

Functional experiments. Whole portal veins were dissected from killed BALB/c mice by ligation with fine surgical thread immediately proximal to the liver and close to the convergence of the splenic vein (length of segment was ~8 mm). After removal of fat deposits, veins were suspended in a 15-ml organ bath containing aerated Krebs solution (see below) at 37°C and attached to a force transducer for isometric tension recording. Tissues were washed with Krebs solution every 15 min until regular spontaneous contractions typical of this blood vessel (22) developed. After a stable recording period of 30 min under control conditions, E-4031 (1-10 µM) was applied to the organ bath for 7 min per concentration. For each vessel three parameters were measured: the peak amplitude above baseline of each spontaneous contraction, total duration of each contraction, and the interval between the each peak contraction. All parameters were measured for individual contractions over a 2-min period and averaged.

Statistics. All data are the mean of n cells ± SE. In all experiments data were taken from at least three different animals. The statistics for Fig. 8 are Bonferroni's t-test after analysis of variance.


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Molecular identification of ERG subtypes expressed in murine portal vein myocytes. As shown in Fig. 1A, ERG1 transcripts were expressed in portal vein but at significantly lower levels than those in brain and heart. It has been reported that the shorter spliced variant with the diverse NH2-terminal cytoplasmic domain, ERG1b is specifically expressed in murine heart (12). We therefore designed specific PCR primers for ERG1a and ERG1b, respectively (see MATERIALS AND METHODS), and performed additional RT-PCR experiments. ERG1b was predominantly expressed in portal vein (Fig. 1A, ERG1a and ERG1b) compared with ERG1a. Subsequently, we determined the expression of ERG2 and ERG3 in portal vein. To identify the expression of murine ERG2, we searched the expressed sequence tag clones using protein-protein BLAST (National Center for Biotechnology Information) that produced three different clones (partial coding sequence) with the sequence identity to rat ERG2; BB632284, BB656358, and BB452732. The amino acid sequences of these clones have over 95% similarity to rat ERG2 and 82-85 and 70-82% similarity to murine ERG1 and ERG3, respectively. We therefore designed specific PCR primers for BB656358. Neither ERG2 nor ERG3 signals were detected in portal vein (Fig. 1A, ERG2 and ERG3). However, amplicons were detected from RT-PCR by using mouse brain RNA as a template. beta -Actin primers were used to confirm that the products generated were representative of RNA (498 bp) and not contaminated with genomic DNA (intron containing 708-bp band, Fig. 1B) because these primers were designed to span an intron as well as two exons (Fig. 1, A and B). Similar results were obtained from at least three separate experiments. Each amplified product was confirmed by DNA sequencing.


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Fig. 1.   Expression of ether-à-go-go-related gene (ERG) channels in murine portal vein. A: RT-PCR detection of ERG1 channels in murine portal veins (PV). PCR products were generated through the use of gene-specific primers for ERG1, ERG1a, ERG1b, ERG2, and ERG3. A 100-bp molecular weight marker was used to estimate the size of the amplicon. Br, brain; He, heart. B: RT-PCR performed in the presence of beta -actin gene-specific primers demonstrates that products are representative of RNA (498 bp). C: cell-based RT-PCR analysis in murine PV cells (PVC). NTC, nontemplate control. D: quantitative RT-PCR for ERG1, ERG1a, and ERG1b expression relative to beta -actin in murine brain, heart, and PV. Values are shown for steady-state transcripts relative to beta -actin in the same preparation. Results are expressed as means ± SE (n = 4 for each).

To avoid the contamination from nonmyocytes, cell-based RT-PCR analyses were performed on freshly isolated murine portal vein cells (PVC). As shown in Fig. 1C, ERG1 and ERG1b signals were easily detected in PVC, whereas ERG1a amplicons were very weak or undetectable in PVC. Similar results were obtained from three separate experiments. These results were consistent with those from tissue-based RT-PCR experiments described above.

The ABI 5700 genetic analyzer (Perkin Elmer Biosystems) was used for accurate quantification of steady-state transcript levels by RT-PCR. Total RNA was prepared from murine brain, heart, and portal vein. RNA was reverse transcribed to cDNA, and steady-state transcripts were determined relative to an endogenous control housekeeping gene (beta -actin). To compare the efficiencies of amplification of ERG1, ERG1a, and ERG1b with those of beta -actin genes, the CT values were plotted against the dilutions of the cDNAs obtained from heart. The slopes of the regression lines for ERG1, ERG1a, ERG1b, and beta -actin gene were 3.64, 3.65, 3.74, and 3.87, respectively, and were within the range of the calculated standard deviations for each pair (P > 0.05; n = 3, not shown). Therefore, the amplification efficiencies of both ERG1 subtypes and beta -actin were considered very similar and allowed for relative quantification of ERG1 transcripts by real-time PCR. The data are expressed as ratios of ERG1, ERG1a, and ERG1b to beta -actin, respectively, and their relative transcriptional expression is shown in Fig. 1D.

In portal vein the values of ERG1 and the two truncated isoforms ERG1a and ERG1b relative to beta -actin were 0.0025 ± 0.00055, 0.00060 ± 0.000057, and 0.0022 ± 0.00053 (n = 4 for each), respectively. The values of ERG2 and ERG3 relative to beta -actin were under 0.0008 in portal vein (not shown). Additional experiments performed by using ERG2 primers specific for BB632284 and BB452732, respectively, also resulted in values below 0.0008 in portal vein (not shown). In brain, ERG1, ERG1a, ERG1b, ERG2, and ERG3 values relative to beta -actin were 0.020 ± 0.0012, 0.014 ± 0.0070, 0.0012 ± 0.00021, 0.0072 ± 0.0010, and 0.45 ± 0.017, respectively (n = 4 for each). In heart, these values were 0.015 ± 0.001, 0.0070 ± 0.00053, 0.0043 ± 0.0012, 0.00080 ± 0.00009, and 0.00091 ± 0.000045, respectively (n = 4 for each). These results suggest that portal vein expresses ERG1, predominantly the ERG1b isoform.

Molecular identification of KCNE subtypes expressed in murine portal vein myocytes. Regulatory proteins encoded by KCNE genes have been shown to alter markedly the kinetics and pharmacological sensitivity of human ether-à-go-go-related gene (HERG) channels (1, 15). We therefore identified the molecular components of KCNE subtypes expressed in murine portal vein (Fig. 2). RT-PCR analyses showed that KCNE2 and -3 were expressed in PVC (Fig. 2A). Quantitative PCR experiments showed that, in portal vein, KCNE3 expression relative to beta -actin (arbitrary units) was 0.012 ± 0.0016 and that the expression of other KCNE transcripts was <0.001 (n = 5 for each; Fig. 2B). As positive controls, the KCNE1-4 expression profiles were determined in brain tissue where these genes are abundantly expressed. The levels of KCNE expression relative to beta -actin were 0.014 ± 0.0049 (KCNE1), 0.063 ± 0.0043 (KCNE1L), 0.065 ± 0.0086 (KCNE2), 0.021 ± 0.0047 (KCNE3), and 0.038 ± 0.0068 (KCNE4), respectively (n = 5 for each). The slopes obtained for the KCNE1-4 primer pairs were similar (3.5-3.7) and were within the range of the calculated standard deviations for each pair (P > 0.05; n = 3). These results show that KCNE3 transcripts are expressed in murine portal vein.


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Fig. 2.   Expression of KCNE subtypes in murine PV. A: cell-based RT-PCR detection of KCNE subtypes in murine PV. PCR products were generated through the use of gene-specific primers for KCNE1-4. Primers were tested on murine brain and heart and sequenced to confirm their identity. B: quantitative RT-PCR for KCNE expression relative to beta -actin in murine PV. Values are shown for KCNE steady-state transcripts relative to beta -actin in the same preparation. Results are expressed as means ± SE (n = 4 for each).

Immunocytochemistry of ERG1 and KCNE3 proteins in murine portal vein myocytes. To determine that the identified ERG1 and KCNE3 transcripts are translated into proteins and expressed on the surface membranes of murine PVC, the cellular localization of their protein was examined by immunocytochemical analysis. Strong ERG1 staining was observed along the cell membrane in PVC (Fig. 3A). In addition, the staining patterns of KCNE3 were observed along cell membranes in PVC (Fig. 3C). ERG1 and KCNE3 signals disappeared by preincubation with the excess antigen, respectively (Fig. 3, B and D). No immunoreactivity was detected in PVC when similar experiments were performed without primary antibodies (not shown). All images were obtained by using the same confocal conditions. Similar staining patterns were obtained in over 15 cells. These data show that PVC expresses ERG1 and KCNE3 proteins.


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Fig. 3.   Expression of ERG1 and KCNE3 proteins in isolated myocytes from the murine PV using immunocytochemical methods. Confocal images show representative isolated smooth muscle cells (PVC). Cells were immunostained with specific antibodies against ERG1 (A) and KCNE3 (C) proteins. As a negative control, cells were preincubated with excess antigen for ERG1 (B) and KCNE3 (D) before addition of primary antibody, respectively. Bar, 20 µm.

Electrophysiological characterization of ERG currents in murine PVC. Currents carried by ERG channels have distinctive characteristics that allow this conductance to be distinguished from other ion channels (see Introduction). In many studies ERG currents are recorded as inward currents with distinctive kinetics when the cell being studied is bathed in an external solution containing a high K+ concentration. In the present study, inward currents with a hooked appearance were elicited in portal vein myocytes bathed in 140 mM K+ containing external solution by membrane hyperpolarization, and the mean peak amplitude at -120 mV was -371 ± 30 pA (n = 21).

It is possible that the inwardly rectifying current recorded in portal vein myocytes under symmetrical conditions was due to the activation of conventional inward rectifier currents (e.g., Kir2.1) that are expressed in vascular myocytes (4). These channels are blocked by low micromolar concentrations of Ba2+. In PVC, the amplitude of the inward current evoked by stepping to -120 from 0 mV was not significantly affected by 100 µM Ba2+, a concentration that would inhibit markedly classic inward rectifier channels (4) (mean amplitude in the absence and presence of 100 µM Ba2+ was -286 ± 27 and -280 ± 25 pA) but was slightly inhibited by 1 mM Ba2+ (mean amplitude at -120 mV was -248 ± 26 pA; n = 3). In comparison, application of the selective ERG blocker E-4031 (1 µM) to murine myocytes bathed in 140 mM K+ external solution produced a rapid inhibition of the peak current evoked by step hyperpolarization from 0 to -120 mV (Fig. 4, A and B). After 3 min of application, the peak current at -120 mV decreased from -356 ± 48 to -151 ± 25 pA (n = 7). Figure 4B shows the mean concentration-effect curve for E-4031 on the inward current recorded under these conditions, and the mean IC50 was 82 nM (n = 4). It can be seen from Fig. 4C that the reversal potential of the E-4031-sensitive current was dependent on the external K+ concentration, indicating that the E-4031-sensitive current was carried mainly by K+ ions, and similar effects were observed in three cells.


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Fig. 4.   Inward currents in murine PV myocytes. A: representative family of currents evoked by stepping the membrane potential from 0 mV to test potentials between +40 and -120 mV in a PV myocyte bathed in an external solution containing 140 mM K+. Left: currents recorded under control conditions. Right: currents recorded in the same cell after 3 min application of 1 µM E-4031. Inset: protocol used in these experiments. B: mean E-4031 concentration-effect curve from 4 cells. Each concentration of E-4031 was applied for 2 min, and the current after each application was presented as a percentage of the control current. Error bars show SE. C: reversal potential of the E-4031-sensitive currents was dependent on the external K+ concentration. Currents were elicited by voltage ramps from -120 to +40 mV in cells bathed in 39, 72.5, and 140 mM external K+. The voltage protocol is shown beneath each current.

Figure 5 shows that the current current-voltage relationship of the maximal E-4031-sensitive current exhibited marked inward rectification consistent with ERG channels in other studies (2, 23, 25). Figure 5A shows that the E-4031-sensitive current at -40 mV was well sustained for the duration of the test step, whereas the E-4031-sensitive current at -100 mV exhibited a rapid decay. Consequently, the current-voltage relationship of the E-4031-sensitive current recorded immediately before stepping back to 0 mV was "U" shaped with a maximal steady-state current of -62 ± 9 pA at -60 mV (Fig. 5B). These data show that murine portal vein myocytes bathed in an external solution containing 140 mM K+ exhibited an inwardly rectifying current that was sensitive to the selective ERG channel blocker E-4031.


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Fig. 5.   Current-voltage relationship of the E-4031-sensitive current. The E-4031-sensitive current was determined at different test potentials using protocol 1 as described in MATERIALS AND METHODS by subtracting the current recorded at each potential in the presence of 1 µM E-4031 from currents recorded at the same potential in the absence of this agent. A: E-4031-sensitive current recorded at 3 different test potentials (-40, -60, and -100 mV). B: mean E-4031-sensitive peak current () and late current (open circle ) recorded at each potential in cells bathed in external solution containing 140 mM K+. The points at which the currents were recorded are highlighted in A (right). Each point is the mean of between 7 and 14 cells ± SE.

Kinetics of the inward current evoked under symmetrical K+ conditions. The E-4031-sensitive inward current was characterized by a pronounced hook as the current initially increased and then decayed exponentially. Figure 6A shows a representative ensemble of currents elicited by membrane hyperpolarization on a faster time base to highlight the voltage-dependent changes in reactivation followed by deactivation. The mean data in Fig. 6B, top, show that time to peak amplitude (TTP) became faster with membrane hyperpolarization, with the mean TTP increasing from 4 ± 0.6 ms at -120 mV to 28 ± 4 ms at -80 mV. The time constant for the decay of the inward current (tau ) was also markedly voltage dependent with mean tau  increasing from 33 ± 10 ms at -120 mV to 103 ± 35 ms at -80 mV (see Fig. 6B, bottom). These values for the voltage-dependent kinetics are similar to previously reported values for native (2, 18, 25) and heterologously expressed ERG channels (20, 21, 23) and are synonymous with the progressive transition of ERG channels from an inactivated state at 0 mV to an initial open state upon hyperpolarization followed by rapid deactivation. The potential dependence of the E-4031-sensitive channel availability was determined in cells bathed in 140 mM K+ using both short (300 ms) and long (5 s) prepulses from the holding potential of 0 mV. Figure 7 shows that a large, E-4031-sensitive inward current was recorded at the test potential (-120 mV) when this was preceded by a depolarizing test step. As the voltage of the prepulse was made more negative, the number of channels available for opening was reduced as more channels became deactivated, and, consequently, the amplitude of the current elicited at -120 mV was smaller. Figure 7B shows a plot of normalized amplitude of the current at -120 mV against the prepulse potential. The data were well fitted by a Boltzmann function that yielded values of V0.5 of -74 ± 3 mV (n = 7) and -51 ± 3 mV (n = 5) for currents generated by short and long prepulses, respectively. Slope values were unaffected by the duration of the prepulse (10 ± 1.6 and 11 ± 1.2, respectively). Overall, these data show that the E-4031-sensitive inward current in murine PVC exhibited kinetics and voltage-dependent channel availability similar to ERG currents native to various cell types (2, 18, 25) and currents generated by heterologously expressed ERG channels (20, 21, 23).


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Fig. 6.   Voltage-dependent kinetics of the hyperpolarization-activated current. A: an example of inward currents recorded in a murine portal vein myocyte elicited by protocol 1 as described in MATERIALS AND METHODS, showing the currents recorded at -120, -100, -80, and -60 mV on an extended time base to highlight the voltage-dependent kinetics of the currents. The time to peak amplitude is shown by the dotted lines, and the deactivation of the current can be tracked by following the indexed lines (z-z', y-y', x-x' and w-w'). The lines overlaying the raw data show the exponential fit to the current decay. B, top and B, bottom show the mean time to peak (ms) and the mean time constant (tau ) for the exponential decay of the current at each potential plotted on a semilog scale, respectively. Voltage dependence of the current decay can be described by an exponential with tau  changing for a 27-mV shift in potential (bottom).



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Fig. 7.   Quasi-steady-state availability of the ERG current. A: representative currents evoked by a 2-step inactivation protocol (shown in inset) in cells bathed in an external solution containing 140 mM K+. Cells were held at 0 mV and stepped to a test potential of -120 mV for 200 ms after a prepulse to various potentials between +40 and -140 mV for either 300 ms (top) or 5 s (bottom). B: mean voltage dependence of the channel availability using either a 300-ms () or 5-s prepulse (open circle ). Each point is the mean of 5-7 cells with error bars representing the SE.

Functional role of IERG in murine portal vein myocytes. The previous experiments utilized the distinctive properties of ERG channels in cells bathed in an external solution containing high K+ concentration to describe the characteristics of a similar current in portal vein myocytes. Experiments were also performed on cells held at -60 mV and bathed in an external solution containing TEA and 4-AP but with a more physiological K+ concentration (5 mM). Figure 8A, left, shows that a rapidly activating and inactivating E-4031-sensitive current could be recorded at potentials positive to the holding potential. Similar data were recorded in four other cells, and the mean current-voltage relationship is shown in Fig. 8A, right. Consequently, IERG may have a role in the functional activity of the portal vein, and isometric tension experiments on whole murine portal veins were therefore performed. Figure 8B shows a representative trace of the spontaneous contractile behavior recorded in five veins. Regular increases in tension were observed every 11.9 ± 0.66 s that were generally composed of a single contractile spike, although sometimes a contractile complex composed of two to three spikes was recorded. Application of 1 µM E-4031 to the tissue bath produced a significant lengthening of the duration of each contractile complex from 6.7 ± 0.44 s to 11 ± 0.59 s (P < 0.01) and also prolonged the interval between each contraction (mean interval in the presence of 1 µM E-4031 was 19 ± 0.9). E-4031 had no significant effect on the peak amplitude that had a mean of 0.27 ± 0.05 g and 0.29 ± 0.01 g in the absence and presence of E-4031 (n = 5 tissues; 41-47 individual contractions). No further effect on interval or duration was observed by applying 10 µM E-4031 (21 ± 1.6 s; n = 3; 12.5 ± 0.9, n = 3, respectively). These data are consistent with E-4031 blocking a channel involved with the normal electrical activity of the murine portal vein.


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Fig. 8.   Predicted functional effect of ERG channel current. A: mean E-4031-sensitive currents evoked by stepping a cell bathed in an extracellular solution containing 5 mM K+ from a holding potential of -60 mV to potentials between -120 and +60 mV for 1 s (left). The mean data from 5 experiments are shown at left. Right: E-4031-sensitive current was determined by subtracting the currents recorded at each potential in the presence of 1 µM E-4031 from currents recorded at the same potential in the absence of E-4031. B: examples of spontaneous, contractile activity in murine portal veins under control conditions and after 5-min application of 1 µM E-4031. C: mean contractile interval and duration in the absence (control) and presence of E-4031. Each bar shows the mean of 5 tissues ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study has shown for the first time the existence of ERG channels in a vascular smooth muscle cell type. Use of mERG1 primers allowed amplification of mRNA transcripts for two isoforms of mERG1 (mERG1a and mERG1b), and the mERG1b isoform was shown by quantitative PCR to predominate. Moreover, the expression genes that regulate ERG channel properties were shown in portal vein cells using primers to KCNE isoforms with KCNE3 expression being markedly higher than other isoforms. Functional translation and successful trafficking of the protein to the membrane were confirmed by immunocytochemistry, and electrophysiological studies showed the existence of an inwardly rectifying, E-4031-sensitive K+ current that exhibited distinctive hooked kinetics when currents were recorded under symmetrical K+ conditions. A functional role for ERG channels in the regulation of electrical events was suggested by the significant effect of E-4031 on spontaneous, rhythmic activity in whole portal veins.

Characteristics of IERG. In murine portal vein cells, a K+ current was recorded that had many similarities with conductances through native and heterologously expressed ERG channels. The currents were blocked by the selective ERG inhibitor E-4031 with an IC50 of about 90 nM that is similar to IC50 values reported previously for native ERG channels (see e.g., Refs. 2, 25). In comparison, micromolar Ba2+ had no effect on the current, and 1 mM Ba2+ only produced a small inhibition similar to other native and heterologously expressed ERG currents (see e.g., Refs. 2, 23). In symmetrical K+ conditions, the E-4031-sensitive currents exhibited very little outward current similar to native ERG currents in opossum esophageal smooth muscle (2), lactotrophs (18), and rat microglia (25) that lead to a pronounced inward rectification that is a feature of this channel. Furthermore, the distinctive hooked kinetics after hyperpolarization from 0 mV were consistent with previous schemes for ERG channels. Thus, at 0 mV, the channels reside in an inactive state because of very rapid inactivation either directly from a closed state or via brief duration openings (20, 21, 23, 25). Membrane hyperpolarization relieves the channel inactivation but also shifts the channel equilibrium from an open to closed state, resulting in the rapid deactivation at negative potentials. The kinetics of channel reactivation and deactivation measured in the present study were quantitatively similar to those of native and heterologously expressed ERG currents (2, 18, 20, 21, 23, 25). The voltage dependence of channel availability in the present study was also comparable with values reported for other native ERG channels (2, 18, 25). These characteristics and the sensitivity to E-4031 distinguish ERG currents from other inward rectifiers such as conventional inward rectifier K+ (Kir) channels that exist in arterial smooth muscle cells (4) and are possibly also present in venous myocytes. These currents have no marked time dependence, are not blocked by E-4031, and are sensitive to low micromolar concentrations of external Ba2+, which we show in the present study has no effect on the inwardly rectifying E-4031-sensitive current. Another form of inward rectifier, previously recorded in rabbit portal vein cells (8), was also present in murine portal vein cells. This current, encoded by HCN genes, is a mixed cation current that slowly activates at hyperpolarized potentials and does not decay, in marked contrast to ERG currents. Moreover, E-4031 did not affect these slowly activating currents in murine portal vein cells (I. Greenwood, unpublished observation). Consequently, in murine portal vein cells, a current can be activated by membrane hyperpolarization in symmetrical K+ conditions that resembles, very strongly, native currents through ERG channels recorded in other cell types (2, 18, 25), as well as expressed ERG channels (20, 21, 23).

An interesting feature of the E-4031-sensitive current in portal vein cells was that the outward current recorded with quasi-physiological K+ concentration in the external solution was relatively transient and did not exhibit the pronounced "bell" shape associated with heterologously expressed mERG channels (11, 12) or native E-4031-sensitive currents in cardiomyocytes (17). The difference in recorded current may be due to discrepancies in experimental protocol and the use of expression systems such as Xenopus oocytes compared with freshly dispersed myocytes. Alternatively, we show that KCNE genes are expressed in murine portal vein cells, and the biophysical properties of expressed HERG are modified markedly by coexpression with the regulatory subunit KCNE2 (MiRP1; Ref. 1). Interestingly, KCNE3 expression was the highest, and the effect of this regulatory protein on ERG channel properties has not been investigated. Furthermore, the reactivation and deactivation kinetics of the ERG current in portal vein myocytes were considerably faster than those for expressed mERG1a and mERG1b channels reported previously (12), which supports the proposal that an auxiliary subunit may influence the kinetics of the observed current. Consequently, the characteristics of the native current may be a product of the coexpression of mERG1 isoforms with regulatory subunits possibly KCNE3. The exact molecular composition of the proteins that underlie the ERG-like current in portal vein myocytes must be the focus of future expression studies.

Putative functional role of IERG. E-4031-sensitive currents compose the rapid component of the cardiac action potential (17) and have also been proposed to contribute to the resting conductance in microglia (25) and esophageal smooth muscle cells (2) by others using solutions and protocols similar to those employed in the present study. In murine portal vein myocytes, when the amplitude of the ERG current was augmented by bathing cells in 140 mM K+, a prominent, noninactivating E-4031-sensitive window current was revealed that had a peak close to the resting membrane potential of portal vein cells (22). These conditions are not physiological, although these data reveal that ERG proteins are expressed in vascular smooth muscle cells and can constitute a noninactivating conductance. Small changes in the contribution of this channel to the resting conductance, possibly due to an increase in extracellular K+, will affect the resting membrane potential because of the high input resistance of smooth muscle cells. When experiments were performed under more physiological conditions (with 5 mM K+ in the external solution), a rapidly activating and inactivating E-4031-sensitive outward current could be evoked in murine portal vein by membrane depolarization. Interestingly, the E-4031-sensitive outward current was not as sustained as ERG currents in cardiomyocytes (17), which may reflect a difference in the channel composition underlying the current (see previous paragraph). Further support for the ERG channel having a functional role in the portal vein was the observation that application of E-4031 to spontaneously contracting whole portal veins caused a significant prolongation of the contraction duration and slowed the interval between each contraction. The mechanisms underlying pacemaking of the portal vein have not been elucidated, but hyperpolarization-activated cation channels analogous to cardiac pacemaker currents are present in this cell type (Ref. 8, present study). Reduced hyperpolarization after inhibition of a resting K+ current would decrease the activity of these pacemaking channels, which may explain the effect of E-4031 on intercontraction interval. Although there are a number of K+ channels in vascular smooth muscle cells (3) and the functional roles of these channels have not been probed in the present study, the ability of E-4031 to prolong the duration of individual contractions suggests a role of IERG in action potential repolarization. Although the precise role of ERG in the membrane potential events of the murine portal vein remains to be elucidated, it is clear that blockade of ERG channels by E-4031 affected significantly the contractility of murine portal veins. It is possible that the rapid kinetics of ERG channels are crucial for a smooth muscle preparation that frequently discharges action potentials and that other K+ currents are more important in quiescent vessels. However, the known role of ERG channels in cardiac arrhythmia and as a focus for type III antiarrhythmic agents suggests that the presence of these channels in the vasculature may have a significant influence on cardiovascular function and possibly inherited vascular abnormalities.


    ACKNOWLEDGEMENTS

We thank Dr. K. Keef for help with contractile recordings and L. Miller and Y. Bayguinov for excellent technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute HL-49254.

Address for reprint requests and other correspondence: B. Horowitz, Dept. of Physiology and Cell Biology, Univ. of Nebraska School of Medicine, Reno, NV 89557-046 (E-mail: burt{at}physio.unr.edu).

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.

May 15, 2002;10.1152/ajpcell.00099.2002

Received 5 March 2002; accepted in final form 13 May 2002.


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DISCUSSION
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