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Am J Physiol Gastrointest Liver Physiol 295: G691-G699, 2008. First published July 31, 2008; doi:10.1152/ajpgi.90348.2008
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NEUROREGULATION AND MOTILITY

Ether-a-go-go-related gene 3 is the main candidate for the E-4031-sensitive potassium current in the pacemaker interstitial cells of Cajal

Elizabeth J. White,1,* Sung Jin Park,1,* Jane A. Foster,2 and Jan D. Huizinga1

1Intestinal Disease Research Programme, Department of Medicine, and 2Brain Body Institute, St. Joseph's Healthcare, McMaster University, Hamilton, Ontario, Canada

Submitted 22 May 2008 ; accepted in final form 24 July 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The interstitial cells of Cajal (ICC), as pacemaker cells of the gut, contribute to rhythmic peristalsis and muscle excitability through initiation of slow-wave activity, which subsequently actively propagates into the musculature. An E-4031-sensitive K+ current makes a critical contribution to membrane potential in ICC. This study provides novel features of this current in ICC in physiological solutions and seeks to identify the channel isoform. In situ hybridization and Kit immunohistochemistry were combined to assess ether-a-go-go-related gene (ERG) mRNA expression in ICC in mouse jejunum, while the translated message was assessed by immunofluorescence colocalization of ERG and Kit proteins. E-4031-sensitive currents in cultured ICC were studied by the whole cell patch-clamp method, with physiological K+ concentration in the bath and the pipette. In situ hybridization combined with Kit immunohistochemistry detected m-erg1 and m-erg3, but not m-erg2, mRNA in ICC. ERG3 protein was colocalized with Kit-immunoreactive ICC in jejunum sections, but ERG1 protein was visualized only in the smooth muscle cells. At physiological K+ concentration, the E-4031-sensitive outward current in ICC was different from its counterpart in cardiac and gut smooth muscle cells. In particular, inactivation upon depolarization and recovery from inactivation by hyperpolarization were modest in ICC. In summary, the E-4031-sensitive currents influence the kinetics of the pacemaker activity in ICC and contribute to maintenance of the resting membrane potential in smooth muscle cells, which together constitute a marked effect on tissue excitability. Whereas this current is mediated by ERG1 in smooth muscle, it is primarily mediated by ERG3 in ICC.

gastrointestinal motility; potassium channels; pacemaker activity

THE RHYTHMIC CONTRACTILITY of gastrointestinal muscle is dependent on the coordinated activities of smooth muscle cells, neurons, and the interstitial cells of Cajal (ICC), which function as pacemaker cells (24). Rhythmic inward currents generated by ICC in the myenteric plexus have been characterized as pacemaker currents that produce an oscillation in membrane potential, known as the slow wave, which regulates the periodic membrane excitability of smooth muscle cells and, thus, also controls aspects of muscle contraction. The basic understanding of the ionic currents and ion channel proteins that contribute to the generation of membrane potential and pacemaker activity in ICC is far from complete (9). The general aim of characterizing the electrophysiology of ICC is to produce a comprehensive profile of ion channels that will clarify their physiological properties, and these ion channels could potentially be targeted to manipulate and improve gastrointestinal motility in pathological conditions.

E-4031 is a class III antiarrhythmic methanesulfonanilide compound that inhibits members of the ether-go-go-related gene (ERG) K+ channel family, including the isoforms ERG1, ERG2, and ERG3 (20). The compound was originally shown to inhibit the cardiac delayed-rectifier K+ channel HERG (human ERG1) (22), and, more recently, E-4031-sensitive currents have been identified in gut smooth muscle (2, 6) and ICC (12, 31). If a difference between the ERG channels expressed in gut muscle, ICC, and cardiomyocytes could be determined, then it may be possible to develop pharmaceuticals to target ICC specifically without disturbing the cardiac action potential.

Because of previous studies that identified ERG1 as a component of the rapid delayed-rectifier current in sinoatrial node pacemaker cells (4), murine portal vein myocytes (16), and opossum esophageal circular smooth muscle (2), ERG1 was the most logical candidate for the E-4031-sensitive current in gut pacemaking. However, Farrelly et al. (6) recently demonstrated HERG immunoreactivity only in smooth muscle cells and PGP9.5-immunoreactive ganglion cells in the myenteric plexus of human jejunum. Therefore, we also assessed ERG2 or ERG3 isoforms as described in the central nervous system (7) and pancreas (14), since electrophysiological characteristics of these channels are largely unknown outside expression systems.

The aim of this study was to search for potentially unique features of the E-4031-sensitive currents using physiological ionic conditions and voltage-step protocols focusing on membrane potentials in the range of slow wave activity generated in ICC and to investigate a potentially unique expression of ERG K+ channel {alpha}-subunits, ERG1, ERG2, and ERG3, and β-subunits, MiRP1 and MinK, in mouse jejunum tissue sections.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All procedures used to obtain mouse tissue were approved by the McMaster University Animal Research Ethics Board. Neonatal (3- to 4-day-old) and adult (6- to 8-wk-old) CD1 mice were killed by cervical dislocation. For primary cultures, the neonatal jejunum muscularis was isolated and cut into small (~1-mm2) pieces in M199 supplemented with 10% FBS, 1% glutamine, and 1% antibiotic-antimycotic (GIBCO, Invitrogen, Burlington, ON, Canada). Tissue explants were cultured for 5–6 days on 20-mm-diameter coverslips at 37°C in 95% O2-5% CO2 before use.

Generation of 35S-labeled m-erg cRNA probes. Total RNA was extracted from jejunum tissue using a combination of TRIzol and the RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada), suspended in nuclease-free water, treated with DNase I, and stored at –80°C. Reverse transcription was carried out in a 30-µl reaction including the DNase I-treated total RNA, 200 ng of random primers, and 500 µM dNTPs; the reaction was incubated at 65°C for 5 min and then chilled on ice. After addition of 40 U of RNaseOUT and 300 U of SuperScript III reverse transcriptase (Invitrogen), the reaction was incubated at 25°C for 5 min and then at 37°C for 1 h and inactivated at 70°C for 15 min. The resultant cDNA was used in PCR to generate probe sequences for m-erg1, m-erg2, and m-erg3. All primers were designed to use an annealing temperature of ~55°C. Specific primers were designed for m-erg1 from GenBank sequence NM_013569 with forward (5'-GTACCAGGAGTTGCCTCGAT-3') and reverse (5'-GTTACAGCACTGTAGGCAGG-3') primers to generate a 222-bp product, for m-erg2 from GenBank sequence NT_039521 with forward (5'-CAATGACAGCCAATCAGGTG-3') and reverse (5'-CCAGCAATCCGCTTCTCTC-3') primers to generate a 124-bp product, and for m-erg3 from GenBank sequence AJ291608 with forward (5'-CAACCAGACTCCATGGTGAA-3') and reverse (5'-GGCAGCTCTCTGAAGTCCTG-3') primers to generate a 164-bp product. The PCR included 1x PCR buffer, 1 mM MgCl2, 0.2 mM dNTPs, forward and reverse primers of the outside reaction at 1 µM each, 1 U of platinum Taq DNA polymerase, and up to 25 µl of nuclease-free water. The thermocycler program included initiation at 94°C for 2 min followed by 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s and a final polish step at 72°C for 2 min.

Sense and antisense 35S-labeled cRNA probes were produced by SP6 or T7 RNA polymerase-mediated in vitro transcription reactions using purified linearized pGEM-T-Easy vector (Promega, Fisher Scientific, Nepean, ON, Canada) containing the m-erg1, m-erg2, or m-erg3 probe sequence generated from PCR products. The in vitro transcription-labeling reaction contained 1x transcription buffer, 10 mM DTT, 12 U of RNAsin, 2.5 mM NTP mix, 0.5 µg of linearized plasmid, 7.5 µl of 35S-labeled UTP (187.5 µCi), and 7.5–10 U of SP6 or T7 RNA polymerase. After 30 min of incubation at 37°C, RNA polymerase (7.5–10 U) was added to this mixture, and incubation continued at 37°C for 30 min. Plasmid DNA template was removed in a reaction containing 20 U of RNAsin and 0.5 U of RQI DNase (Promega, Fisher Scientific), which was incubated at 37°C for 10 min. Unincorporated NTPs were removed using Probe-Quant G-50 MicroColumns (GE Health, Baie d'Urfé, PQ, Canada). A scintillation counter was used to measure 35S labeling.

Combined Kit immunostain and in situ hybridization for ERG mRNA. Cyrosections (10 µm) were fixed with 4% formaldehyde, rinsed in PBS, and incubated in 5 µg/ml ACK4 with 80 U of RNaseOUT (Invitrogen) diluted in PBS for 30 min. Slides were washed in PBS, incubated in goat anti-rat IgG-biotin for 30 min, washed in PBS, incubated in avidin-biotin complex for 20 min with concentrations as described for the Vectastain ABC system (Vector Laboratories, Burlingame, CA), rinsed in PBS, and incubated in diaminobenzidine (Sigma Aldrich, Oakville, ON, Canada) for 7 min. Immunostained slides were immediately used for in situ hybridization. Sections were fixed again with 4% formaldehyde, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0), dehydrated in ethanol, and delipidated in chloroform. 35S-labeled probes were diluted in hybridization buffer (pH 7) to a final approximate count of 5 x 105 cpm [containing 1.2 M NaCl, 20 mM Tris (pH 8.0), 2 mM EDTA (pH 8.0), 20% dextran sulfate, 0.02% sheared single-strand DNA, 0.1% total yeast RNA, 0.02% yeast tRNA, 2x Denhardt solution, 0.5x total volume formamide, 0.01x total volume 10% SDS, 0.01x total volume sodium thiosulfate, and 0.02x total volume 5 M DTT]. Glass coverslips were applied, and the slides were incubated at 55°C in a humidified chamber for 16–18 h. Slides were treated with 20 µg/ml RNase solution for 30 min and then incubated in RNase buffer solution for 30 min at room temperature, 2x saline-sodium citrate (SSC) for 1 h at 50°C, 0.2x SSC for 1 h at 55°C, and 0.2 SSC for 1 h at 60°C. Slides were dehydrated in an ethanol series with 0.3 M ammonium acetate and 100% ethanol and air-dried. For determination of the cellular localization of hybridized probes, slides were dipped in Amersham Hypercoat LM-1 emulsion (GE Health), stored in darkness for 4 wk at 4°C, and then developed in D19 (Kodak, Rochester, NY), fixed in nonhardening fixer (Kodak), counterstained in toluidine blue, and mounted in Permount. Slides were examined using a Zeiss Axiovert microscope.

Immunofluorescence studies for Kit, ERG isoforms, and β-subunits. Mouse jejunum was cut into 7-µm-thick cryosections, which were stored at –80°C. Sections were thawed, dried at room temperature for 30 min, and then immunostained. Tissue sections were fixed with cold 4% paraformaldehyde for 20 min and then rinsed with PBS and blocked with 5% normal goat serum in PBS for 1 h. Each tissue section was examined for Kit and ERG or β-subunit (MiRP1 and MinK) immunofluorescence using antibodies and concentrations described in Table 1. Briefly, tissues were incubated overnight in primary antibody at 22°C, washed in PBS, incubated in secondary antibody for 1 h at 22°C, washed in PBS, incubated with 10 µM DRAQ5 (Biostatus, Leicestershire, UK) for 10 min, and rinsed in PBS; then coverslips were applied with antifade mounting medium (Biomeda, Foster City, CA). Images were captured using an LSM 510 confocal microscope multitracking program with laser excitation wavelengths of 488 nm (Ar), 543 nm (HeNe1), and 633 nm (HeNe2) and x20, x63, and x100 objectives. An image resolution of 512 x 512 or 1,024 x 1,024 pixels was used, and identical confocal microscope acquisition settings were used to capture images for all three ERG isoforms. Images were captured and illustrated as z stacks, but all colocalization analysis was carried out on a minimum of three z slices (each optical slice = 1 µm) within each z stack using ImageJ (National Institutes of Health, Bethesda, MD). Colocalization data were quantified using Mander's colocalization coefficient showing the intensities of red pixels (Kit+ regions in the image) with a green intensity above 0 (ERG+ signal) divided by the sum of red pixels with intensity above 0. This value is expressed as a percentage of colocalized pixels relative to the total Kit-immunoreactive red pixels in the selected region. After analysis, images were adjusted for brightness and contrast using Corel Draw 11 (Corel, Ottawa, ON, Canada) for illustration purposes.


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  		Table 1. Antibodies used for tissue immunofluorescence

 
Electrophysiology. Whole cell currents were measured by patch clamp from single cultured ICC situated close to neonatal explants. Patch pipettes were pulled on a micropipette puller (model P-87, Sutter) from thin-walled glass capillary tubing with filament; pipette resistance was 3–5 M{Omega}. Data were acquired using an EPC-9 amplifier (HEKA Elektronik) controlled by a personal computer running EPC Instruments software (Pulse/PulseFit) at 1 kHz (ramp pulse) or 10 kHz (step pulse) and filtered at 2.9 kHz with an analog four-pole filter. Capacitance and series resistance compensation was performed with the EPC-9 amplifier. Series resistance was compensated up to 80%. Cells were visualized with an inverted microscope (Axiovert 25, Zeiss). The recording chamber was constantly perfused with the extracellular solution (ECS) at a rate of 1 ml/min. A perfusion device (Valvelink 8) was used to apply test solutions directly to cells. The recording chamber was grounded via an agar-salt bridge (3% agar and 1 M KCl), minimizing liquid junction potentials produced by test solutions. The cells were continuously bathed with ECS at room temperature (23–25°C). ECS contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 dextrose, with pH adjusted to 7.35–7.40 with NaOH. Intracellular solution contained (in mM) 70 KCl, 70 potassium aspartate, 4.5 magnesium ATP, 10 EGTA, and 5 HEPES, with pH adjusted to 7.25–7.30 with KOH. The test drug, E-4031, was obtained from Sigma-Aldrich. Data were analyzed using Origin software (version 6.0, Microcal Software, Northhampton, MA).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ERG mRNA expression in the small intestine muscle layer. In situ hybridization combined with bright-field immunohistochemistry spatially localized m-erg1, m-erg2, and m-erg3 mRNA in mouse jejunum tissue in relation to Kit+ ICC. For positive controls, m-erg1 mRNA signal was confirmed in heart tissue, and m-erg2 and m-erg3 mRNA signals were confirmed in brain (data not shown). In mouse jejunum tissue, m-erg1 and m-erg3 mRNA signals were present throughout the smooth muscle cell layers and within Kit+ cells in the myenteric plexus (Fig. 1, A and C). The negative control sense probes for m-erg1 and m-erg3 showed minimal signal (Fig. 1, D and F). The m-erg2 signal was not present (Fig. 1B), and nonspecific signal was observed with the negative control sense probe (Fig. 1E). mRNA signals from mouse jejunum tissue sections subjected to only in situ hybridization produced signal patterns similar to those of the combined immunohistochemistry-in situ hybridization protocol for m-erg1, m-erg2, and m-erg3 antisense and sense probes (data not shown).


Figure 1
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  		Fig. 1. Colocalization of m-erg mRNA and Kit protein in adult mouse jejunum tissue sections. Note the presence of m-erg1 and m-erg3 mRNA signals, visualized as black grains and highlighted by black arrowheads, in smooth muscle cells and overlapped with Kit-immunoreactive cells (visualized by diaminobenzidine brown precipitate) in the myenteric plexus and the absence of m-erg2 signal. Antisense in situ hybridization signals are shown in A–C and sense probe signals are shown in D–F for m-erg1, m-erg2, and m-erg3, each combined with Kit immunohistochemistry. Scale bars, 25 µm. LM, longitudinal muscle; AP, Auerbach's (or myenteric) plexus; CM, circular muscle; MUC, mucosa.

 
ERG protein and β-subunit expression. Immunofluorescence was used to investigate the presence of immunoreactivity for ERG1, ERG2, and ERG3 proteins and the β-subunits MiRP1 and MinK in relation to Kit-immunoreactive ICC in adult mouse jejunum tissue sections. The β-subunits MiRP1 and MinK were not detectable within the muscularis by immunofluorescence (data not shown). ERG1 was immunoreactive within smooth muscle cells, whereas 27 ± 14.2% (SD) of Kit-immunoreactive red pixels colocalized with ERG1-immunoreactive green pixels when the complete muscularis was selected (Fig. 2A, regions i and ii). In case colocalization artifact was created by selection of Kit-immunoreactive regions that overlapped with the ERG1-immunoreactive regions in smooth muscle cells, this colocalization was compared with selection of only the ICC cell bodies. Selection of a specific ICC as the region of interest produced 6 ± 5.2% (SD) colocalization of Kit and ERG1 (Fig. 2A, region ii). In the peptide-preincubated ERG1 antibody control, the proportion of ERG1 pixels colocalized with Kit in the complete muscularis was 4.5 ± 3.4% (SD) (Fig. 2B), and the immunoreactivity in the smooth muscle cells was eliminated. ERG2 was detected within the smooth muscle cells and colocalized with 11.5 ± 5.7% (SD) of Kit-immunoreactive red pixels in the muscularis layer (Fig. 2C), but colocalization was similar [11.4 ± 6.4% (SD)] in the peptide-preincubated ERG2 antibody control (Fig. 2D). This suggested that the low signal in ICC was nonspecific, although the ERG2 signal in the smooth muscle cells was greatly reduced in the peptide-preincubated ERG2 antibody control. ERG3 was located in smooth muscle cells and ICC in the myenteric plexus, with 47.9 ± 12.2% (SD) of Kit-immunoreactive red pixels colocalizing with ERG3 signal in the complete muscularis layer (Fig. 2E). Selection of specific ICC as a region of interest (similar to region ii in Fig. 2A) produced a colocalization value of 95.4 ± 6.7% (SD). With preincubation of the anti-ERG3 antibody with peptide antigen, colocalization of Kit-immunoreactive red pixels was reduced to 7.9 ± 4.5% (SD) (Fig. 2F), and signal was reduced in the smooth muscle cells.


Figure 2
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  		Fig. 2. Ether-a-go-go-related gene (ERG) protein expression in adult mouse jejunum costained with Kit and DRAQ5 nuclear stain. ERG1 and ERG2 were predominant in smooth muscle cells; ERG3 was distributed throughout the muscularis in smooth muscle cells and nerve plexus, including interstitial cells of Cajal (ICC). A, C, and E: jejunum tissue sections immunostained with ERG1, ERG2, and ERG3, respectively, with Kit immunofluorescence and nuclear stain shown in separate and merged images. B, D, and F: sections in which each ERG primary antibody was preincubated with peptide (pep) antigen, with Kit immunofluorescence and DRAQ5. DRAQ5 far-red nuclear stain was pseudocolored blue. Regions i and ii, areas analyzed for colocalization of Kit and ERG1 in complete muscularis or ICC cell body only, respectively. Similar analysis was applied for ERG3, but only region i was analyzed for ERG2. Scale bars, 10 µm.

 
Effect of E-4031 on electrical properties of the small intestine musculature. With use of a standard microelectrode to penetrate a smooth muscle cell, the ICC-initiated slow-wave activity was readily observed in a tissue preparation of the mouse small intestine musculature (Fig. 3A). The methanesulfonanilide compound E-4031 caused a significant depolarization of the musculature at 1 µM (7 ± 2 mV, n = 6), probably due to block of ERG channels in smooth muscle cells as well as ICC; further depolarization was seen with 2 µM E-4031. In concert with the depolarization, changes in the shape of the slow wave were noted, likely due to effects of E-4031 on ICC; at 2 µM E-4031, prolongation of the slow wave became significant and action potentials appeared superimposed on the slow wave. Hence, E-4031-sensitive currents have a marked effect on tissue excitability.


Figure 3
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  		Fig. 3. Intracellular activity and E-4031-sensitive whole cell membrane currents under physiological ionic conditions. A: intracellular activity of mouse small intestine as recorded with a microelectrode using previously published methods (10). E-4031 at 1 µM primarily affected membrane potential and at 2 µM, in addition, prolonged the slow wave and increased the appearance of action potentials superimposed on the slow waves (due to depolarization). B: membrane currents from a voltage-clamped ICC. Cells were stepped for 700 ms to potentials between –80 and 50 mV in 10-mV increments from a holding potential of –60 mV. Each step was followed by a step to –50 mV. Recordings were performed under physiological ionic conditions (top left). The same voltage-clamp step was applied to the cell after exposure to 2 µM E-4031 (top right). Current-voltage (I-V) relations are shown for currents at the start (square) and end (circle) of steps in control cells (open symbol) and in the presence of 2 µM E-4031 (filled symbol). Currents from each cell were normalized to capacitance before data from 7 cells from 7 different cultures (bottom) were averaged. C: E-4031-sensitive currents digitally subtracted from cells shown in B. E-4031-sensitive currents were obtained from the difference between the membrane currents recorded before and after application of 2 µM E-4031. Mean I-V relations for currents at the start ({blacksquare}) and end (bullet) of voltage steps (n = 7). Values are means ± SE; n is number of observations.

 
Electrophysiological evidence of E-4031-sensitive K+ currents. The whole cell patch-clamp technique was utilized previously on isolated ICC to detect E-4031-sensitive currents in cultured ICC using commonly employed, but nonphysiological, K+ concentrations in the experimental solutions (12, 31). To understand the characteristics of the current under physiological ionic conditions and to compare the currents with other cell types under similar conditions, 5 mM extracellular K+ and 140 mM intracellular (pipette) K+ were used. Under these conditions, depolarization from –60 mV evoked outward current starting at –30 mV that was partially inhibited by E-4031 (Fig. 3B).

The E-4031-sensitive currents at each test potential were obtained by digital subtraction of the current traces in the presence of E-4031 from those in the absence of E-4031 (Fig. 3C). The threshold for activation of outward current was between –30 and –20 mV. Interestingly, the E-4031-sensitive currents (Fig. 3C) did not show dramatic inactivation as seen in some expression systems (19, 21). On repolarization (at the end of the voltage steps), small tail currents, which tend to be very large in expression systems because of recovery of inactivation, were seen in ICC (Fig. 3C). The E-4031-sensitive current between –30 and –50 mV was inward in direction, resulting in a negative slope of the current-voltage (I-V) plot (Fig. 3C).

K+ selectivity of the ERG-like current. To assess the K+ selectivity of the E-4031-sensitive currents, current profiles in the presence of 0 and 140 mM extracellular K+ were compared (Fig. 4). The reversal potential was –1.9 ± 3.2 mV (n = 5) with 140 mM K+, in which the expected equilibrium potential for K+ is 0 mV (Fig. 4B). Removal of extracellular K+ moved the reversal potential to less than –80 mV, and the distinct negative slope of the E-4031-sensitive I-V plot was no longer present (Fig. 4A). Thus the channel was K+ selective, and the negative slope of the E-4031-sensitive I-V plot originated from K+ current.


Figure 4
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  		Fig. 4. K+ selectivity of current in 0 and 140 mM K+ extracellular solutions (ECS). A: I-V relation of E-4031-sensitive current in 0 mM K+ external solution ([K+]es). B: I-V relation of E-4031-sensitive current in 140 mM K+ external solution. Composition of 0 mM K+ ECS was as follows (mM): 140 NaCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 dextrose, with pH adjusted to 7.35–7.40 with NaOH. Values are means ± SE; n is number of observations.

 
Activation kinetics. The activation kinetics of the E-4031-sensitive current were measured at the start of the depolarizing pulses (Fig. 5). The activation time course between –20 and 40 mV was analyzed by two methods. The first method fitted the rising phase of a current trace as a single-exponential function. The mean current traces (n = 5; Fig. 6A) were fitted to obtain a time constant, which was plotted as a function of membrane potential shown in Fig. 5B. The activation time constants of the E-4031-sensitive currents were voltage dependent, and activation was rapid at more positive voltages. The mean time constants were 13.4 ± 0.6 ms at –20 mV and <10 ms at –10 to 40 mV (n = 7). The second method used the time to peak from the original current trace. The times to peak were also plotted as a function of membrane potential shown in Fig. 5C. The time to peak of the E-4031-sensitive currents varied strongly with membrane potential, in a manner similar to the time constant. Time to peak was 74.4 ± 15.8 ms at –20 mV and decreased to <25 ms at –10 to +40 mV.


Figure 5
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  		Fig. 5. Activation kinetics of E-4031-sensitive currents in ICC. A: voltage-clamp protocol (top) and corresponding E-4031-sensitive tail current (bottom) traces. B: time constants of activation. Rising phase of currents during depolarizing pulses was fitted with a single-exponential equation to obtain time constants (n = 7). C: time to peak value was calculated from the rising phase of current during depolarization (n = 7). D: voltage dependence of activation of E-4031-sensitive current. Voltage-clamp protocol (top) and corresponding E-4031-sensitive tail current (bottom) traces in response to the same voltage protocol in Fig 4 are shown. E: peak amplitudes of E-4031-sensitive tail currents elicited on return to –50 mV normalized with reference to the amplitude at +50 mV and plotted against the test potential. Curve was fitted by a single-exponential Boltzmann equation: 1/{1 + exp[(V1/2 – Vm)/k]}, where Vm is test potential, V1/2 is voltage at which activation is half-maximal, and k is the slope factor, yielding values for V1/2 and k. Values are means ± SE; n is number of observations.

 

Figure 6
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  		Fig. 6. Inactivation and deactivation kinetics of the E-4031-sensitive current in ICC. A: voltage-clamp protocol (top) and corresponding E-4031-sensitive tail current (bottom) traces. B: initial peak currents at 30 mV plotted vs. prepulse potentials normalized with reference to their amplitude at +30 mV. Continuous curve was fitted by the Boltzmann equation, yielding values for V1/2 and k (n = 5). C: voltage-clamp protocol (top) and corresponding E-4031-sensitive deactivating tail current (bottom) traces. D: time constants of deactivation. Falling phase of current step to membrane potentials between –40 and –70 mV was fitted with a single-exponential equation to obtain time constant (n = 5). Values are means ± SE; n is number of observations.

 
The voltage dependence of activation was studied by activation of the E-4031-sensitive currents through 700-ms depolarizing steps to potentials between –30 and +50 mV (Fig. 5D). The voltage dependence was evaluated by measurement of the amplitude of the tail current elicited on return to –50 mV (n = 5), which reflects the degree of activation at the preceding depolarizing test potential. The activation variable was then obtained by normalization of the amplitude of the tail current at each test potential with reference to its peak amplitude at +50 mV and plotted as a function of membrane potential to provide the steady-state activation curve (Fig. 5E).

Inactivation kinetics. To study the voltage dependence of steady-state inactivation of E-4031-sensitive currents, currents were evoked from –80 to 30 mV. Inactivation was allowed for a period of 700 ms; then the voltage was stepped up to 30 mV (Fig. 6A). When the voltage step to 30 mV was launched from –60 mV, significant current was generated; when it was launched from –20 mV, the current evoked was much smaller, indicating significant inactivation. To construct a typical inactivation curve, the peak currents at 30 mV were normalized and plotted vs. the prepulse potentials (the potentials from which the depolarization to 30 mV was launched) to provide the steady-state inactivation curve (Fig. 6B). The inactivation curve was well fitted by a single-exponential Boltzmann equation, with fit parameters as follows: V1/2 = –40.3 ± 0.5 mV and k = 5.7 ± 0.4 (n = 5), where V1/2 is the voltage at which activation is half-maximal and k is the slope factor.

Deactivation kinetics. Deactivation kinetics were examined by measurement of the voltage dependence of the time course of the E-4031-sensitive current decay. The cells were voltage stepped to 50 mV for 700 ms to fully activate the channel and then stepped to different membrane potentials from –70 to –40 mV, which is the membrane potential range of the slow wave (Fig. 6C). Time constants of deactivation were determined by fitting a single-exponential equation to the initial current decay at each potential. The initial phase of deactivation is well fitted by a single-exponential equation. Time constants were plotted as a function of the test potential in Fig. 6D. The rate of deactivation was dependent on membrane potential, with the currents decaying more rapidly as the membrane potential became more negative. The time constant was <60 ms at the membrane potentials examined.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the elucidation of ionic components that contribute to the pacemaking electrical slow wave of ICC in the myenteric plexus, it is essential to characterize the electrophysiological and molecular properties of the channels involved. Despite the culture environment, ICC in the neonatal mouse jejunum explant culture produce rhythmic inward currents and express Kit protein, both of which are essential characteristics of ICC in the adult mouse. Thus ICC in newborn mice have the components necessary for pacemaker function that are typical of the adult mouse. The examination of ERG channel expression in adult tissue confirmed that the channels identified functionally at a developmentally earlier stage are present at the molecular level in the mature animal. In the present study, we show that the E-4031-sensitive current in ICC in physiological ionic solutions shares features with the ERG3 isoform of the ERG K+ channel family. This finding is consistent with the molecular identification of ERG3 protein by immunoreactivity and erg3 mRNA by in situ hybridization. The identification of erg1 mRNA in ICC suggests that this isoform could contribute to the E-4031-sensitive current, but we could not detect ERG1 protein in ICC, and the electrophysiological characteristics of channel activation, inactivation, and deactivation were different from typical ERG1 features. In ICC, the electrophysiological evidence obtained in physiological solutions suggests that the E-4031-sensitive currents restrict ICC excitability by limiting membrane depolarization and by limiting the duration of the slow-wave potential, thus contributing to the control of muscle excitability.

Under physiological ionic conditions, ICC displayed a marked depolarization-activated E-4031-sensitive current that shared many features with ERG3 currents but differed from typical ERG1 currents. In most cells natively or artificially expressing the ERG1 channel, the currents inactivate immediately on activation, and they recover from inactivation by repolarization. In ICC, the activation is relatively fast, with a time constant of activation of 13 ms at –20 mV; similarly, ERG3 channels expressed in oocytes display a time constant of activation of 33 ms (20), whereas the corresponding values were ~200 ms for ERG1 in mouse cardiac cells and 770 ms for ERG1 expressed in HEK 293 cells (30). Time constants for ERG1 and ERG2 currents expressed in oocytes were >300 ms (20). The I-V relationship of many E-4031-sensitive outward currents displays a bell shape, with a maximum at ~0 mV, which is thought to be due to strong inactivation of the current at more depolarized potentials (8, 18). The absence of this inactivation phenomenon in ICC is similar to observations in murine portal vein myocytes (16) and subendocardial Purkinje myocytes (25). Consequently, during the recovery from inactivation, the outward "resurgent" current evoked on hyperpolarization was not as pronounced in ICC as in the ERG1-expressing mouse sinoatrial node cells (25), whereas in Xenopus oocytes under physiological ionic conditions the ERG3 channel showed a minor resurgent current compared with ERG1 and ERG2 (20). Another factor contributing to the relatively minor resurgent current was the rapid deactivation (inactivation of the resurgent current) observed in ICC with a time constant of 56 ms at –40 mV, while the comparable deactivation time constants at –40 mV were 119 and 200 ms in guinea pig atrium and HERG-expressing HEK 293 cells, respectively (17, 30). The deactivation time constant of ERG3 currents expressed in Chinese hamster ovary cells was measured at 18.7 ms at –120 mV (28), whereas we measured the time constant at 19.1 ms at –70 mV for the E-4031-sensitive current in ICC (12). The comparable time constants for ERG1 and ERG2 at –120 mV were 74 and 89 ms, respectively (28). Marked E-4031-sensitive currents evoked on hyper- or repolarization are thought to promote action potential repolarization in sinoatrial node cells and, subsequently, increase the frequency of action potentials. This is likely less of a factor in ICC. At the molecular level, there is evidence for ERG1 mRNA in ICC, but no ERG1 protein is detected. These data are consistent with immunofluorescence studies in human jejunum, which also demonstrated strong ERG1 immunoreactivity in smooth muscle cells (6). The impression of ERG1 immunoreactivity in ICC previously reported using a bright-field immunohistochemical technique (31) could not be confirmed by the present study, likely because of accuracy limitations in serial-section staining compared with the present colocalization immunofluorescence technique. Nevertheless, the presence of ERG1 protein cannot be completely excluded because of the detection of m-erg1 mRNA and a related observation of m-erg1 mRNA expression enrichment in the ICC associated with Auerbach's plexus (3). Perhaps the translation of the ERG1 transcript could be negatively regulated by a microRNA mechanism to prevent the expression of ERG1 protein in ICC. Alternatively, there is potential for an ERG1 protein in ICC with a unique carboxyl terminus that remains undetectable with commercially available antibodies.

Since smooth muscle cells depolarized from a resting membrane potential of –57 mV in response to E-4031 (Fig. 3A), the activation threshold of the E-4031-sensitive currents in smooth muscle cells is likely lower than that of ICC. Indeed, marked E-4031-sensitive currents were evoked at the resting membrane potentials of smooth muscle cells in esophageal smooth muscle (2). The activation threshold measured in ICC (Fig. 3B), however, was measured at room temperature, and the ICC were isolated from tissue; hence, the value in vivo may be different.

Differences between kinetics of the currents studied here and kinetics studied in other systems may also be due to variation in intracellular factors or other channel subunits. We did not detect the ERG channel β-subunits MiRP1 and MinK, which can alter ERG channel activity (1, 11). Other possible explanations for the differences are recording conditions (temperature, extracellular pH, extracellular cation concentrations, and voltage protocol), which can have important effects on the ERG channel gating (26, 29, 30). In addition, differences in current characteristics may be due to cell-specific regulation of the channel protein through the cyclic nucleotide-binding domain, phosphorylation sites, or the Per-Arnt-Sim domain, which modifies gating characteristics (13, 27).

An interesting feature of the E-4031-sensitive currents observed in ICC was the inwardly directed K+ current between –30 and –50 mV, apparently occurring against the electrochemical gradient and in a time-dependent manner, resulting in a negative slope of the I-V plot (Fig. 3C). Other K+ channel blockers (4-aminopyridine and tetraethylammonium) did not result in a negative slope of the I-V curve of blocker-sensitive current (S. J. Park, unpublished observations). This current could be generated by the Na+-K+ pump, which can use active transport to produce K+ influx against the electrochemical gradient. In general, Na+-K+ pump currents are outward in direction (23). U87-MG cells exhibited K+ influx against the electrochemical gradient (5), which the authors suggested could be due to the absence of Na+ in the intracellular solution, with the Na+-K+ pump functioning as an extracellular K+/intracellular K+ exchanger (15). K+ influx can also occur if the channel is strongly rectifying in this region. In murine portal vein myocytes, the I-V curve of the E-4031-sensitive currents also exhibited this unique negative slope (16). The inward current in ICC occurs at –50 to –30 mV and may contribute to the plateau potential of the slow wave.

This study provides evidence for a unique ERG K+ current in ICC that contributes to the characteristics of gut pacemaker activity. Differential ERG channel expression in ICC compared with intestinal smooth muscle cells and cardiac muscle cells may make ERG3 a unique target to regulate ICC excitability without affecting the cardiac action potential. Since it is the pacemaker activity from ICC that propagates into the musculature and, in part, defines muscle excitability, drugs affecting specifically the ERG3 channel in ICC may have potential to optimize prokinetic drug function for patients.


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The research was supported by Canadian Institutes of Health Research (CIHR) Operating Grant MOP-12874 (to J. D. Huizinga). E. J. White was supported by a CIHR-Canadian Digestive Health Foundation-partnered doctoral research award. S. J. Park was supported by a CIHR-Canadian Association of Gastroenterology postdoctoral fellowship.


   ACKNOWLEDGMENTS
 
Jing Ye and Yaohui Zhu provided the data presented in Fig. 3A.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Huizinga, Dept. of Medicine, McMaster Univ., 1200 Main St. West, HSC-3N8, Hamilton, ON, Canada L8N 3Z5 (e-mail: huizinga{at}mcmaster.ca)

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.

* E. J. White and S. J. Park contributed equally to this work. Back


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  1. Abbott GW, Sesti F, Splawski I, Buck ME, Lehmann MH, Timothy KW, Keating MT, Goldstein SA. MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. Cell 97: 175–187, 1999.[CrossRef][Web of Science][Medline]
  2. Akbarali HI, Thatte H, He XD, Giles WR, 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]
  3. Chen H, Ordog T, Chen J, Young DL, Bardsley MR, Redelman D, Ward SM, Sanders KM. Differential gene expression in functional classes of interstitial cells of Cajal in murine small intestine. Physiol Genomics 31: 492–509, 2007.[Abstract/Free Full Text]
  4. Clark RB, Mangoni ME, Lueger A, Couette B, Nargeot J, Giles WR. A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells. Am J Physiol Heart Circ Physiol 286: H1757–H1766, 2004.[Abstract/Free Full Text]
  5. Ducret T, Vacher AM, Vacher P. Voltage-dependent ionic conductances in the human malignant astrocytoma cell line U87-MG. Mol Membr Biol 20: 329–343, 2003.[CrossRef][Web of Science][Medline]
  6. Farrelly AM, Ro S, Callaghan BP, Khoyi MA, Fleming N, Horowitz B, Sanders KM, Keef KD. Expression and function of KCNH2 (HERG) in the human jejunum. Am J Physiol Gastrointest Liver Physiol 284: G883–G895, 2003.[Abstract/Free Full Text]
  7. Guasti L, Cilia E, Crociani O, Hofmann G, Polvani S, Becchetti A, Wanke E, Tempia F, Arcangeli A. Expression pattern of the ether-a-go-go-related (ERG) family proteins in the adult mouse central nervous system: evidence for coassembly of different subunits. J Comp Neurol 491: 157–174, 2005.[CrossRef][Web of Science][Medline]
  8. Lees-Miller JP, Kondo C, Wang L, Duff HJ. Electrophysiological characterization of an alternatively processed ERG K+ channel in mouse and human hearts. Circ Res 81: 719–726, 1997.[Abstract/Free Full Text]
  9. Lyford GL, Farrugia G. Ion channels in gastrointestinal smooth muscle and interstitial cells of Cajal. Curr Opin Pharmacol 3: 583–587, 2003.[CrossRef][Web of Science][Medline]
  10. Malysz J, Donnelly G, Huizinga JD. Regulation of slow wave frequency by IP3-sensitive calcium release in the murine small intestine. Am J Physiol Gastrointest Liver Physiol 280: G439–G448, 2001.[Abstract/Free Full Text]
  11. McDonald TV, Yu Z, Ming Z, Palma E, Meyers MB, Wang KW, Goldstein SA, Fishman GI. A minK-HERG complex regulates the cardiac potassium current IKr. Nature 388: 289–292, 1997.[CrossRef][Medline]
  12. McKay CM, Ye J, Huizinga JD. Characterization of depolarization-evoked ERG K currents in interstitial cells of Cajal. Neurogastroenterol Motil 18: 324–333, 2006.[CrossRef][Web of Science][Medline]
  13. Morais Cabral JH, Lee A, Cohen SL, Chait BT, Li M, Mackinnon R. Crystal structure and functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain. Cell 95: 649–655, 1998.[CrossRef][Web of Science][Medline]
  14. Muhlbauer E, Bazwinsky I, Wolgast S, Klemenz A, Peschke E. Circadian changes of ether-a-go-go-related-gene (Erg) potassium channel transcripts in the rat pancreas and beta-cell. Cell Mol Life Sci 64: 768–780, 2007.[CrossRef][Web of Science][Medline]
  15. Nonaka T, Warden DH, Matsushita K, Stokes JB. K+ self-exchange by the Na+ pump: regulation by Pi and metabolic perturbations. Am J Physiol Cell Physiol 269: C170–C178, 1995.[Abstract/Free Full Text]
  16. Ohya S, Horowitz B, Greenwood IA. Functional and molecular identification of ERG channels in murine portal vein myocytes. Am J Physiol Cell Physiol 283: C866–C877, 2002.[Abstract/Free Full Text]
  17. Sanguinetti MC, Jurkiewicz NK. Delayed rectifier outward K+ current is composed of two currents in guinea pig atrial cells. Am J Physiol Heart Circ Physiol 260: H393–H399, 1991.[Abstract/Free Full Text]
  18. Sanguinetti MC, 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/Free Full Text]
  19. Schonherr R, Heinemann SH. Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel. J Physiol 493: 635–642, 1996.[Abstract/Free Full Text]
  20. Shi W, Wymore RS, Wang HS, Pan Z, Cohen IS, McKinnon D, 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]
  21. Smith PL, Baukrowitz T, Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature 379: 833–836, 1996.[CrossRef][Medline]
  22. Spector PS, Curran ME, Keating MT, Sanguinetti MC. Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Open-channel block by methanesulfonanilides. Circ Res 78: 499–503, 1996.[Abstract/Free Full Text]
  23. Thomas RC. Electrogenic sodium pump in nerve and muscle cells. Physiol Rev 52: 563–594, 1972.[Free Full Text]
  24. Thomsen L, Robinson TL, Lee JC, Farraway LA, Hughes MJ, Andrews DW, Huizinga JD. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med 4: 848–851, 1998.[CrossRef][Web of Science][Medline]
  25. Tseng GN. IKr: the hERG channel. J Mol Cell Cardiol 33: 835–849, 2001.[CrossRef][Web of Science][Medline]
  26. Vereecke J, Carmeliet E. The effect of external pH on the delayed rectifying K+ current in cardiac ventricular myocytes. Pflügers Arch 439: 739–751, 2000.[CrossRef][Web of Science][Medline]
  27. Warmke JW, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci USA 91: 3438–3442, 1994.[Abstract/Free Full Text]
  28. Wimmers S, Bauer CK, Schwarz JR. Biophysical properties of heteromultimeric erg K+ channels. Pflügers Arch 445: 423–430, 2002.[CrossRef][Web of Science][Medline]
  29. Yao JA, Du X, Lu D, Baker RL, Daharsh E, Atterson P. Estimation of potency of HERG channel blockers: impact of voltage protocol and temperature. J Pharmacol Toxicol Methods 52: 146–153, 2005.[CrossRef][Medline]
  30. Zhou Z, Gong Q, Ye B, Fan Z, Makielski JC, Robertson GA, January CT. Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J 74: 230–241, 1998.[Web of Science][Medline]
  31. Zhu Y, Golden CM, Ye J, Wang XY, Akbarali HI, Huizinga JD. ERG K+ currents regulate pacemaker activity in ICC. Am J Physiol Gastrointest Liver Physiol 285: G1249–G1258, 2003.[Abstract/Free Full Text]




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