Altered gene expression and increased bursting activity of colonic smooth muscle ATP-sensitive K+ channels in experimental colitis

Xiaochun Jin,1 Anna P. Malykhina,1 Florea Lupu,2 and Hamid I. Akbarali1

1Department of Physiology and 2Oklahoma Medical Research Foundation, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73034

Submitted 6 November 2003 ; accepted in final form 7 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The ATP-sensitive K+ channel (KATP) is a complex composed of an inwardly rectifying, pore-forming subunit (Kir 6.1 and Kir 6.2) and the sulfonylurea receptor (SUR1 and SUR2). In gastrointestinal smooth muscle, these channels are important in regulating cell excitability. We examined the molecular composition of the KATP channel in mouse colonic smooth muscle and determined its activity in the pathophysiological setting of experimental colitis. Following 7 days of dextran sulfate sodium (DSS) treatment in drinking water, colonic inflammation was scored by histology and physical signs. In whole cell recordings, levcromakalim-induced currents were significantly larger in inflamed cells. In cell-attached patch recordings of single-channel events, levcromakalim enhanced the bursting duration in inflamed cells. The single-channel conductance of ~42 pS was not altered with inflammation. mRNA for both Kir 6.1 and 6.2 were detected by RT-PCR. Kir 6.1 was localized to the plasma membrane, whereas Kir 6.2 was mainly detected in the cytosol by immunohistochemistry. Quantitative PCR showed that Kir 6.1 gene expression was upregulated by almost 22-fold, whereas SUR2B was downregulated by threefold after inflammation. Thus decreased motility of the colon during inflammation may be associated with changes in the transcriptional regulation of Kir 6.1 and SUR2B gene expression.

sulphonylurea receptor; inflammation; dextran sulfate; real-time PCR; voltage clamp


ULCERATIVE COLITIS IS AN inflammatory bowel disease characterized by recurrent episodes of colonic inflammation and tissue degeneration (22–24). In humans, as well as in several different animal models of intestinal inflammation, the contractile force generated by the smooth muscle is significantly reduced, resulting in altered motility leading to diarrhea and/or constipation (4, 8, 25, 34). The cellular mechanisms for these changes is unclear but is likely due to the release of a complex array of inflammatory mediators that alter cell excitability by affecting membrane ion channels.

In several different animal models of colonic inflammation, voltage-dependent Ca2+ currents of smooth muscle have been shown to be suppressed due to either decrease in membrane expression (19) or a possible alteration in the channel regulation (17). Previously, in a dextran sulfate sodium (DSS) model of experimental colitis in mice, we observed that in addition to decreased Ca2+ currents, colonic inflammation also results in selective changes in K+ currents of colonic smooth muscle (3). The activation of ATP-sensitive K+ channel currents (KATP) in response to lemakalim, a potassium channel opener, were markedly enhanced, whereas the biophysical properties of the transient outward current remained unaltered. KATP channels have been previously described in gastrointestinal smooth muscle (11, 18); however, their role in the pathophysiology of smooth muscle is not clear.

KATP is a heterooctomer comprised of two subunits, a sulfonylurea receptor (SUR) and the inwardly rectifying, pore-forming K+ channel, Kir 6.x. The two isoforms, Kir 6.1 and Kir 6.2, are inwardly rectifying channels encoded by two separate genes that associate with either of the two isoforms of the SUR receptor, SUR1 and SUR2. SUR2 is alternatively spliced producing SUR2A and SUR2B (5, 7, 27, 28). It is now well recognized that Kir 6.2/SUR1 and Kir 6.2/SUR2A form the major KATP complex in the pancreatic {beta}-cell and the heart, respectively (13, 14, 28); however, the subunits comprising those of gastrointestinal smooth muscle are unclear. Koh and colleagues (16, 18) have suggested that Kir 6.2/SUR2B underlie the KATP channel in the murine colonic smooth muscle on the basis of the presence of transcripts identified in single cells. However, the small single-channel conductance of 27 pS (18) appears to be more consistent with Kir 6.1. The characterization of the KATP complex is generally defined on the basis of the single-channel conductance that distinguishes the isoforms of the Kir subunit and pharmacologically for the SUR subunit (28). Heterologously expressed Kir 6.1 demonstrates a conductance of ~35 pS, whereas Kir 6.2 has a conductance of ~80 pS (13, 14, 26, 36). In some smooth muscle cells, it is likely that coexpression of Kir 6.1/Kir 6.2 leads to an intermediate conductance (9, 26, 33).

Recent studies (32) demonstrating the effects of various knockout of either of the genes encoding the KATP channel illustrate the pathophysiological role of Kir 6.2/SUR1 in relation to persistent hyperinsulinemic hypoglycemia of infancy, whereas Kir 6.2–/– mice demonstrated depressed ST elevation during myocardial ischemia/reperfusion. Kir 6.1–/– mice show a phenotype similar to Pirnzmetal angina in humans, which may be due to loss of KATP function in vascular smooth muscle (5, 20). In gastrointestinal smooth muscle, the physiological role of the KATP channel may be related to modulation of cell excitability (11, 18). However, whether changes in KATP channel function occur in pathophysiological conditions is not known. In the present study, we examined KATP channels in the mouse experimental colitis model and demonstrate that the major sarcolemmal isoform of the Kir channel in mouse colon is composed of Kir 6.1 and SUR2B and the channel demonstrates increased bursting activity in response to the KATP channel opener after inflammation. Inflammation also enhanced the gene expression of Kir 6.1.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Induction and assessment colitis.

Colonic inflammation was induced by administering 5% DSS in drinking water ad libitum for 7 days. All protocols were approved by the Institutional Animal Care Committee. The mice were killed, and the distal colon was excised and placed in Tyrode's solution. To assess the severity of colitis, the daily disease activity index (DAI) was calculated by determining the daily weight of each animal, the presence of blood occult (detected by hemoccult strips; Smith Kline Diagnostics, San Jose, CA) or gross blood in the feces and stool consistency. Each of the parameters was assigned a score based on previously described criteria (31), which was used to calculate an average DAI for each animal. Briefly, the scores ranged from 0 to 4, with 0 being no weight loss, normal stool consistency, and no rectal bleeding, whereas 4 was assigned when the weight loss exceeded 20% with diarrhea and gross bleeding. Histological assessment of colonic lesions was also determined. After the removal of the colon, at least two cross sections from the distal diseased area were immediately fixed in 10% formalin. The tissue was then embedded in paraffin and stained with hematoxylin and eosin.

Immunohistochemistry.

Mice colon samples were embedded in Tissue-Tek OCT and snap-frozen in liquid nitrogen-cooled isopentane. Cryosections (7-µm thick) were blocked with 3% bovine serum albumin in PBS (containing 0.01% saponin) for 1 h at room temperature. The slides were subsequently incubated with primary antibodies: goat anti-Kir 6.1 (antigen sc-11224, Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-Kir 6.2 (antigen sc-11225, Santa Cruz Biotechnology) (20 µg/ml) for 1 h at RT. After sections were thoroughly washed with PBS (0.01% saponin), they were incubated with secondary antibody, Cy3 conjugated rabbit anti-goat IgG (Zymed, 1:100), fixed with 4% paraformaldehyde for 5 min and mounted with Vectashield (Vector) containing 0.001% TO-PRO (Molecular Probes) for nuclear staining. Sections were analyzed by confocal laser scanning microscopy using a Nikon C1 system. Controls were carried out by preabsorption of the primary antibodies with the blocking peptide.

Cell isolation.

Colonic smooth muscle cells were isolated from 5- to 6-wk-old BALB/c mice as described previously (12). Mice were euthanized and the distal colon was quickly removed. The colon was opened along the mysenteric border, and muscle strips were dissected away from the mucosa and submucosa in low Ca2+ Tyrode's solution. Strips of colonic muscle, which include both circular and longitudinal layers, were transferred to 5 ml of Tyrode's solution containing 2 mg collagenase, 2 mg bovine serum albumin, and 1.5 mg trypsin. Incubation in the enzyme solution was carried out at 37°C for 10–12 min. The partially digested tissues were then incubated in enzyme-free solutions containing serum albumin and were gently agitated with a wide-bore fire-polished pipette to release smooth muscle cells. Isolated cells were stored on ice and used within 6 h of dispersion. All electrophysiological recordings were performed at room temperature (22–25°C).

Electrical recordings.

Standard whole cell, cell-attached, and inside-out patch configurations were used. The voltage-clamp amplifier was an Axopatch 200A (Axon Instruments). Data were low-pass filtered at 5 kHz and sampled at a rate of 10 to 100 µs per sample. The pipettes were prepared on a Flaming-Brown horizontal puller (model P-87; Sutter Instruments) and fire polished. Resistance of the pipettes was ~5 M{Omega} for whole cell configuration and 8–10 M{Omega} for cell-attached or inside-out single-channel recordings when filled with pipette solution.

Solution.

Solutions used for electrophysiological recordings and cell isolation are listed in Table 1. The low-Ca2+ Tyrode's solution was equilibrated with 95% O2-5% CO2. All of the bathing solutions were equilibrated with 100% O2. The pH was adjusted to 7.4 with KOH. For inside-out patch recordings, the same external solution was used as that in cell-attached recordings with the addition of 1 mM ADP-Mg.


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Table 1. Solutions used for electrophysiological recordings and cell isolations

 
Single-channel analysis.

Single-channel currents were analyzed by using Fetchan and Pstat subroutines in the pClamp 8.2 software (Axon Instruments) and Origin for Windows software (Microcal Software, Northampton, MA). For analysis of the single-channel kinetics, the amplitudes of current, distribution of open and closed times, and burst analysis were measured by constructing histograms after events-list analysis from raw records. Events were detected by setting a threshold at one-half of the amplitude of the open-channel current. The opened- and closed-time histograms were fitted with exponential components by the method of maximum likelihood. The number of exponential densities used for fitting the histograms was determined in pStat on the basis of examination of the F statistic (P > 0.99).

Bursts were defined as an opening or series of openings separated by closures shorter than a critical time. An optimal interburst interval was calculated from closed-time distribution using the algorithms in PClamp 8.2 software. Burst analysis were then performed to define the duration of openings within burst, duration of closings within burst, and total burst durations. Channel activity was calculated as channel open probabilities (NPo). NPo was measured by integrating idealized records of channel opening and closing transitions and dividing this by the time integral of single-channel current.

Intracellular microelectrode recordings.

Colonic muscle strips were dissected away from the mucosa and cut into small strips. Each strip was pinned mucosal side up and placed in the recording chamber containing Krebs solution (in mM): 120.9 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 NaH2PO4, 2.5 CaCl2, 1.2 MgCl2, and 11 glucose (pH 7.4) and oxygenated in 95% O2-5% CO2 solution. Strips were superfused at 3 ml/min and maintained at 37°C in the presence of nifedipine (1 µM) and atropine (1 µM). Microelectrodes have resistances of 30–60 M{Omega} when filled with 3 M KCl. The transmembrane potential was measured with an amplifier IE-210 (Warner Instruments, Holliston, MA), and the signals were digitized by using an analog-to-digital convertor and analyzed by AxoScope 8.2 (Axon Instruments).

Total RNA extraction, conventional and real-time RT-PCR.

Colonic muscle strips (devoid of mucosa) were frozen in liquid N2, and total RNA was extracted by using the Micro-to-Midi total RNA isolation kit (Invitrogen) according to the manufacturer's instructions. All RNA samples were quantified by spectrophotometer and run on denaturing RNA gels to check the quality and integrity of the RNA samples. First-strand cDNA was synthesized by using the Super Script first-strand synthesis system for RT-PCR (Invitrogen). Each cDNA sample was synthesized by using 50 units of Superscript II RT at 42°C for 50 min in the presence of 5 µg of total RNA, 0.5 µg of oligo(dT) primers, and 10 mM dNTPs in a total of 20 µl reaction. PCRs were performed on a Robocycler PCR machine (Stratagene, La Jolla, CA) using 3 µl of cDNA, 2.5 units of Taq DNA polymerase, 10 mM dNTPs, and 10 µM each forward and reverse primers in 50-µl volume. Samples with minus RT or water templates were used as controls. PCR was performed under the following conditions: 1 cycle at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 30 s, and extension at 72°C for 1 min. Thirty microliters of RT-PCR products were separated on 2% agarose/1x Tris-acetate-EDTA gels, and DNA bands were visualized by ethidium bromide staining.

Real-time PCR was performed on the SmartCycler (Cephid) using SYBRgreen as the intercalating dye. Four independent control and five inflamed total RNA samples were examined. For each mRNA sample, colon samples from three mice were used to get sufficient mRNA. For each sample, PCR was conducted in a 25-µl volume of reaction mixture containing SYBRgreen (1:125,000 dilution; Molecular Probes), Ready-to-Go-PCR beads (Amersham) that include dNTPs and Taq polymerase, 2.5 µl both forward and reverse primer, 2 µl MgCl2 (25 mM), and 2 µl template cDNA prepared from equal amounts of total RNA from control and inflamed samples. The PCR protocol consisted of three steps with an initial denaturation (95°C for 120 s), followed by 35 cycles of denaturation (95°C for 15 s), annealing (57–60°C for 15 s), and extension (72°C for 15 s). A melt curve was derived at the end of the PCR by heating the PCR product from 60 to 95°C. A single peak in the melt curve is indicative of the specificity of the PCR product. The absence of peaks in water controls suggested lack of primer-dimer formation. This was further confirmed by gel electrophoresis of the RT-PCR products. The semiquantitative comparison between control and inflammation was calculated according to a previously published method (10). The cycle threshold value, (CT count), for each sample was normalized by subtracting it from the CT value of the housekeeping gene {beta}-actin to establish {Delta}CT ({Delta}CT = target gene CT{beta}-actin CT). To determine the relative enhanced expression of the target gene by inflammation, inflamed sample {Delta}CT was compared with control sample {Delta}CT to obtain {Delta}{Delta}CT ({Delta}{Delta}CT = inflamed {Delta}CT control {Delta}CT). The fold change was measured as 2{Delta}{Delta}CT.

Primer design.

Primers used are listed in Table 2. Primers for real-time PCR experiments were designed to amplify smaller-sized products. An additional set of primers for Kir 6.1 were designed to span an intron (primer set 3) to verify any possible genomic DNA contamination. The {beta}-actin primers were also designed to span an intron in addition to two exons (30).


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Table 2. Primer sequences

 
Chemicals.

Diazoxide, glibenclamide, bovine serum albumin, and trypsin were purchased from Sigma (St. Louis, MO). Collagenase was obtained from Yakult (Tokyo, Japan). Levcromakalim was obtained from Tocris (Ellisville, MO). Antibodies for Kir 6.1 (antigen sc-11224) and Kir 6.2 (antigen sc11225) were purchased from Santa Cruz Biotechnology.

Statistical analysis.

Statistical difference was evaluated by using Student's paired or unpaired t-test. Differences with values of P < 0.05 between control and test group were considered to be significant. Values in the text are as given as means ± SE.


    RESULTS
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 MATERIALS AND METHODS
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 DISCUSSION
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Mice treated with 5% DSS in drinking water developed diarrhea with watery stools within 7 days. Histological analysis showed diffuse inflammation of the mucosa with scattered ulcerations and lymphocytic infiltration. There was typical loss of goblet cells and crypts (Fig. 1A). The DAI was calculated by measuring weight, presence of occult blood in stools, and stool consistency. Each parameter was given a score adapted from previously published criteria (31) and divided by 3. The DAI shows that physical signs were apparent by day 7 (Fig. 1B). When excised and placed in Tyrode's solution, the colon samples from DSS-treated mice were invariably quiescent and flaccid compared with controls.



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Fig. 1. Inflammation-induced changes in gross architecture in mice colon samples and associated changes in physical signs. A: cross section of distal colon in control and after 7 days of dextran sulfate sodium (DSS) treatment (original magnification x150). Hematoxylin and eosin stains show disruption of the mucosal epithelium and infiltration of lymphocytes. B: disease activity index (DAI) calculated for 7 consecutive days in DSS-treated mice. DAI was calculated as detailed in MATERIALS AND METHODS.

 
Whole cell voltage clamp recordings.

To identify KATP in colonic smooth muscle cells, conventional whole cell recordings were made in isolated single cells dialyzed with low ATP (0.1 mM) and initially bathed in normal HEPES solution at a holding potential of –70 mV from control and DSS-treated mice colonic cells, henceforth referred to as inflamed cells.

The average capacitance was 62.4 ± 3.8 pF (n = 22) in control and 60.0 ± 3.4 pF (n = 23) in inflamed cells. Changing the external solution from 5 mM K+ to that containing 140 mM K+ resulted in a small, steady, inward current measuring –1.9 ± 1.08 pA/pF in control cells, and –4.8 ± 2.6 pA/pF in inflamed cells. Perfusion with levcromakalim (20 µM) in this symmetrical K+ gradient further increased the inward currents to –7.7 ± 1.1 pA/pF (n = 7) in control cells and –26.3 ± 8.6 pA/pF (n = 9) in inflamed cells (Fig. 2), representing an almost fourfold difference in the response to the potassium channel opener. Currents in both control and inflamed cells were reversed by either glibenclamide (10 µM) or by returning to 5 mM K+ bathing solution.



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Fig. 2. Levcromakalim-induced currents in control and inflamed cells. A: whole cell recordings of inward currents induced in control (top) and inflamed (bottom) cells. Currents were obtained at a holding potential of –70 mV in symmetrical K+ (140 mM) solutions. Note difference in scale bars. B: normalized amplitude of inward currents induced by levcromakalim in control and inflamed cells.

 
KATP currents are time independent and weakly voltage dependent. To determine the voltage dependence of the levcromakalim-activated currents, voltage-step protocols were applied. Membrane currents were recorded in response to steps from a holding potential of –70 mV to test potentials ranging from –120 mV to +10 mV in high-K+ bathing solutions. With a low extracellular Ca2+ concentration, 1 mM tetraethylammonium in the bath, and hyperpolarizing voltage steps, activation of Ca2+-activated K+ currents was minimized. Figure 3A shows an example of original current records obtained from a control cell. The holding current in high-K+ solution was –100 pA (Fig. 3A), which increased to approximately –750 pA after perfusion with levcromakalim (10 µM). Test depolarizations produced time-independent currents. Fig. 3B shows a current-voltage relationship for levcromakalim-induced currents in a control and an inflamed cell. In the presence of 10 µM levcromakalim, hyperpolarization to –120 mV resulted in currents measuring –26.9 pA/pF in a control cell and –53.3 pA/pF in an inflamed cell.



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Fig. 3. Voltage-dependent effects of levcromakalim-induced currents. Step voltages applied from 0 mV to –120 mV in 10 mV increments from a holding potential of –70 mV in high-K+ solutions. A: current traces in high K+ and in the presence of 10 µM levcromakalim from a control cell. B: current-voltage relationship from a control and an inflamed cell for levcromakalim-induced currents. C: concentration-dependence of levcromakalim. Maximal amplitudes of levcromakalim-induced currents measured at –120 mV step hyperpolarization and normalized as fold change over basal current at this potential (*P ≤ 0.05).

 
To determine the concentration dependence of levcromakalim between control and inflamed cells, current-voltage relationship (I-V) curves were constructed in the presence of 0.5, 1.0, and 10 µM. For each cell, the amplitude of levcromakalim-induced current was normalized as fold increase over basal current at –120 mV hyperpolarization. Fig. 3C shows that there was almost a sixfold increase in the currents at 0.5 µM levcromakalim in inflamed cells, whereas the currents only increased by onefold in controls.

Single-channel recordings.

The single-channel properties of the KATP channel were determined in cell-attached patches. To avoid activation of the large-conductance Ca2+-activated K+ channel, 200 nM charybdotoxin was included in the patch pipette, and currents were measured at negative potentials. Cells were bathed in high-K+ solution containing 10 µM levcromakalim. KATP channels demonstrated brief burst-like openings, separated by long, closed intervals. This bursting activity appeared to be increased in inflamed cells (Fig. 4A). The amplitudes at each potential, obtained from amplitude histograms, such as those shown in Fig. 4C, were plotted against voltage to obtain the single-channel conductance. Whereas single-channel activity was markedly increased in inflamed cells, there were no significant differences in the single-channel conductance from control and inflamed cells. The conductance obtained from the slope of the I-V curve was 43.3 ± 1.7 pS (n = 8) in control cells and 42.1 ± 2.5 pS (n = 9) in inflamed cells (Fig. 4, B and D). Single-channel currents were abolished in the presence of glibenclamide (10 µM). The single-channel conductance was similar in inside-out patches.



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Fig. 4. Cell-attached patch recordings of single ATP-sensitive K+(KATP) channels. A: raw traces obtained in the presence of 10 µM levcromakalim at –40, –60, –80, and –100 mV in control (left) and inflamed (right) cells. B: current-voltage relationship for single-channel amplitude for the same cells as in A. C: amplitude histogram from control and inflamed cells at –80 mV. D: average slope conductance in control and inflamed cells from 8–9 patches.

 
Inflammation resulted in longer burst durations. Measuring the open probability against time at a holding potential of –80 mV in the presence of 10 µM levcromakalim shows that the interburst intervals are significantly reduced in inflamed cells (Fig. 5). This was consistently observed in several different control and inflamed cells (n = 4). To establish whether the concentration-response relationship was shifted at the single-channel level, cell-attached patch recordings were made at a constant holding potential of –80 mV. The open probability was measured at 0, 0.1, 1, 10, and 20 µM levcromakalim over a period of 120 s. As shown in Fig. 6, in the absence of levcromakalim, single-channel activity was not detected in control cell patches (n = 6). However, in inflamed cells, openings were observed in only two of six patches. The open probability of these openings was low (NPo = 0.07 ± 0.005). No openings were detected in the other four patches. At 0.1 µM, the open probability increased to 0.15 ± 0.067 (n = 6) in the inflamed cells, but again, no channel activity was observed in the control cells. At 1 µM levcromakalim, the NPo in inflamed cells increased to 0.25 ± 0.003 (n = 4). Detectable openings in control patches were observed at 10 µM levcromakalim.



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Fig. 5. Cell-attached patch recordings of KATP channels in control (top) and inflamed (bottom) cell (A) demonstrating bursting pattern. Note long bursts in inflamed cell. B: open probability measured against time for control (left) and inflamed cell (right).

 


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Fig. 6. Concentration dependence of levcromakalim on single-channel events. A: raw traces at 0, 0.1, 1, 10, and 20 µM in control (left) and inflamed (right) cell. B: concentration-dependent effect of levcromakalim on open probability from control and inflamed cells. Numbers of patches examined are given in parenthesis. *P ≤ 0.05; **P ≤ 0.01.

 
We then measured the inflammation-induced changes in the kinetics of bursting behavior. Intraburst open times, mean closed times, and burst duration histograms were calculated for each patch at a potential of –80 mV in the presence of 10 µM levcromakalim. Figure 7 shows an example of the histograms obtained from a control and an inflamed cell. These were well fit with single exponentials. The values for open and closed-time constants within bursts were not significantly different between control and inflamed cells. The open-time constant was 1.36 ms in control and 1.75 in inflamed. The closed-time constants were 0.94 and 1.04 ms in control and inflamed cells, respectively. This indicates that inflammation does not influence the open and closed times for single-channel events within a burst. However, the time constant for burst durations, which was fitted by a single exponential as well, was dramatically prolonged by inflammation from 7.77 to 144.7 ms. Table 3 summarizes the mean burst duration and mean duration of closings within bursts obtained from 10 control and 6 inflamed patches. The mean value of burst duration was significantly increased by 15-fold with inflammation. Although the mean duration of closings per burst was increased with inflammation, on average this only represented 26% of the burst, whereas it was 43% in controls. These data suggest that inflammation results in 1) an increase in burst duration, 2) an increase in open probability, and 3) decrease in the interburst interval but does not alter the intraburst mean open and short closed times.



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Fig. 7. Bursting kinetics of KATP in control (top) and inflamed (bottom) cell. Histograms for the duration of openings within bursts (left), closing within bursts (middle), and total burst durations (right) were all fit by single exponentials. Time constants ({tau}) are presented in inset.

 

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Table 3. Mean burst duration and closings per burst measured in 6 and 10 patches

 
To examine whether the changes in ion channel activity at the single-channel and whole cell level affect whole tissue segments, we then measured the response to levcromakalim in muscle strips. The resting potentials in control and inflamed tissues were –51.8 ± 1.9 (n = 8) and –51.0 ± 3.0 mV (n = 10), respectively. As shown in Fig. 8, the extent of hyperpolarization was markedly greater in inflamed tissues than controls for the concentrations measured. At 0.1 µM, levcromakalim-induced hyperpolarization measured –4.4 ± 1.3 mV (n = 5) in controls and –18.7 ± 2.2 mV (n = 5) in inflamed tissues.



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Fig. 8. Membrane potential recordings of colonic smooth muscle. Microelectrode impalements were made in whole tissue segments. Tissues were perfused with 0.1 and 1 µM levcromakalim. A and C, top: concentrations are recordings from control tissues; bottom: inflamed colon. Bar graph (B) shows extent of hyperpolarization induced by levcromakalim from control (n = 5) and inflamed (n = 5) colon (*P ≤ 0.05).

 
Identity of the KATP isoforms in the mouse colon.

Previously, Koh et al. (18) identified transcripts for Kir 6.2 and SUR2B in the mouse colon but not for Kir 6.1. We reexamined the presence of Kir 6.1 and 6.2 by conventional RT-PCR. Figure 9A shows the presence of transcripts for both Kir 6.1 and 6.2 in the mouse colon. To negate any possibility of genomic contamination, we also designed primers spanning an intron between exons 2 and 3 of Kir 6.1 (primer set 3, Table 2). Figure 9B shows that a single band of 342 bp was obtained corresponding to the predicted size of Kir 6.1 mRNA. This was confirmed in three different groups of cDNA from control and inflamed tissues.



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Fig. 9. Expression of Kir 6.1 and Kir 6.2 in murine colonic smooth muscle. A: RT-PCR of transcripts for Kir 6.1 and 6.2. Lane 1 is 100-bp markers. B: mRNA for Kir 6.1 detected by using primer set 3 (see MATERIALS AND METHODS) that spans an intron. A single band of the predicted size of 342 bp was obtained in both control and inflamed sample, negating any presence of genomic DNA contamination. C: mRNA for sulphonylurea receptor (SUR)2A/2B detected by using primer set 7 (see Table 2) that encompass the 176-bp insertion in mouse SUR2A. Lanes 1 and 2 represent control colon tissues, lanes 3 and 4 represent inflamed colon tissues, and lane 5 is water control without transcript. A single band of SUR2B (490 bp) is present in both control and inflamed sample but not of SUR2A (665 bp).

 
The expression of SUR1, SUR2A, and SUR2B was examined by RT-PCR. To distinguish between SUR2A and SUR2B, primers (set 7, Table 2) were designed to encompass the 176-bp insertion specific for m-SUR2A (15). Figure 9C shows that the amplicon size was equivalent to SUR2B in both control and inflamed cDNA. Neither SUR1 nor SUR2A was expressed in control or inflamed colon. The presence of SUR2A would have revealed a band of 665 bp.

To define the protein localization of the Kir 6.1 and Kir 6.2 isoforms, cross sections of the mice colon were processed for immunostaining with anti-Kir 6.1 and anti-Kir 6.2 antibodies. As shown in Fig. 10, Kir 6.1 was localized to the plasma membrane of both longitudinal and circular smooth muscle as shown in the transverse sections of the colon. On the other hand, Kir 6.2 staining appeared diffuse and more intense around the nucleus. There was no particular evidence of membrane localization. Thus it appears that Kir 6.1 is the major isoform in the plasma membrane consistent with the small single-channel conductance.



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Fig. 10. Immunohistochemical localization of Kir 6.1 and Kir 6.2 in transverse sections of murine colon. Negative controls were carried out by preincubating the primary antibodies with appropriate peptides. LM, longitudinal muscle; CM, circular muscle; blue, nuclear staining; scale bar, 100 µM.

 
Quantitative analysis of Kir 6.1 and SUR2B.

To determine the changes in the gene expression of Kir 6.1 and SUR2B, we examined mRNA levels by real-time PCR. In an initial analysis, we determined that neither of the primers for Kir 6.1 nor SUR2B produced primer dimers. This was measured both by gel electrophoresis and by the melting curves. cDNA was prepared from four groups of control mice and five groups of DSS-treated mice. In each group, two to three mice colons were pooled for RNA isolation. For each sample of mRNA, the CT for {beta}-actin was also determined and used to establish {Delta}CT as described in MATERIALS AND METHODS. The {Delta}CT count for controls was 10.7 ± 0.08 (n = 4) for Kir 6.1 and 9.8 ± 0.36 (n = 4) for SUR2B. When measured for mRNA from inflamed smooth muscle, the {Delta}CT count for Kir 6.1 was 6.28 ± 0.14 (n = 5) and SUR2B was 11.4 ± 0.56 (n = 5). An example of the fluorescence signal curves is shown in Fig. 11A for control and inflamed mRNA. Melt curves showed single peaks for control and inflamed samples (Fig. 11B). A quantitative comparison between control and inflamed cells was measured by normalizing the {Delta}CT counts (see MATERIALS AND METHODS). mRNA for Kir 6.1 was increased by almost 22-fold, whereas that for SUR2B was decreased by almost threefold (Fig. 12).



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Fig. 11. Real-time PCR measurements for Kir 6.1 and SUR2B from one set of control and inflamed mouse colon mRNA. A: SYBRgreen fluorescence signal. B: melt curves for Kir 6.1 (top) and SUR2B (bottom). Note single peaks for control and inflamed samples and absence of primer-dimer formation in water controls.

 


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Fig. 12. Quantitative comparison of gene expression for Kir 6.1 and SUR2B from control (n = 4) and inflamed mRNA (n = 5). A: cycle threshold (CT) for each sample was normalized against the CT value of {beta}-actin and presented as {Delta}CT. B: fold changes for Kir 6.1 and SUR2B between control and inflamed samples was measured as 2{Delta}{Delta}CT (see MATERIALS AND METHODS). *P ≤ 0.05; **P ≤ 0.01.

 

    DISCUSSION
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 MATERIALS AND METHODS
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In this study, we demonstrate the changes in KATP channel in a murine experimental model of colitis. Our findings show that the major isoform of the membrane KATP is composed of Kir 6.1/SUR2B in the mouse colon. After inflammation, KATP currents were increased in response to levcromakalim, resulting in larger hyperpolarizations in colonic muscle segments. An increase in whole cell current amplitude and an increase in bursting duration at the single-channel level reflect the enhanced Kir 6.1 gene expression.

Previously, Koh et al. (18) measured the single-channel conductance of ~27 pS in the mouse colon and identified the transcript for Kir 6.2 and SUR2B, but not Kir 6.1 in smooth muscle cells. However, the small single-channel conductance of ~42 pS and previous reports that KATP in gastrointestinal smooth muscle is inhibited by protein kinase C (11, 16, 33) are more consistent with the major isoform being Kir 6.1. We detected transcripts for both Kir 6.1 and Kir 6.2. By using primers spanning an intron, we confirmed that there was no genomic contamination. The protein expression of both transcripts was identified by immunohistochemical localization. Kir 6.1 is strongly expressed on the plasma membrane, consistent with the single-channel conductance, whereas Kir 6.2 appeared to be restricted to the cytosol, particularly around the nucleus. The significance of Kir 6.2 in the cytosol is not clear at present but could reflect defects in protein trafficking of the immature channel in the colon. Inflammation did not alter the conductance of the channel, indicating that increased current was not due to change in the expression of the Kir isoforms.

The finding that KATP channel activation leads to an increased hyperpolarization of inflamed tissues is consistent with an increase in whole cell currents and increased burst durations in cell-attached patches. The increase in currents would be expected with an increase in expression of the pore-forming Kir 6.1 with inflammation. An analysis of the bursting behavior indicated that neither the mean closed time nor the mean open time for a single-channel event within a burst was altered by inflammation. However, the total mean close durations within a burst was significantly reduced. This could reflect either an increase in the number of channels, or increase in the frequency of openings. The increase in mRNA for Kir 6.1, as determined by quantitative PCR, suggests that transcriptional regulation of the pore-forming Kir subunit may result in an increase in the bursting duration observed with inflammation.

Activation by levcromakalim was necessary to demonstrate the increased responses. Because the major binding site for the K+ channel opener lies within the SUR subunit, it is surprising that SUR2B was not upregulated by inflammation but rather was slightly downregulated. Similar observations have been made with regard to changes in Kir 6.1 and SUR2 in cardiac myocytes and kidney cells after ischemia. Akao et al. (2) noted an upregulation of Kir 6.1, but not SUR2, after myocardial ischemia/reperfusion. The mRNA for Kir 6.1 increased by ~2.5-fold in both ischemic and nonischemic regions of the rat left ventricle after LAD ligation (2). In the heart, the functional activation of the KATP channel during myocardial ischemia results in the shortening of the action potential leading to a decrease in Ca2+ influx, thus protecting against cardiac damage (21). Similarly, Sgard et al. (29) also demonstrated an increase in the mRNA for Kir 6.1 but not SUR2 in rat kidney after ischemic injury. It is not clear how the altered transcriptional regulation of the two subunits affects the functional channel complex. One possibility may be that there is sufficient copy number of the SUR subunit present after inflammation, thus allowing for a functional heterooctomer channel complex. Alternatively, variants of the SUR may be expressed that were not detected by the primers used in the present study. The SUR subunit in smooth muscle is SUR2B, as demonstrated by RT-PCR and by sensitivity to diaoxide (18). Neither SUR1 nor SUR2A could be readily detected in our study, similar to that reported by Koh et al. (18) in the mice colon. Furthermore, inflammation did not appear to induce transcription of SUR1 or SUR2A. Although our studies show that SUR2B transcription was not increased, there are several tissue-specific variants due to alternative splicing of SUR2 receptors that may exist in a single cell. These isoforms may have different sensitivities to the agonist. One such variant that involves alternative splicing of exon 17 has differential sensitivity to ATP (6). It is therefore conceivable that inflammation may alter the expression of specific SUR variants.

In addition to the increased expression of Kir 6.1, responses to levcromakalim may also be shifted by a change in the metabolic state of the inflamed cells. Electrophysiological studies from inside-out patches have suggested that the effects of K+ channel openers may be potentiated in the presence of ADP (1). Thus either an increase in ADP or a change in ATP-to-ADP ratio in inflamed cells could enhance the response to levcromakalim, in conjunction with increased Kir 6.1. Further studies to test this possibility would require experiments using inside-out patches. It is noteworthy, however, that the increased response to levcromakalim was also observed in whole cell recordings in which the concentrations of ATP were identical between control and inflamed cells while these were dialyzed through the patch pipette.

In summary, the present results provide electrophysiological and molecular evidence of changes in K+ channels after inflammation of the colon. In concert with the reported downregulation of the Ca2+ influx through voltage-dependent Ca2+ channels (3, 17, 19), enhanced K+ is likely to result in decreased excitability contributing to the clinical expression of decreased motility of colonic smooth muscle after inflammation. There is also now increasing evidence of changes in transcriptional regulation of channels after insult and injury. This has been particularly well characterized for neuronal sodium channels after injury and inflammation and has been termed transcriptional channelopathy (35). Our present findings suggest that inflammation-induced changes in ATP-sensitive K+ channels likely belong to this class of disorders.


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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46367 and DK-59777.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. I. Akbarali, Dept. of Physiology, Univ. of Oklahoma Health Science Center 940 Stanton L. Young Blvd., Oklahoma City, OK 73104 (E-mail: hamid-akbarali{at}ouhsc.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.


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