Differential modulation of voltage-dependent K+ currents in colonic smooth muscle by oxidants

Madhu Prasad1,2,3,4 and Raj K. Goyal2,4

Departments of 1Surgery and 2Research and Development, Veterans Affairs Medical Center, West Roxbury 02132; and 3Department of Surgery, Brigham and Women's Hospital, 4Harvard Medical School, Boston, Massachusetts 02115

Submitted 9 April 2003 ; accepted in final form 10 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The effect of oxidants on voltage-dependent K+ currents was examined in mouse colonic smooth muscle cells. Exposure to either chloramine-T (Ch-T), an agent known to oxidize both cysteine and methionine residues, or the colon-specific oxidant monochloramine (NH2Cl) completely suppressed the transient outward K+ current (Ito) while simultaneously enhancing the sustained delayed rectifier K+ current (Idr). In contrast, the cysteine-specific oxidants hydrogen peroxide (H2O2) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) exhibited partial and slow suppression of Ito by inducing a shift in channel availability of -18 mV without affecting Idr. After enhancement by NH2Cl or Ch-T, Idr was sensitive to 10 mM tetraethylammonium but not to other K+ channel blockers, suggesting that it represented activation of the resting Idr and not a separate K+ conductance. Extracellular dithiothreitol (DTT) partially reversed the effect of H2O2 and DTNB on Ito but not the actions of NH2Cl and Ch-T on either Idr or Ito. Dialysis of myocytes with GSH (5 mM) or DTT (5 mM) prevented suppression of Ito by H2O2 and DTNB but did not alter the effects of NH2Cl or Ch-T on either Idr or Ito. Ch-T and NH2Cl completely blocked Ito generated by murine Kv4.1, 4.2, and 4.3 in Xenopus oocytes, an effect not reversible by intracellular DTT. In contrast, intracellular DTT reversed the effect of H2O2 and DTNB on the cloned channels. These results suggest that Ito is suppressed via modification of both methionine and cysteine residues, whereas enhancement of Idr likely results from methionine oxidation alone.

colon; colitis; redox; ion channel


DURING COLITIS, activated polymorphonuclear neutrophils (PMN) in the colonic mucosa and submucosa release reactive oxygen species (ROS) into the bowel wall. ROS such as superoxide (), hydroxyl radical (OH-), hypochlorous acid (HOCl), and hydrogen peroxide (H2O2) directly mediate many of the injurious effects of inflammation on colonic tissues (4, 28). In addition to their direct effect on cellular function, conventional products of the PMN oxidative burst combine with ammonia (NH3) present at high levels within the colon (10-70 mM luminal) to produce a group of colon-specific amine-based oxidants such as monochloramine (NH2Cl).

The effects of ROS on colonic epithelial cells have been widely studied. Oxidants increase junctional permeability, stimulate epithelial Cl- secretion, and thus contribute to the impaired salt and water absorption characteristic of colitis (6, 11). The role of ROS as mediators of smooth muscle dysfunction present during colitis is not well defined, although muscle strips from the colon of humans and animals with colitis exhibit variable changes in contractile activity (15, 22, 30, 38).

Emerging evidence indicates that chemical oxidation and reduction may serve to modulate the activity of ion channels (18, 25, 36, 45). This may be of particular significance in pathological conditions associated with oxidative stress. Colonic myocytes from dogs with chemically induced colitis exhibit reduced Ca2+ and K+ current density (26, 27). A separate report (1) demonstrates suppression of L-type Ca2+ current and concurrent activation of glibenclamide-sensitive K+ current in colonic smooth muscle cells from mice with colitis induced by dextran sulfate.

We have previously shown (34) that NH2Cl enhances activity of large-conductance, Ca2+-activated K+ channels (BKCa) in rabbit colonic smooth muscle cells. In contrast to this finding, we recently reported (33) that NH2Cl completely suppresses the transient outward K+ channel (Ito) in colonic myocytes of the mouse, an effect that is reproduced in cloned homotetramers of the channel {alpha}-subunit in Xenopus oocytes. The present series of experiments demonstrates differential modulation of two voltage-dependent K+ currents, Ito and the delayed rectifier K+ current (Idr), by ROS in single smooth muscle cells of the mouse colon. Like NH2Cl, the oxidizing agent chloramine-T (Ch-T) abolishes Ito while simultaneously enhancing Idr. Although the cysteine-specific oxidants H2O2 and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) partially inhibit Ito, they do not affect Idr. Our results suggest that individual ROS can exert divergent effects on distinct K+ channels as a function of time, dose, and site of action.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of single cells. Mice (C57/BL6) were housed in accordance with guidelines established by the Animal Care Committee at the Veterans Affairs Medical Centers in Boston, Massachusetts, and Portland, Oregon, and also the Oregon Health and Science University. After animals were killed, the abdomen was opened and the distal 2 cm of colon were excised and placed immediately in a Sylgard-coated petri dish containing low-Ca2+ Tyrode solution gassed with 95% O2-5% CO2. The colon was opened along its mesenteric border; fecal pellets and residue were removed and pinned mucosa side down. Muscle tissue was divided into small fragments and allowed to equilibrate at room temperature (20-25°C) in the gassed solution for 30 min.

Single smooth muscle cells were prepared by enzymatic digestion of the intact tissue. Briefly, the dissected colonic tissue was triturated with a wide-bore Pasteur pipette for 15 min in a cocktail containing collagenase (0.5 mg/ml), trypsin (0.1 mg/ml), and bovine serum albumin (1 mg/ml) in low-Ca2+ Tyrode solution at room temperature. After dispersal, the solution of cells was stored at 4°C in enzyme-free, low-Ca2+ Tyrode solution. For experiments, cells were placed in a perfusion chamber mounted on the stage of an inverted phase-contrast microscope (Carl Zeiss). Individual myocytes were allowed to settle to the bottom of the chamber and then superfused with HEPES-buffered Tyrode solution (pH 7.4) at room temperature. All experiments were carried out within 4 h after cell dispersal.

Oocyte recordings and molecular biology. Xenopus laevis females were obtained from Nasco (Fort Atkinson, WI). No animal underwent more than two operations, with procedures separated by 3 wk or more. Harvested oocytes were dissociated in 1% collagenase A in OR-2 (in mM: 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES), washed several times and injected with 50 nl of mRNA (20-50 ng), and assayed 2-5 days after injection.

Kv4.1 (GenBank accession no. NM 008423) was the generous gift of Dr. Lawrence Salkoff (St. Louis, MO); Kv4.2 (AAD16972 [GenBank] and Kv4.3 (AAD16973 [GenBank] were generously provided by Dr. Wayne Giles (Calgary, AB, Canada). Constructs were subcloned into the oocyte expression vector pBF. In vitro mRNA was synthesized from pBF constructs, using SP6 polymerase (Invitrogen, Gaithersburg, MD).

Solutions and chemicals. Dissection of smooth muscle and enzymatic dispersal of single cells was carried out in low-Ca2+ Tyrode solution containing (in mM) 135 NaCl, 2.7 KCl, 0.33 NaH2PO4, 11 NaHCO3, 1 MgCl2, 0.01 CaCl2, and 5.5 glucose. The external solution employed to record K+ currents contained (in mM) 135 NaCl, 5.4 KCl, 0.33 NaH2PO4, 5 HEPES, 1 MgCl2, 1.8 CaCl2, and 5.5 glucose and was adjusted to pH 7.4 with NaOH. Nifedipine (1 µM) was included to prevent Ca2+ entry into the cells through L-type Ca2+ channels and facilitate isolation of Ca2+-independent K+ currents. Muscle cells were dialyzed with an internal solution containing (in mM) 100 K-aspartate, 30 KCl, 5 HEPES, 5 EGTA, 2 MgATP, 0.1 GTP, adjusted to pH 7.2 with NaOH. This maintained free cytosolic [Ca2+] at <1 nM (calculated with Patcher's Power Tools version 1.41 for Igor Pro; Wavemetrics, Lake Oswego, OR). Some recordings were made with internal solution containing DTT (5 mM), GSH (5 mM), or catalase (200 U/ml). We confirmed that these agents did not alter the pH of the internal solution at the concentrations used.

Oocyte recordings were made in standard ND-96 solution containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, and 5 HEPES, adjusted to pH 7.4 with NaOH. DTT was directly injected into oocytes in some experiments. This was performed by introducing a sharp micropipette into the oocyte and injecting 50 nl of 1 M DTT during the course of a recording (estimated 10 mM final [DTT]). Data were obtained only from those cells in which holding current remained unaffected by this manipulation.

NH2Cl and taurine monochloramine (tau-NHCl) were synthesized as described previously (34). These compounds as well as DTT, H2O2, and DTNB were added to the perfusing solution in the concentrations indicated. Solvent pH was not affected by addition of these compounds in the concentrations indicated.

Electrophysiological methods. Membrane currents were recorded in smooth muscle cells with the use of standard gigaseal patch-clamp techniques in conventional whole cell configuration (19). Pipettes were pulled from borosilicate tubes 1.5 mm in diameter (WPI, Sarasota, FL) with the use of a Flaming/Brown-type puller (P87; Sutter Instruments, Novato, CA) and then fire-polished on a microforge. Pipette resistances were 2-4 M{Omega} when filled with intracellular recording solution. Currents were recorded with an EPC-9 patch-clamp amplifier (Heka Elektronik, Lambrecht/Pfalz, Germany) controlled by PULSE software (Heka) and were digitized onto the hard disk of a PowerMac G4 personal computer (Apple Computers, Cupertino, CA) for analysis by custom-designed programs written in the Igor programming environment. A standard P/4 procedure was employed to nullify linear leak and capacitance currents. Series resistance was monitored online and compensated by at least 70% in all recordings.

Two-electrode voltage-clamp recordings were made in oocytes. Currents were measured by using a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA), digitized with an ITC-16 data-acquisition interface (InstruTech, Port Washington, NY), and stored on the hard drive of an Apple PowerPC desktop computer. As with the myocyte recordings, stimulus protocols were controlled by PULSE software, data analysis was performed using Igor, and linear leak and capacitance were corrected using a P/4 protocol.

All values are presented as means ± SE. Student's t-test was used at the 0.05 confidence level to determine the significance between any two means.

Drugs. Ch-T, DTT, GSH, DTNB, H2O2, nifedipine, and catalase were obtained from Sigma (St. Louis, MO). Collagenase A was from Boehringer Mannheim. Collagenase used to prepare single smooth muscle cells was from Yakult Pharmaceutical (Tokyo, Japan). All other chemicals were reagent grade. Stock solutions of DTNB (10 M) and nifedipine (0.1 M) were prepared in DMSO and ethanol, respectively. Currents were not modified by vehicle alone. None of the compounds utilized in this study changed the pH of the internal or external solutions. NaOCl (200 µM), NH4Cl (10-20 mM), and taurine (1-5 mM) were found to have no effect on the currents examined in this study.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We (33) and others (24) have previously shown the presence of two distinct voltage-dependent K+ currents, Ito and Idr, in mouse colonic myocytes under Ca2+-buffered conditions. Ito activates and inactivates rapidly at subthreshold potentials and is inhibited by external 4-aminopyridine (4-AP; 5 mM), whereas Idr activates above -10 mV, does not inactivate, and is inhibited by tetraethylammonium (TEA; 10 mM). In the present series of experiments, use of nifedipine as a Ca2+ channel blocker in place of the divalent cations Co2+ or Mn2+ resulted in marked acceleration of Ito inactivation kinetics, likely due to a surface charge phenomenon known to occur in the presence of divalents (14).

Effects of Ch-T and NH2Cl on voltage-dependent K+ currents. Whole cell Ca2+-independent currents were recorded in the presence of Ch-T or NH2Cl. We have previously demonstrated that NH2Cl completely blocks Ito at low micromolar concentrations (33). Although those experiments were performed in the presence of TEA (10 mM) to selectively isolate Ito, TEA was not included in the present studies.

Exposure of cells to Ch-T, which oxidizes both protein methionine and cysteine residues, resulted in complete blockade of Ito, similar to the effect of NH2Cl (Fig. 1A). In contrast to their effect on Ito, both Ch-T and NH2Cl enhanced Idr by more than twofold at potentials positive to 0 mV (294 ± 38 pA for control vs. 591 ± 42 pA for Ch-T and 647 ± 56 pA for NH2Cl, n = 6; Fig. 1, A-C). Even after activation by oxidants, Idr remained sensitive to TEA and insensitive to 4-AP, indicating that this did not represent impaired fast inactivation of Ito or stimulation of a separate K+ conductance.



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Fig. 1. Effect of chloramine-T (Ch-T) and NH2Cl on voltage-dependent, Ca2+-independent K+ currents in mouse colonic smooth muscle cells. A: cells held at -80 mV and then stepped to +20 mV for 500 ms exhibited a rapidly activating and inactivating transient outward current (Ito) and a sustained component (Idr). Exposure to 100 µM Ch-T for 5 min completely abolished Ito and enhanced Idr, effects not reversible by simple washout. Even after activation by Ch-T, Idr is inhibited by TEA (10 mM). B: family of outward currents obtained in colonic myocyte before and after treatment with Ch-T (100 µM) for 5 min. Cells were held at -80 mV and then stepped in 10-mV increments from -100 mV to +60 mV for 500 ms every 10 s. As with Ch-T, NH2Cl simultaneously blocked Ito while enhancing Idr. C: current-voltage (I/V) plot demonstrates the effect of Ch-T ({bullet}) and NH2Cl ({circ}) on Ito and Idr. Both agents abolished Ito and enhanced Idr. Idr was TEA sensitive before and after exposure to the oxidants. Data are expressed as means ± SE for 8 experiments.

 

Time dependence of NH2Cl and Ch-T action on both voltage-dependent K+ currents was very similar. The effect of both oxidants on Ito was considerably more rapid than on Idr, with time to half-maximal effect (t1/2) equal to 2.0 ± 0.4 min (Ito) vs. 4.2 ± 0.7 min (Idr) for NH2Cl and 2.2 ± 0.4 min (Ito) vs. 6.4 ± 1.1 min (Idr) for Ch-T. NH2Cl was more potent than Ch-T in its blockade of Ito (EC50: 520 ± 80 nM NH2Cl vs. 8.1 ± 1.3 µM Ch-T, n = 6 each) and also enhancement of Idr (EC50: 4.2 ± 0.9 µM NH2Cl vs. 72 + 6 µM Ch-T, n = 6 each; Fig. 2A).



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Fig. 2. A: dose dependence of NH2Cl and Ch-T effects on Ito and Idr. Both agents exhibited dose-dependent modulation of Idr ({circ}) and Ito ({bullet}). The effect of NH2Cl on Idr was less potent than its effect on Ito. Ch-T exhibited similar characteristics. The effect of NH2Cl on both Idr and Ito was more potent than corresponding effects of Ch-T (see RESULTS for details). B: effect of Ch-T on Ito in Xenopus oocyte expressing homotetrameric mKv4.3. Ch-T blocked Ito generated by the cloned channel, suggesting that it has a direct effect on the pore-forming {alpha}-subunit. Note that outward tail current flattened after exposure to Ch-T, confirming abolition of K+ current.

 

Suppression of Ito by Ch-T did not result from changes in activation or inactivation kinetics, voltage-dependent availability, or recovery from inactivation (Table 1). This finding was similar to our previous findings with NH2Cl (33).


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Table 1. Biophysical and kinetic parameters of Ito after exposure of smooth muscle cells to Ch-T

 

Like NH2Cl, Ch-T inhibited Ito generated by expression of cloned murine (m)KV4.1, 4.2, and 4.3 in Xenopus oocytes, suggesting that Ch-T blocked Ito by an effect on the poreforming channel {alpha}-subunit (Fig. 2B; data not shown for mKv4.1 and 4.2).

The reversal potential of tail currents as a function of external [K+] confirmed that the outward current enhanced after treatment with Ch-T and NH2Cl represented a K+ conductance (Fig. 3, A and B). Reversal potential of tail currents plotted as a function of external [K+] revealed a slope of 60 ± 4 mV (n = 6), which closely approximates that predicted by the Nernst equation for a pure K+ conductance (Fig. 3B). It was possible that the enhanced sustained outward K+ current following exposure to Ch-T or NH2Cl resulted from activation of a K+ conductance distinct from Idr. Several K+ channel blockers were therefore tested for their effectiveness as inhibitors of this current following oxidant activation. Whereas TEA (10 mM) completely inhibited the oxidant-induced increase in Idr, iberiotoxin (10 nM), apamin (3 µM), 4-AP (5 mM), and dendrotoxin-{alpha} (200 nM) had no effect (Fig. 3C). Thus the current enhanced by Ch-T and NH2Cl was indeed the 4-AP-insensitive Idr and did not represent activation of a separate K+ current. Enhancement of Idr by NH2Cl and Ch-T in the presence of 4-AP demonstrated that the activated current did not result from changes in fast inactivation of Ito (Fig. 3D).



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Fig. 3. A: tail currents elicited in colonic myocyte exposed to NH2Cl with use of a standard double-pulse protocol at varying external [K+]. Note absence of Ito, leaving only enhanced Idr after treatment with NH2Cl. Cells were held at -80 mV and then stepped to +20 mV for 250 ms, followed by steps in 10-mV decrements from +20 mV to -110 mV for 100 ms. For clarity, only reversal of tails is shown as a function of [K+]. The numerical data represent reversal potential (Erev) of tail currents as means ± SE for 6 experiments at each indicated [K+]. B: Tail current Erev plotted against log external [K+], revealing slope consistent with pure K+ conductance. This finding demonstrates that enhanced outward current measured after treatment with NH2Cl or Ch-T represents a K+ current (see RESULTS for details). C: Ch-T-enhanced Idr was sensitive to TEA (10 mM) but not to any other blocker tested. This finding suggests that the K+ current enhanced by Ch-T and NH2Cl represents increased activity of the native Idr, and not activation of a separate K+ conductance. cont., Control; Glib., glibencamide; IBTX, iberiotoxin; APM, apamin; DTX, dendrotoxin. Data are expressed as means ± SE for 6 cells each (*P < 0.05). D: Ch-T enhanced Idr in the presence of 4-aminopyridine (4-AP). This finding demonstrates that increased magnitude of Idr does not result from attenuating inactivation of Ito and confirms that the activated current is TEA sensitive. Cells were held at -80 mV and stepped to +20 mV for 500 ms every 30 s. Data represent means ± SE for 6 experiments. E: effect of taurine NHCl (tau-NHCl). This membrane-impermeant molecule did not affect either Ito or Idr. When the same cell was exposed to NH2Cl, Ito was abolished and Idr was enhanced, suggesting that both currents are modulated from within the cytosol. F: smooth muscle cells perfused with Ca2+ exhibited slower inactivation kinetics (compare with E). Idr were isolated by selective blockade of Ito by 4-AP (5 mM). Idr were not sensitive to 1 µM nifedipine.

 

Tau-NHCl, a cell-impermeant NH2Cl analog, did not enhance Idr, indicating that NH2Cl appeared to act from within the cytosol (Fig. 3E). Nifedipine has been shown to block the human heart delayed rectifier (hKv1.5) expressed in HEK-293 cells (46). In contrast to the findings with hKv1.5, Idr selectively isolated by blocking Ito with 4-AP (5 mM) in mouse colonic smooth muscle cells was not sensitive to nifedipine (Fig. 3F).

Effect of H2O2 and DTNB on voltage-dependent K+ currents. Ch-T is known to oxidize both methionine and cysteine residues on proteins (35). Although it has been established that NH2Cl oxidizes sulfhydryl groups on cysteine amino acids, its methionine functionality has not been defined (17). To determine whether the reciprocal effects of NH2Cl and Ch-T on Idr and Ito were due to oxidation of only cysteine residues, we characterized the effects of the cysteine-specific oxidizing agents H2O2 and DTNB on Idr and Ito. Cells exposed to H2O2 exhibited only partial reduction of Ito at concentrations as high as 30 mM (Fig. 4, A and B). This contrasted with complete inhibition of Ito observed in the presence of Ch-T and NH2Cl. Higher concentrations of H2O2 or DTNB caused breakdown of the seal between patch pipette and cell. Neither H2O2 nor DTNB was able to completely suppress Ito in any cell tested, with maximum inhibition usually 50-60% (Fig. 4B). This effect was less rapid than the inhibition induced by Ch-T and NH2Cl (Fig. 4C). The t1/2 values for inhibition of Ito by H2O2 and DTNB were 8.5 ± 1.3 and 10.2 ± 2.2 min, respectively, significantly slower than with either NH2Cl or Ch-T (n = 6). Suppression of Ito by H2O2 and DTNB was not reversible by simple washout, suggesting that this effect was likely mediated by covalent modification. DTNB and H2O2 were equally potent in suppressing Ito (Fig. 4D), although they were markedly less effective than either Ch-T or NH2Cl.



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Fig. 4. Effect of cysteine-specific oxidants H2O2 and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) on Ito and Idr. A: cells were held at -80 mV and stepped for 500 ms to +20 mV. H2O2 (10 mM) attenuated but did not abolish Ito. This effect was not reversible by simple washout. Idr was unaffected by H2O2 treatment. B: I/V plots demonstrate partial blockade of Ito by DTNB ({blacktriangleup}) and H2O2 ({blacksquare}). Neither agent affected Idr. Data are presented as means ± SE for 8 cells. C: comparison of time course of H2O2 ({blacksquare}) and NH2Cl ({bullet}) inhibition of Ito. Each curve represents experiments in which cells were exposed to either H2O2 or NH2Cl individually. The effect of NH2Cl was significantly more rapid and complete than the effect of H2O2. Data are presented as means ± SE for 8 cells. Dotted lines represent midpoint of maximal inhibition. D: dose dependence of H2O2-({blacksquare}) and DTNB-induced ({bullet}) inhibition of Ito. EC50 was ~3 mM for both. EC50 values were calculated as 50% of maximum inhibition because current was not completely suppressed.

 

In contrast to the effect of NH2Cl or Ch-T, DTNB and H2O2 shifted availability of Ito (V1/2) to more negative test potentials without altering the slope factor (-68 ± 3 mV for control vs. -85 ± 3 mV for H2O2 and -84 ± 2 mV for DTNB, n = 8 each; Fig. 5A). Although channel availability was reduced as a function of voltage, the recovery from inactivation was unchanged after exposure to H2O2 or DTNB and did not therefore contribute to suppression of Ito (t1/2 for recovery = 80 ± 7 ms for control vs. 78 ± 8 ms for 10 mM H2O2 and 77 ± 7 ms for 5 mM DTNB, n = 8). Neither H2O2 nor DTNB altered kinetics of Ito activation or inactivation (Fig. 5B).



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Fig. 5. Effect of DTNB and H2O2 on biophysical and kinetic properties of Ito. A: voltage-dependent availability of Ito (V1/2) before and after treatment with DTNB (10 mM). A family of currents was generated in myocytes held at -80 mV and stepped for 3 s from -100 mV to +20 mV in 10-mV increments, followed by a second pulse to +20 mV for 500 ms. Data at right have been plotted as a function of test potential and then fit to a Boltzmann function. DTNB ({blacksquare}) induces a shift in V1/2 to more negative potentials by 17 mV. Data are presented as means ± SE for 8 cells. Findings are similar with H2O2 ({blacktriangleup}). B: treatment with H2O2 reduced the amplitude of Ito but did not affect kinetics of activation or inactivation as illustrated by the superimposed normalized traces at right.

 

DTNB and H2O2 suppressed Ito generated by homotetramers of mKV4.1, 4.2, and 4.3 in Xenopus oocytes, suggesting an action on the channel {alpha}-subunit (Fig. 6, A and B). As in colonic smooth muscle, the effect of these cysteine-selective compounds on the cloned channels was smaller in magnitude than with Ch-T or NH2Cl. Voltage-dependent availability of the cloned channels was shifted by H2O2 and DTNB to more negative potentials, similar to smooth muscle cells (-63 ± 3 mV for control vs. -78 ± 3 mV for H2O2, n = 8; Fig. 6C).



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Fig. 6. Effect of DTNB and H2O2 on murine (m)Kv4.3. A: currents measured in oocytes expressing homotetramers of mKv4.3 were partially inhibited by H2O2 (10 mM). Subsequent exposure to Ch-T rapidly and more completely blocked the current. B: time dependence and degree of inhibition of current by H2O2. The effect of Ch-T was more rapid and complete, similar to the effect in colonic smooth muscle cells. C: voltage-dependent availability of homotetrameric mKv4.3 before and after exposure to 10 mM H2O2 ({blacktriangleup}) or 10 mM DTNB ({blacksquare}). As with native Ito in colonic myocytes, cysteine-specific oxidants shifted V1/2 by 15 mV to more negative potentials. Data have been fit to a Boltzmann function, as in Fig. 5. Data are presented as means ± SE for 8 cells.

 

In distinct contrast to the enhancing action of Ch-T and NH2Cl, H2O2 and DTNB did not affect Idr, suggesting that enhancement of Idr by Ch-T and NH2Cl might not be mediated by cysteine oxidation (Fig. 4B vs. Fig. 1, A-C).

Effect of reducing agents on voltage-dependent K+ currents. Inhibition of Ito and enhancement of Idr by oxidants were not reversible by simple washout, suggesting that these changes might be due to covalent chemical transformation. Reducing agents have been reported to reverse oxidant-induced alterations in the properties of several native and cloned ion channels (4, 13, 32, 39). We therefore tested whether the cysteine-specific reducing agent DTT would effectively modify oxidant-induced changes in Ito or Idr.

DTT alone (5-10 mM) had no effect on Ito or Idr (Fig. 7A). External DTT did not restore Ito after inhibition by NH2Cl or Ch-T (Fig. 7, A and B). Furthermore, external DTT did not reverse activation of Idr induced by NH2Cl or Ch-T (Fig. 7, A and B). These findings suggested that the action of Ch-T, like NH2Cl, might occur from within the cytosol. It is also possible that these agents might modify DTT-resistant amino acid residues.



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Fig. 7. Effect of external DTT on oxidant-influenced changes in Idr and Ito. A: extracellular DTT alone did not affect either current, as shown by representative traces from a single experiment. After DTT was washed out, Ch-T blocked Ito and enhanced Idr, effects that were not reversed by extracellular DTT. B: DTT alone had no effect on either Ito or Idr. After washout of DTT, Ch-T enhanced Idr and suppressed Ito. Subsequent exposure to DTT failed to restore currents to control levels.

 

In contrast to the results in cells exposed to Ch-T or NH2Cl, extracellular DTT partially restored Ito (by 38%) after blockade by the cysteine-specific agents H2O2 or DTNB (Fig. 8, A and B). In data not shown, external DTT partially reversed the shift in V1/2 induced by these agents (-85 ± 3 to -78 ± 2 mV for H2O2 and -84 ± 2 to -78 ± 2 mV for DTNB).



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Fig. 8. Effect of extracellular DTT on H2O2-induced changes in Ito. A: blockade of Ito by H2O2 was partially restored by external DTT, as shown by traces from a single cell. B: time dependence of the effect of DTT on H2O2-treated cells. Data are presented as means ± SE for 8 cells.

 

The ability of oxidants to modulate Ito and Idr in cells dialyzed internally with DTT or GSH was examined. Internal DTT (5 mM) and GSH (5 mM) had no independent effect on Ito or Idr. Both DTT and GSH prevented inhibition of Ito by H2O2 and DTNB (Fig. 9A). Ito was resistant to suppression by H2O2, but not DTNB, in cells dialyzed with catalase, an enzyme that breaks down H2O2 into H2O and (Fig. 9B). This suggests that suppression of Ito by these cysteine-specific oxidants occurred from within the cytosol and that their effect on Ito likely resulted from cysteine oxidation. In contrast, neither DTT nor GSH attenuated the suppression of Ito caused by Ch-T and NH2Cl, and did they not prevent the activation of Idr by these agents, implying that a cysteine-independent mechanism was likely responsible for these changes (Fig. 9C).



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Fig. 9. Effect of intracellular reducing agents on oxidant-induced modulation of Idr and Ito. A: dialysis of myocytes with GSH (5 mM) prevented suppression of Ito by H2O2 (left). Similarly, cells dialyzed with DTT (5 mM) were resistant to suppression of Ito by 5 mM DTNB (right). Internal DTT prevented DTNB-induced inhibition of Ito, and internal GSH blocked suppression of Ito by H2O2 in data not depicted here. B: H2O2 did not suppress Ito in smooth muscle cells dialyzed with catalase (200 U/ml); however, the current remained susceptible to DTNB. Catalase specifically detoxified H2O2 but not DTNB. This finding demonstrates that the effect of H2O2 on Ito is mediated from within the cytosol. C: intracellular GSH (5 mM) did not alter the effect of Ch-T on either Idr or Ito (left). Similarly, intracellular DTT (5 mM) did not prevent NH2Cl-induced inhibition of Ito or enhancement of Idr (right). Although relevant data are not depicted here, GSH did not affect the actions of NH2Cl, and DTT did not alter the effects of Ch-T, on either Ito or Idr. These findings suggest that the actions of Ch-T and NH2Cl on Idr and Ito are likely mediated by residues not amenable to cysteine reduction.

 

The effect of internal DTT on Ito generated by channel clones resembled the results in muscle cells. Although injection of DTT by itself did not alter currents, introduction of DTT into oocytes reversed attenuation of mKv4.3 current induced by H2O2 (Fig. 10). Although not depicted here, results were the same with mKv4.1 and 4.2. The effect on DTNB-treated cells was similar, indicating that H2O2 and DTNB likely inhibited Ito from the cytosolic aspect. Injection of DTT into oocytes did not reverse inhibition induced by NH2Cl or Ch-T, indicating that these oxidants likely affect DTT-resistant amino acid residues affecting channel function (Fig. 11). The effects of redox agents on Ito and Idr are summarized in Table 2.



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Fig. 10. Effect of cysteine-reactive agents on mKv4.3. Oozcytes expressing mKv4.3 were held at -80 mV and then stepped to +40 mV for 100 ms every 60 s. A: H2O2 partially suppressed current generated by homotetrameric channel (compare a and b). Current was almost completely restored after oocytes were injected with DTT (c). B: time course of H2O2 and DTT effects. a, b, and c are representative traces from a single cell, reflecting time points as indicated. Extracellular DTT did not reverse H2O2-induced suppression of current. Injection of DTT did not affect currents.

 


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Fig. 11. Effect of DTT on NH2Cl-induced suppression of mKv4.3. Oocytes expressing mKv4.3 were held at -80 mV and stepped for 100 ms to +40 mV every 30 s. A: NH2Cl completely abolished current generated by mKv4.3 (compare a and b). Current was not restored after intracellular introduction of DTT (c). B: time course of experiment. Traces a, b, and c represent time points as indicated. Normalization was performed by subtraction of residual current after exposure to 4-AP (5 mM).

 

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Table 2. Summary of effects of ROS on voltage-dependent currents in mouse colonic smooth muscle cells

 


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The major findings of the present study are that, in smooth muscle cells of the mouse colon, 1) Ch-T, which oxidizes both methionine and cysteine amino acids, abolishes Ito while simultaneously enhancing Idr; 2) NH2Cl exerts similar effects on both of these currents but is more potent in its action; 3) the cysteine-selective oxidants H2O2 and DTNB partially suppress Ito but, in marked contrast to the effect of NH2Cl and Ch-T, do not affect Idr; and 4) the cysteine-specific reducing agents DTT and GSH do not independently alter either Ito or Idr, and they restore Ito after inhibition by H2O2 or DTNB, but they have no effect on changes in Ito or Idr induced by NH2Cl or Ch-T. Taken together, these findings suggest that 1) individual oxidants can exert markedly different effects on the various K+ channels in a single smooth muscle cell and 2) chemically distinct oxidants may modulate specific K+ channels in different ways. Both Ito and Idr are known to play important roles in the rhythmic electrical activity of the colon; thus changes in their behavior induced by different oxidants might differentially affect motility in colitis.

Ch-T is a mild oxidizing agent that reacts with the exposed thioether group on methionine residues and cysteine residue sulfhydryl groups at pH 7.0-8.5 (37). Ch-T abolishes fast inactivation of the Na+ current in myelinated neurons and skeletal muscle fibers of the toad while enhancing slow inactivation of cloned Na+ channels (42, 43). Ciorba et al. (10) have demonstrated that Ch-T slows the inactivation of cloned Shaker B/C K+ channels expressed in Xenopus oocytes, thereby enhancing Ito, and that this effect results from oxidation of both methionine and cysteine residues. Ch-T has been found to partially suppress Ito and enhance Idr in rabbit atrial myocytes (40). The results presented here differ from these findings in several important aspects. First, Ito in colonic smooth muscle cells is completely, and not partially, blocked by Ch-T and NH2Cl. Second, external DTT and internal DTT (or GSH) do not prevent or reverse the effect of Ch-T or NH2Cl on Ito, suggesting that their effect is not mediated by oxidation of cysteine amino acids. Third, Ch-T activates a glibenclamidesensitive Idr in rabbit atrial myocytes, whereas Idr in mouse colonic smooth muscle cells is TEA sensitive but glibenclamide insensitive. Finally, apparent activation of Idr at low concentration of Ch-T in atrial muscle, unlike colonic muscle, is sensitive to 4-AP and therefore represents inhibition of fast inactivation of Ito. It has been suggested that some of the actions of Ch-T are mediated by the spontaneous production of HOCl in aqueous systems (28). It is unlikely that HOCl mediates the findings in the present study because this compound had no effect on either Ito or Idr in control experiments.

NH2Cl and Ch-T are similar in their time course of action on Ito and Idr, and although not addressed in this study, potency differences between these agents may be related to differences in permeability or chemical reactivity. The data presented here show that Ch-T, like NH2Cl, has no effect on activation kinetics, inactivation kinetics, voltage-dependent channel availability, or recovery from inactivation. Moreover, Ch-T completely blocks currents generated by homotetramers of mKv4.1, 4.2, and 4.3 in Xenopus oocytes, which suggests that its effect on Ito in smooth muscle cells may be mediated by oxidation of amino acids on the pore-forming channel {alpha}-subunit. Koh et al. (24) have shown that Ito in murine colonic myocytes results specifically from expression of the shal-family transcripts encoding mKv4.1, 4.2, and 4.3.

Several novel auxiliary proteins influence the activity of Ito. K+ channel interacting proteins (KChips), K+ channel activating factor, and K+ channel inactivation suppressor modulate channel kinetics but not current magnitude (3, 20, 31). KChips do enlarge the magnitude of Ito when coexpressed with Kv4.1, 4.2, or 4.3; however, this results from increased surface expression. Although an effect of NH2Cl and Ch-T on KChip activity cannot be ruled out, it is unlikely that KChip-induced alteration in surface expression of channels would be responsible for the complete blockade of Ito observed during the time frame of the experiments presented here.

Ca2+ is known to indirectly influence Ito inactivation via modulation of calmodulin kinase II (CaMK-II) and protein phosphatase 1 (PP-1) (2, 23). It is doubtful that inhibition of Ito by any of the ROS used in this study result secondarily from changes in CaMK-II or PP-1 because inactivation of the current is not altered by treatment with oxidants. In addition, Ito has been measured under stringent low-Ca2+ conditions in the present series of experiments. Complete blockade of Ito by NH2Cl and Ch-T resembles the effect of the K+ channel blocker 4-AP. None of the auxiliary proteins modulating Ito activity induces a similar suppression.

Nifedipine has been shown to block the human heart delayed rectifier current (hKv1.5) expressed in HEK-293 cells (46), and it is therefore possible that enhancement of Idr after exposure to Ch-T or NH2Cl results from relief of channel blockade by nifedipine induced by these agents. This is not the likely basis for oxidant enhancement of Idr in mouse colonic myocytes because the current was not sensitive to nifedipine in these cells.

The cysteine-selective oxidizing agents H2O2 and DTNB also inhibit Ito. Suppression of Ito by H2O2 and DTNB requires concentrations exceeding 3 mM, occurs four- to fivefold more slowly than with Ch-T or NH2Cl, and causes only partial blockade of the current. Although the precise concentration of H2O2 produced by PMNs in vivo is not known, it has been estimated that local [H2O2] can exceed 1-2 mM, and thus suppression of Ito by H2O2 observed in this study may occur during acute inflammation (9). Intracellular dialysis of smooth muscle cells with cysteine-specific reducing agents prevents the action of DTNB and H2O2, suggesting that these compounds exert their effects on Ito from within the cytosol. Oxidation of cysteine residues by H2O2 and DTNB also suppresses currents produced by mKv4.1, 4.2, and 4.3 in Xenopus oocytes, suggesting an effect on the channel {alpha}-subunit. Treatment with H2O2 and DTNB shift V1/2 of the native current in smooth muscle cells as well as the cloned channel in oocytes to more negative potentials (by 15-17 mV). A recent report (29) demonstrated that treatment of hippocampal neurons with H2O2 induces a similar shift in voltage-dependent availability of Ito by -15 mV, with concurrent suppression of Idr. Inhibition of Ito by arachidonic acid in hippocampal neurons was prevented by intracellular dialysis with cysteine-specific reducing agents, closely resembling the effect in mouse colonic smooth muscle (7).

Unlike Ch-T or NH2Cl, neither DTNB nor H2O2 alters activity of Idr, illustrating another fundamental difference between the two classes of oxidants tested here. It is possible that concentrations of DTNB or H2O2 exceeding those employed in this study might modulate Idr; however, breakdown of gigaseals at higher concentrations (>30 mM) prevented such analysis.

Ch-T enhances the BKCa channel by oxidation of methionine residues (41). In contrast, oxidation of cysteine residues suppresses both native and cloned BKCa (12, 44). In a previous report (34), we showed that NH2Cl enhances BKCa in rabbit colonic smooth muscle. Taken together, these data suggest that Ch-T and NH2Cl modulate at least three separate K+ currents in colonic smooth muscle cells in the same manner: Ito, Idr, and BKCa. These effects appear unrelated to cysteine oxidation. NH2Cl mimics the actions of Ch-T and behaves differently from cysteine-specific agents. Although the actions of NH2Cl were not specifically examined in this study, it is possible that, like Ch-T, NH2Cl oxidizes methionine as well as cysteine residues.

In addition to ubiquitous ROS such as H2O2 and HOCl, the high concentration of NH3 in the colon lends itself to the generation of unique amine-based oxidants such as NH2Cl (16). The data presented here suggest that colon-specific oxidants may exert distinct effects on smooth muscle ion channels.

Several reports have indicated that contractile force generation in the colonic wall is altered in colitis (8, 21, 30). Neurons and interstitial cells of Cajal, which both indirectly affect contractile force by their influence on smooth muscle cells, may also be affected by ROS produced during colitis. The results presented here demonstrate that ROS produced in colitis exert a direct effect on the smooth muscle membrane by modulating K+ channels that underlie rhythmic electrical activity. Alteration in the electrical activity of smooth muscle by ROS produced in colitis may play a major role in directly attenuating contractile force as seen in colitis.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. James Maylie and Dr. John Adelman for advice and technical assistance.

GRANTS

This work was supported by an Advanced Research Career Development Award from the Department of Veterans Affairs (to M. Prasad), a Basic Research Award from Glaxo Institute of Digestive Health (to M. Prasad), and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-31092 (to R. K. Goyal).


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Prasad, Dept. of Surgery, VA Medical Center, 1400 VFW Parkway, West Roxbury, MA 02132 (E-mail: mprasad{at}partners.org).

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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