Inwardly rectifying chloride channel activity in intestinal pacemaker cells

Yaohui Zhu, Andrea Mucci, and Jan D. Huizinga

Intestinal Disease Research Programme, Department of Medicine, McMaster University, Hamilton, Ontario, Canada

Submitted 9 July 2004 ; accepted in final form 1 November 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Cl channels are proposed to play a role in gut pacemaker activity, but little is known about the characteristics of Cl channels in interstitial cells of Cajal (ICC), the intestinal pacemaker cells. The objective of the present study was to identify whole cell Cl currents in ICC associated with previously observed single-channel activity and to characterize its inward rectification. Whole cell patch-clamp studies showed that ICC express an inwardly rectifying Cl current that was not sensitive to changes in cation composition of the extracellular solutions. Currents were not affected by replacing all cations with N-methyl-D-glucamine (NMDG+). Whole cell currents followed the Cl equilibrium potential and were inhibited by DIDS and 9-anthracene carboxylic acid. Ramp protocols of single-channel activity showed that inward rectification was due to reduction in single-channel open probability, not a reduction in single-channel conductance. Single-channel data led to the hypothesis that strong cooperation exists between 30-pS channels that show less cooperation at potentials positive to the reversal potential. Hence, an inwardly rectifying Cl channel plays a prominent role in determining pacemaker activity in the gut.

pacemaking activity; gastrointestinal motility; interstitial cells of Cajal


RHYTHMIC ACTIVITY OF SINGLE cells or multicellular networks is a common feature in all organisms. Many neurons in the brain and specialized cells in the heart and gut are characterized by endogenous rhythmic activity that relies on a complex interplay between distinct ion channels. Although Cl channels are most often associated with stabilization of the resting membrane potential through passive flux and volume control, they are gaining prominence because it is now understood that they play a role in many critical, specialized electrophysiological properties of excitable cells (7). In smooth muscle cells and, presumably, interstitial cells of Cajal (ICC), the Cl equilibrium potential is positive to the resting membrane potential, which makes it possible that selective opening of Cl channels contributes to cell depolarization. Recently, evidence has been presented that Cl channels may contribute to the depolarization phase and the plateau phase of rhythmic membrane potential changes (slow waves) in ICC. First, pharmacological data suggested that Cl channels play a role in rhythmic inward currents generated by chemically isolated ICC (25). Then rhythmic single-channel activity was observed in cell-attached patch-clamp recordings on ICC synchronous with rhythmic membrane potential oscillations (6). Furthermore, strong evidence was presented that Cl channels were prominent in the generation of the plateau phase of the slow wave (4, 8, 9) and the pacemaker potential in ICC measured in situ (9). The objective of the present study was to identify whole cell currents in ICC associated with the previously observed single-channel activity and to characterize the inwardly rectifying properties of whole cell and single-channel activity. The results indicate that inward rectification is a prominent feature of whole cell Cl currents and single-channel activity consistent with a major role of Cl channels in the generation of pacemaker activity in the gut musculature.


    MATERIALS AND METHODS
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Cell Culture

Explant preparations from the jejunum of CD1 neonatal mice were isolated by sharp dissection without enzymatic digestion (Fig. 1a) as described elsewhere (6, 14, 23). Cells were used within 3–6 days.



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Fig. 1. Culture and identification of interstitial cells of Cajal (ICC) associated with Auerbach plexus (ICC-AP). a: ICC-AP network in mouse small intestine tissue fixed with acetone. ICC were identified by using c-kit antibodies and a secondary antibody coupled to Texas Red. Only the area of the myenteric plexus was scanned by using confocal microscopy. b: Explant technique makes ICC accessible to patch clamping. ICC are shown just outside the explant stained without fixation using c-kit antibodies coupled to the fluorophore Alexa Fluor 488. c: An example of an ICC identified in culture using methylene blue before patch clamping was attempted. ICC stain dark after uptake of methylene blue for 30 min. d: ICC identified using methylene blue after a successful patch-clamp experiment. The ICC were darkly stained by methylene blue for 45 min with subsequent washout of methylene blue and possessed the typical structural features of branches that were relatively thick, short, and bifurcated in contrast to nerve cells that have long, thin processes. e and f: Double staining with c-kit antibodies coupled to Alexa Fluor 488 (e) and methylene blue (f). Incubated with ACK2-Alexa Fluor 488 for 15 min and methylene blue for 30 min. Cells with marked uptake of methylene blue were c-kit positive. Cells faintly stained with methylene blue were not considered ICC. Scale bars represent 10 µm.

 
Drugs

Alexa Fluor 488 was from Molecular Probes (Eugene, OR) and E4031 was from Alomone Labs (Jerusalem, Israel). Methylene blue, 9-anthracene carboxylic acid (9-AC), DIDS, NMDG, and all other reagents were purchased from Sigma (St. Louis, MO).

Electrophysiology

The standard whole cell patch-clamp configuration was employed to record membrane currents and the cell-attached and excised inside-out patch configurations were used for single channel identification. Cultured cells were continuously superfused with Tyrode solution containing (in mM): 135 NaCl, 5.4 KCl, 2 CaCl2, 0.8 MgCl2, 0.33 NaH2PO4, 5 HEPES, and 5.5 glucose (pH 7.35 with NaOH). To block K+ currents, the bath solution was (in mM) 135 XCl, 2 CaCl2, 0.8 MgCl2, 0.33 NaH2PO4, 5 HEPES, 5–10 tetraethylammonium (TEA), 3–5 4-amino-pyridine (4-AP), 5.5 glucose (pH 7.35 with XOH), with X being Na+ or Cs+ or N-methyl-D-glucamine (NMDG+). To change the extracellular Cl concentration, isethionic acid was substituted for Cl (in mM): 100 Na-isethionate, 35 NaCl, 5.4 KCl, 0.33 NaH2PO4, 5 HEPES, 5.5 glucose (pH 7.35 with HCl). The pipette solution contained (in mM) 100 K-aspartate, 30 KCl, 5 HEPES, 5 ATP-Na2, 1 MgCl2, 0.1 GTP, and 5 EGTA, unless otherwise stated. For inside-out recordings, the same solutions were used for both bath and pipette, except that Ca2+ was reduced to 1 µM in pipette. For cell-attached recordings, the extracellular solution was culture medium 199 containing (in mM) 116 NaCl, 26 NaHCO3, 1 NaH2PO4, 0.4 Na-acetate, 5.4 KCl, 1.4 CaCl2, 0.8 MgSO4, 5.6 glucose. Modifications to the solutions are indicated in the text. All experiments were conducted at room temperature (~23°C). Membrane potentials were corrected for the liquid junction potential (Vp-jp). To conform with standard practice, the measured currents from the cell-attached and inside-out configurations have been reversed, and a membrane potential of –65 mV was assumed as resting membrane potential in the cell-attached configuration (6). The optimal pipette tip resistance ranged from 4–5 M{Omega} (whole cell) 7–10 M{Omega} (single channel).

Statistical Analysis

Results were expressed as means ± SE with n being the number of cells. The paired Student's t-test was used to evaluate differences between mean values. P values of ≤0.05 were considered to indicate a statistically significant difference.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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ICC Identification

Recordings were obtained from single, mechanically active ICC identified by vital staining with c-kit antibody coupled to Alexa Fluor 488 (Fig. 1b) or by morphological criteria before patching and methylene blue staining afterward (Fig. 1, cd). Methylene blue (10–6 M) was freshly dissolved in the extracellular solution and perfused on the cells at room temperature for 30–40 min. Methylene blue accumulated preferentially in ICC as noted previously in colonic tissue (11). Methylene blue accumulation was followed through the microscope; the cell body and branches were slowly filled and low concentrations of methylene blue (10–8 M) induced or increased contractile activity of ICC (n = 10). Typical contractile activity (branches and cell body contracting often independently) was another criterion for confirming the identity of the ICC. Contractile activity was abolished by methylene blue at a concentration of 10–6 M, but this could be reversed if washout of methylene blue was done a few minutes after the start of perfusion. When ICC stained in this way were also subjected to c-kit immunostaining, an identical cell population was stained (Fig. 1, ef).

Whole Cell Configuration

An inwardly rectifying current, later to be identified as a Cl current, was discovered during studies into the ether-à-go-go related gene (ERG) K+ current we reported recently (29). The ERG K+ current displayed as a fast-activating but transient-inward current evoked by hyperpolarizing pulses with the use of an extracellular solution containing high extracellular K+ as well as TEA and 4-AP to block most K+ outward currents (Fig. 2). While doing experiments under these conditions, another inward current was observed that was not inactivating and not sensitive to the human ERG K+ channel blocker E4031 (26). The currents were evoked by hyperpolarizing pulses from 20 to –120 mV, and the inwardly rectifying sustained currents were reversed at a potential of –46 ± 8 mV close to the equilibrium potential of Cl (–40 mV) (n = 10; Fig. 2).



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Fig. 2. Typical profile of whole cell currents recorded in ICC with voltage protocols of depolarizing and hyperpolarizing pulses. Bath solution (in mM): 135 NaCl, 5.4 KCl, 2 CaCl2, 0.33 NaH2PO4, 5 HEPES, 0.8 MgCl2, 5.5 glucose (pH 7.35 with NaOH). Pipette solution (in mM): 100 K-aspartate, 30 KCl, 5 HEPES, 5 ATP-Na2, 1 MgCl2, 0.1 GTP, 5 EGTA. The current-voltage (I-V) relationship showed a reversal potential of –48 mV. Equilibrium potential (Eq)-K = –70 mV; Eq-nonspecific cations (NSC) = 0.5 mV; Eq-Cl = –39 mV. A: profile of outward currents evoked by depolarizing pulses and inward currents evoked by hyperpolarizing pulses under physiological conditions. The I-V relationship is dominated by an outwardly rectifying K+ current. B: when the bath solution was changed to (in mM) 140 KCl, 0.1 CaCl2, 1 MgCl2, 5 HEPES, 5.5 glucose (pH 7.35 with KOH), as well as 5 mM TEA and 3 mM 4-AP and hyperpolarizing pulses given from 0 to –120 mV, most outward currents were blocked and an inwardly rectifying sustained current was revealed with a reversal potential shifting to the right at –45 mV. The I-V relationship shows an inwardly rectifying component with a reversal potential at –45 mV, close to the equilibrium potential of Cl.

 
No difference between current amplitude evoked by hyperpolarizing pulses from 0 or –70 mV holding potential or changing cation and anion compositions. To further distinguish the unidentified inward current from the ERG inward current, the holding potential was set at –70 mV. The ERG inward current was not activated when a hyperpolarizing pulse was given from –70 to –120 mV (29). Under these conditions, the peak current amplitude of the unidentified current in response to a voltage pulse from –70 to –120 mV was –218 ± 47 pA (Fig. 3). When pulses were given from 0 to –120 mV, the peak current amplitude was –269 ± 49 pA (Fig. 3) (n = 7). Replacing extracellular Na+ with K+, the current amplitudes were –325 ± 32 pA and –405 ± 47 pA, with hyperpolarizing pulses from holding potentials of –70 and 0 mV, respectively (Fig. 3). The reversal potentials did not change significantly when switching between extracellular Na+ and K+.



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Fig. 3. Effects of holding potential and cation exchange on current amplitude. Bath solution (in mM): 135 NaCl or 135 KCl ({bullet} = NaCl; {circ} = KCl), 0.1 CaCl2, 1 MgCl2, 5 HEPES, 5.5 glucose (pH 7.35 with KOH), 10 TEA, and 3 4-AP. The intracellular (pipette) solution contained 140 mM K and 15 mM Cl. Details of pipette solution (in mM): 135 K gluconate, 4.5 KCl, 10 NaCl, 5 HEPES, 5 ATP-Na2, 1 MgCl2, 0.1 GTP, 10 EGTA. AD: holding potential –70 mV with outside (bath) concentration of Na+ ([Na+]o) = 135 mM, Eq-NSC = 0 mV, Eq-K = –70 mV, Eq-Cl = –59.6 mV (The current amplitude was approximately –280 pA at –120 mV and the reversal potential was approximately –60 mV) (A); and with [K+]o= 140 mM, Eq-NSC = 0 mV, Eq-K = 0 mV, Eq-Cl = –59.6 mV. the current amplitude was approximately –380 pA at –120 mV and the reversal potential was approximately –62 mV (B). C: I-V plots from A and B. D: I-V plots from 7 cells. EH: holding potential 0 mV with [Na+]o = 135 mM, Eq-NSC = 0 mV Eq-K = –70 mV, Eq-Cl = –59.6 mV. The current amplitude was approximately –285 pA at –120 mV and the reversal potential was approximately –58 mV (E); and with [K+]o= 140 mM, Eq-NSC =0 mV, Eq-K = 0 mV, Eq-Cl = –59.6 mV. The current amplitude was approximately –450 pA at –120 mV and the reversal potential was approximately –52 mV (F). G: I-V plots from E and F. H: I-V plots from 7 cells.

 
The non-ERG current was also studied by replacing intracellular K+ with Cs+ to block major K+ currents (Fig. 4). With Na+ as main extracellular cation and the equilibrium potential of Cl set at –63 mV, the reversal potential was –70 ± 5 mV (n = 5). Replacing extracellular Na+ with K+, the reversal potential was –63 ± 2 mV (n = 6). Note the marked inward rectification shown in Fig. 4. Inward rectification was not affected by Mg2+ because omitting it from the pipette solution did not influence the current profiles (n = 10, data not shown).



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Fig. 4. Inward currents in the presence of intracellular Cs+. Bath solution (in mM): 135 NaCl or 135 KCl, 2 CaCl2, 0.33 NaH2PO4, 5 HEPES, 0.8 MgCl2, 5.5 glucose (pH 7.35 with NaOH). Pipette solution (in mM): 135 Cs gluconate, 10 NaCl, 5 HEPES, 5 ATP-Na2, 1 MgCl2, 0.1 GTP, 10 EGTA. Eq-NSC = 0 mV, Eq-Cl = –63 mV. AB: [Na+]o = 135 mM. Current amplitudes and reversal potentials were similar at holding potentials of –70 mV (A) and holding potentials of 0 mV (B). Note that the intracellular substitution of K+ with Cs+ did not significantly alter inward rectifying components (compared with Fig. 3). I-V curve in B: n = 5. CD: [K+]o = 140 mM. Current amplitudes and reversal potentials were similar at holding potentials of –70 mV (C) and holding potentials of 0 mV (D). Substitution of K+ for Na+ did not have a marked effect. [Cs]i, intracellular concentration of cesium. I-V curve in D: (n = 6).

 
When the Cl equilibrium potential was changed by decreasing the extracellular Cl concentration from 146 to 45 mM using isethionate, the current-voltage (I-V) relationship shifted positively, from –51 ± 4 to –14 ± 3 mV, after the Cl equilibrium potential (n = 5; Fig. 5). Complete omission of extracellular Cl markedly decreased currents indicating that extracellular Cl was essential for normal channel activity. When the Cl equilibrium potential was changed by using 116 mM NMDG-Cl in the pipette instead of K-aspartate and KCl, making the extra- and intracellular Cl concentrations ([Cl]i) equimolar, the reversal potential of the inwardly rectifying current was –5 ± 3 mV (n = 6; Fig. 6). When extracellular Na+ was exchanged for NMDG+, the reversal potential did not change significantly and was 5 ± 2 mV (n = 8).



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Fig. 5. Changing the Cl equilibrium potential by replacing 100 mM NaCl with 100 mM Na-isethionate in the bath solution caused a marked shift in reversal potential in accordance with the change in Cl equilibrium potential. Bath solution (in mM): 135 NaX (where X = Cl and/or isethionate), 5.4 KCl, 2 CaCl2, 0.8 MgCl2, 0.33 NaH2PO4, 5 HEPES, 5.5 glucose (pH 7.35 with NaOH). Pipette solution (in mM): 100 K-aspartate, 30 KCl, 5 HEPES, 5 ATP-Na2, 1 MgCl2, 0.1 GTP, 5 EGTA. A and C: with 135 mM extracellular NaCl. B and D: with 100 mM extracellular Na-isethionate and 35 mM NaCl. Eq-Cl = –10.6 mV. E: I-V relationships under NaCl ({blacksquare}) and Na-isethionate ({square}) conditions (n = 5).

 


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Fig. 6. Current profile with equimolar-Cl concentrations and NMDG+ as main cation. Bath solution (in mM): 116 NaCl, 20 CsCl, 2 CaCl2, 0.33 NaH2PO4, 5 HEPES, 0.8 MgCl2, 5.5 glucose. Pipette solution (in mM): 116 NMDG-Cl, 20 CsCl, 5 HEPES, 5 ATP-Na2, 1 MgCl2, 0.1 GTP, 10 EGTA. Eq-NSC = 48 mV; Eq-Cl = 0 mV. A: with the above ionic composition, the current was strongly rectifying and the reversal potential was –5 mV. I-V curve (n = 6). B: when extracellular NaCl was replaced by 116 mM NMDG-Cl (changing Eq-NSC to 2.4 mV), the reversal potential was 4 mV, and no significant change of the current profile occurred. I-V curve: n = 8.

 
Blockade by DIDS and 9-AC. DIDS and 9-AC strongly inhibited the inwardly rectifying currents (Fig. 7) . The peak inward current, evoked by a hyperpolarizing voltage step from 0 to –120 mV, was reduced by 10 µM DIDS 20 ± 8%, and by 100 µM DIDS 67 ± 12% (n = 9). The same current was inhibited by 100 µM 9-AC 33 ± 6% and by 300 µM 9-AC 73 ± 16% (n = 5). The reduction of the outward current at +20 mV was 27 ± 7% by 100 µM DIDS (n = 9) and 43 ± 9% by 9-AC 300 µM (n = 5).



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Fig. 7. DIDS and 9-anthracene carboxylic acid (9-AC) markedly inhibited the inwardly rectifying currents. Bath solution (in mM): 116 NMDG-Cl, 20 CsCl, 2 CaCl2, 0.33 NaH2PO4, 5 HEPES, 0.8 MgCl2, 5.5 glucose (pH 7.35 with NaOH). Pipette solution (in mM): 116 NMDG-Cl, 20 CsCl, 5 HEPES, 5 ATP-Na2, 1 MgCl2, 0.1 GTP, 10 EGTA. A: with the use of the above ionic composition, a typical current profile was obtained. B: addition of DIDS 100 µM strongly inhibited the currents. C: I-V relationship in control condition ({blacksquare}) and after addition of DIDS ({square}). D: with the use of the above ionic composition, a typical current profile was regained after washout of DIDS. E: addition of 9-AC 300 µM strongly inhibited the currents. F: I-V relationship in control condition ({blacksquare}) and after addition of 9-AC ({square}). G: inhibition of the peak current by DIDS (n = 9) and 9-AC (n = 5) at potentials +20 and –120 mV.

 
Single-channel activity in the whole cell configuration. In eight whole cell recordings in which the inwardly rectifying Cl channel was dominant, abrupt changes in current amplitude were observed, signifying a sudden increase or decrease in the number of channels contributing to the current. Figure 8 shows a typical example. At membrane potentials between –120 mV and –60 mV, three or more distinct current levels were identified at each potential (Fig. 8). These current jumps amounted to conductance increases of ~150, ~450, and ~750 pS. This suggested single channel values of 150 and 300 pS. The reversal potential of these currents was –40 mV, similar to the reversal potential of the Cl whole cell currents. At voltages positive to the reversal potentials, discrete conductance levels were ~30, ~90, and ~150 pS. This suggested single-channel activity of 30 and 60 pS. In seven similar experiments, the reversal potential of the whole cell currents occurred at –42 ± 6 mV, and that of the single-channel activity at –40 ± 5 mV. Apparent single channel activities of 90 or 150 pS were dominant negative to the reversal potential and 30 or 60 pS positive to the reversal potential.



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Fig. 8. Single-channel activity recorded simultaneously with inwardly rectifying whole cell currents. Bath solution: 140 NaCl, 5.4 KCl, 2 CaCl2, 0.8 MgCl2, 0.33 NaH2PO4, 5 HEPES, 5.5 glucose. Pipette solution (in mM): 100 K-aspartate, 40 KCl, 5 HEPES, 5 ATP-Na2, 1 MgCl2, 0.1 GTP, 5 EGTA. Eq-NSC = 0.4 mV, Eq-Cl = –34 mV, Eq-K = –83 mV. A: whole cell recordings with single-channel activity superimposed. The holding potential was 0 mV, and voltage steps were imposed from –120 to + 30 mV. B: single-channel activity at –120 mV. The symbols ({blacksquare}, {bullet}, and {blacktriangleup}) link B to D. BL, whole cell baseline. Note an additional conductance level at approximately –385 pA that is not included in the I-V relationship for simplicity. C: I-V relationship of the whole cell current. Line shown is an optimal fit using 3rd order polynomial (y = –0.0001 x 3 – 0.0242 x 2 + 2.0396 x 88.48; R2 = 0.9968). D: I-V relationships of the three lowest dominant conductance levels of the single-channel activity. Lines shown are fitted using linear fits on data negative to the reversal potential. Data points positive to the reversal potential do not fit on this line suggesting rectification on the basis of single-channel conductance or decrease in channel cooperation (see RESULTS and DISCUSSION). ({blacksquare} = chord conductance 150 pS, R2 = 0.875; {bullet} = 450 pS, R2 = 0.9648; {blacktriangleup} = 750 pS, R2 = 0.9739).

 
Characteristics of this single-channel activity observed in whole cell recordings were remarkably similar to the characteristics of the Cl channel activity we reported previously in patched ICC (6). This led us to hypothesize that the inwardly rectifying Cl currents observed here as whole cell currents might be generated by what we described earlier as a high conductance Cl channel (6). This channel was described as showing cooperation, ie., the tendency of two or more channels to open and close simultaneously, possibly due to a common gating mechanism. Hence, the observation that the apparent single-channel conductance was larger at hyperpolarizing potentials compared with depolarizing potentials could be interpreted in two ways. One possibility was that the observed inward rectification was due to a drop in single-channel conductance. Another possibility was that the smaller single-channel conductances positive to the reversal potential were due to a reduction in channel cooperation going from negative to positive membrane potentials. This hypothesis was further investigated.

Single-channel activity (ramp and pulse protocols). Single-channel activity was studied in the inside-out patch configuration using ramp protocols from +60 to –90 mV (Fig. 9). Pipette and bath solutions contained 140 mM NMDG-Cl, making Cl the dominant current carrier and the Cl equilibrium 0 mV. Abundant channel activity was observed between 0 and –90 mV with a reversal potential of 0 mV. Much less activity was seen positive to the reversal potential indicating that open probability was markedly decreased at depolarized potentials. In 144 ramp sweeps in 10 cells, a 210 pS conductance level was identified at –60 mV holding potential in 120 sweeps but at +60 mV, only in 12 sweeps.



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Fig. 9. Rectification of single-channel activity in the inside-out configuration. There were 140 mM NMDG-Cl in bath and pipette solutions. See MATERIALS AND METHODS for details. Currents are shown without additional filtering. When the patch potential was changed from 0 to +60 mV, channel activity was seen in some sweeps at conductance levels of ~30 or ~60 pS. After 100 ms, the patch potential changed gradually from +60 to –90 mV (over a time period of 400 ms) as shown. In 90% of the sweeps, no channel activity occurred at positive potentials. However, at negative potentials, channel openings occurred at a conductance level of ~60 pS or multiples thereof. Top: voltage protocol; middle: compilation of 32 sweeps; bottom: 2 of the 32 sweeps.

 
In the cell-attached configuration (n = 5), the bath solution was chosen to contain 140 mM NMDG-Cl, and the pipette solution contained 140 mM KCl. With an assumed resting membrane potential of –65 mV and an assumed [Cl]i of 30 mM, the equilibrium potential for Cl was –39 mV. With the use of ramp protocols, the single-channel activity (Fig. 10 AC) displayed a reversal potential of –30 ± 4 mV. Prominent channel activity of 60, 120, and 180 pS occurred between 0 and –120 mV, but little activity occurred positive to 0 mV holding potential. Occasionally, channel activity did not diminish immediately when the membrane potential reached positive values (Fig. 10, DF). Such experiments presented the opportunity to observe whether or not the single-channel conductance changed. Examination of the 120 pS conductance level in Fig. 10, E and F, revealed that the I-V relationship remained linear into the depolarization range, indicating that the single-channel conductance did not decrease. All other identifiable conductance levels in the five experiments studied showed a linear I-V relationship when they extended into the depolarizing range. It was also observed that channel closure occurred when the membrane potential reached values to 50 mV, indicating that it was channel open probability that decreased with depolarization and not single-channel conductance. Hence, the conclusion may be warranted that it is not a change in channel conductance but a change in open probability that determines the inward rectification.



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Fig. 10. AC: rectification of single-channel activity in the cell-attached configuration. Bath solution: 140 mM NMDG-Cl; pipette solution: 140 mM KCl (for details see MATERIALS AND METHODS). Currents are shown without additional filtering. Holding potential is –60 mV, followed by a ramp from +30 to –120 mV over 400 ms. The single tracing in B shows channel activity with a reversal potential of –30 mV similar to Eq-Cl assuming an intracellular Cl concentration = 30 mM and a cell membrane potential of –65 mV. No detectable activity occurred at positive potentials. The lower tracings are a composite of 3 sweeps and show large current transitions corresponding to conductance levels of 60, 120, 180 pS. DF: rectification of single-channel activity is not due to a decrease in single-channel conductance. Inside-out configuration with 140 mM K solution in bath and pipette. Currents are shown without additional filtering. Holding potential is 0 mV, followed by hyperpolarization to –60 mV for 100 ms followed by a ramp from –60 to +90 mV over 300 ms. On hyperpolarization to –60 mV, channel activity was seen immediately corresponding to conductance levels of 60 and 120 pS. In this particular experiment, channels did not close on reaching positive potentials, offering the opportunity to observe that the single-channel conductance per se did not decrease because the current amplitude had a linear time course. Above +50 mV, channels started to close dropping from the 120 pS to the 60 pS conductance level or to complete closure. E: is a composite of three sweeps. F: one of the sweeps.

 
To obtain further data on channel cooperation, single-channel activity was studied using pulse protocols. Figure 11 shows an example of burst-type Cl channel activity occurring between –65 and –125 mV. The estimated equilibrium potential of Cl was –39 mV (see legend of Fig. 11) and the reversal potential was –40 mV. At membrane potentials negative to the reversal potential, dominant conductance levels were 90, 120, 300, and 360 pS, with differences in conductance levels, or apparent single-channel conductance values, of 180, 210, 240, or 390 pS or more. At potentials on the positive side of the reversal potential, 60, 150, and 180 pS differences in conductance levels were more commonly observed. This is best illustrated by a comparison of Fig. 11B (–115 mV) and Fig. 11G (–25 mV). At –115 mV, a conductance of 210 pS dominates, whereas at –25 mV, a 60 pS conductance is most prevalent. These data are consistent with the hypothesis that the cooperative behavior of a 30-pS channel decreases when the voltage changes from the negative to the positive side of the reversal potential (n = 5).



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Fig. 11. Single-channel activity observed in the cell-attached configuration using a pulse protocol revealed decline in channel cooperation on depolarization. For solution details see MATERIALS AND METHODS. Cell membrane potential was assumed to be –65 mV. Conductance levels were calculated as (current amplitude)/(membrane potential – reversal potential). Amplitude histogram bin sizes were –125 to –95 mV = 0.25, –65 mV = 0.15, –35 and –25 mV = 0.10. All amplitudes were calculated using a recording of 25 s duration except for tracing D. AE: traces negative to the reversal potential with calculated conductance levels and current amplitude histograms. Chord conductance levels observed indicate large conductance levels with few (see D) smaller conductance levels present. FG: traces positive to the reversal potential. The channel activity has been reduced to smaller conductances, possibly due to decreased channel cooperation. All traces were recorded within a time frame of 5 min, ruling out the time factor as responsible for the change in channel kinetics. H: I-V relationships are shown of single-channel activity corresponding to 120 pS ({blacktriangleup}) and 360 pS ({bullet}). No transitions of 360 pS conductance occurred positive to the reversal potential.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study identifies for the first time an inwardly rectifying whole cell Cl current in ICC. Furthermore, it provides evidence that the Cl channel identified previously in ICC on the basis of single-channel activity (6) is associated with this inwardly rectifying whole cell current. These data provide support for the hypothesis that ICC exhibit an inwardly rectifying Cl current that can be rhythmically activated (Fig. 11). This Cl current will be an important part of the ICC ion channel repertoire to initiate and regulate its electrical activity. It will contribute to generating the rhythmic inward currents and determine the plateau potential, and it will have a stabilizing influence on ICC excitability.

The inwardly rectifying current preferentially uses Cl ions on the basis of the following evidence. First, the reversal potential does not follow the equilibrium potentials of K+, Na+, or Ca2+. Because the current is not affected by replacing extracellular cations with NMDG+, which does not permeate cation channels, it is unlikely to be mediated by a nonselective cation (NSC) channel. NMDG+ was also used as the only cation in the internal pipette solutions effectively precluding a possible contribution from NSC channels including cationic inward rectifiers. Under conditions in which the equilibrium potential for NSC currents was distinct from the Cl equilibrium potential, the reversal potential of the inwardly rectifying whole cell current was similar to the Cl equilibrium potential. Changing Cl for isethionate or aspartate, the inwardly rectifying whole cell current changed reversal potential according to change in the Cl equilibrium potential. The current was blocked by DIDS and 9-AC. The current was easily distinguished from the inwardly rectifying ERG K+ current that was recently identified in ICC (29); the current amplitude was not affected by changing the holding potential from 0 mV to –70 mV with a voltage pulse going to –120 mV, which would eliminate the ERG K+ current. The current was also not affected by E4031. Hence, ICC can generate an inwardly rectifying Cl current.

The following hypothesis is derived from the single-channel data. A 30-pS channel shows strong cooperative behavior in which 2–10 channels are simultaneously activated and inactivated. The inward rectification is based on the observation that open probability at membrane potentials positive to the reversal potential is markedly less compared with potentials negative to the reversal potential. Reduction in single-channel conductance is not part of inward rectification, but a reduction in channel cooperation occurs simultaneously with the reduction in open probability. This hypothesis is based on the following observations. With the use of ramp protocols, open probability is markedly less at positive membrane potentials. In whole cell recordings and cell-attached single channel recordings, apparent large conductance single-channel activity is much more prominent at hyperpolarizing potentials. Channel cooperation is stronger at potentials negative to the reversal potential compared with positive to the reversal potential, because the average apparent single-channel conductance is larger at hyperpolarizing potentials. Channel cooperation has been observed in other "high conductance" Cl channels (7, 10, 18, 28).

An inwardly rectifying Cl channel was recently identified in native cardiac myocytes with a proposed role in the regulation of cardiac electrical activity. It was only expressed in a small percentage of cells, which made the authors conclude that it may only be physiologically important under pathological conditions (1). An alternative possibility is that the patched cells do not all have the right intracellular conditions for the channel to be activated. It may also be, as the authors suggest, that the channel is most important in the cardiac pacemaker cells that constituted a small percentage of the cells. There are several differences between the inwardly rectifying Cl channel observed in cardiac myocytes and the one reported here. Volume changes evoke this current in myocytes (1), but the inwardly rectifying Cl current is not evoked by volume changes in ICC (S. J. Park and J. D. Huizinga, unpublished observation). In cardiac myocytes, the current is described as slowly activating, which is not seen in ICC. Duan et al. (1) showed that the this current was not inhibited by SITS. Here we show a clear sensitivity to the disulfonic stilbene derivative DIDS. Duan et al. (1) suggested that the inwardly rectifying Cl current in cardiac myocytes was transmitted through ClC-2. Because of the different characteristics, this is unlikely the case for ICC. The channel in ICC may be similar to a poorly characterized high conductance, inwardly rectifying Cl channel in glial cells (15). This channel also shows long opening times and the highest open probability in the physiological voltage range. Single-channel activity observed in the present study has many features in common with Cl channel activity recorded from rabbit colonic smooth muscle cells (20). Although this channel was not identified as an inwardly rectifying channel, no channel openings were observed positive to 40 mV with marked open probability around the resting membrane potential.

Cl channels play an important role in the physiology of ICC. It is likely that both Cl channels (6) and NSC channels (22, 27) contribute to the generation of the spontaneous rhythmic inward currents in ICC and, hence, slow-wave activity in the musculature. Tokutomi et al. (25) were the first to provide evidence for a role of Cl channels in the generation of pacemaker activity in the mouse small intestine. In the guinea pig antrum, it was shown that the plateau phase of the pacemaker activity was generated by Ca2+-activated Cl channels (4, 8). Kito and Suzuki (9) recorded pacemaker potentials from cells in the myenteric plexus of the mouse small intestine, which they identified as ICC through injection of Lucifer yellow. These pacemaker potentials were abolished by combined application of Ni2+ and DIDS, which led to the conclusion that pacemaker activity was made up of activities from a voltage-dependent Ca2+-permeable channel and a Ca2+-activated Cl channel. Interestingly, with either Ni2+ or DIDS application, rhythmic pacemaker activity remained, albeit altered, indicating that both the Ca2+ permeable channel and the Cl channel were rhythmically activated to produce pacemaker activity. Another function of the Cl channel is the facilitation of nerve-mediated inhibition. Hyperpolarization of ICC induced by inhibitory enteric nerves is mediated by inhibition of Cl channels (21). Conclusive evidence for the molecular identity of the Cl channels is not yet available; hence, it is not known whether the physiological roles described above are mediated by the same or different Cl channels.

The general consequences of the presence of an inwardly rectifying Cl conductance in ICC are the following. Constitutively active Cl channels will be involved in setting the membrane potential at a more depolarized level. Activation of Cl channels will contribute to the cell membrane conductance; it decreases the cell input resistance and time constant. It is important to note that the depolarization caused by Cl channels is self-limiting; positive voltages will strongly diminish its influence. The Cl current will have a stabilizing influence; when strongly hyperpolarizing influences, such as inhibitory junction potentials, tend to reduce excitability, the presence of a Cl current will drive the membrane potential back to its resting potential. There are several factors that complicate the elucidation of the precise physiological functions of the Cl channel. There are, no doubt, complex interactions with other channels active at the resting membrane potential, such as the ERG-type K+ channel (29) and the NSC channel (27). It may well be that the relative importance of the Cl and NSC channels in generating inward currents depends on the experimental conditions or changing physiological conditions. It seems possible that blockade of Cl influx affects influx of Na+ or K+. It is possible that Cl channels become permeable to cations (2, 3). Gating mechanisms of the Cl channel need further investigation; complete omission of Cl from the extracellular fluid by another anion is always followed by a marked decrease in current amplitude at the whole cell level (present study) and the single channel level (6). It appears that extracellular Cl is essential for normal channel gating as observed with other Cl channels (16).

The mechanisms of activation and inactivation of Cl currents by intracellular components has not been elucidated yet. Ca2+ likely contributes. The whole cell currents described in the present study increased one to twofold when the bath solution was changed from 0 to 2.5 mM Ca2+ (Y. Zhu and J. D. Huizinga, unpublished observation). Ca2+ is unlikely the only modulator. Variability in intracellular activity in the cells patched will be a cause of intercellular variability in current amplitude (whole cell) or open probability (single-channel activity). Similarly, the hyperpolarization-activated inward current in the heart shows significant variability in biophysical characteristics dependent on the cell type that is also contributed to regulation by intracellular activity (17).

In the present study, methylene blue was colocalized with c-kit antibody-stained ICC, making methylene blue suitable for identifying ICC after successful patch recording without the need for a fluorescence microscope. Methylene blue is rather nonspecific in staining cellular structures. However, when given time, it is taken up irreversibly by ICC associated with Auerbach plexus but not ICC associated with deep muscular plexus (24) because of accumulation in the sarcoplasmic reticulum (5, 12, 13). Washout of the dye eliminates nonspecific staining but accumulated dye remains visible.

In summary, characteristics of an inwardly rectifying Cl current are presented at the whole cell and single channel levels in ICC. This is a contribution to the further understanding of the ionic basis of pacemaker activity in the gut, which underlies peristaltic motor activity. Because Cl channels are also identified in pacemaking elsewhere (19), it may provide insight into general mechanisms of pacemaking.


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Operating costs were supported by the Canadian Institutes of Health Research.


    ACKNOWLEDGMENTS
 
Tissue cultures and Figs. 1, a and b were obtained skillfully by Jing Ye. Input from Catherine Golden is gratefully acknowledged.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Huizinga, McMaster Univ., HSC-3N5C, 1200 Main St. West, Hamilton, ON L8N 3Z5, Canada (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.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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