1. High-conductance chloride channels generate pacemaker currents in interstitial cells of Cajal 
Jan D. Huizinga, Yaohui Zhu, Jing Ye, Areles Molleman 
Gastroenterology 
Volume 123, Issue 5, Pages (November 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Cell superfusion set-up. Cell superfusion can be performed without disturbing the ion concentrations at the reference electrode. C identifies the patch pipette. The bath is perfused through A. The reference electrode is at position E. The outflow occurs through suction at position D. At position B, directly in front of the patch, a fast flow superfusion allows the patch to be bathed in experimental solutions without solution change at the reference electrode. The method is based on a design by Barajas-Lopez et al.14.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Recording of rhythmic channel activity from a positively identified ICC. (A) ICCs were identified by morphology, rhythmic contractile activity, and staining by ACK2 c-kit antibody coupled to fluorescent Alexa 488. Individual ICCs grew in culture out of small clumps of cells (explants) taken from the intestine of a 2- to 3-day-old mouse. This image was taken by a confocal microscope. Note that some c-kit–positive cells were attached to the explant whereas others were isolated and relatively far away from the explant. The cell recorded from (C) is identified by a black arrow. (B) The same image obtained with a bright field objective. Note that only relatively few cells are positive using the c-kit antibody. (C) The dish was placed in the electrophysiology set-up by using a different inverted microscope. The picture was taken while the recording was obtained, the large black triangle is the pipette out of focus. By using a cell-attached patch with a high K solution in the pipette and culture medium (145 mmol/L Na) in the bath solution, periodic channel activity was revealed as shown. The applied patch potential was −80 mV. See the Materials and Methods section for complete composition of solutions. Scale bars: 10 pA, 2 s.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Whole-cell rhythmic currents. ICCs, identified as in Figure 2, showed rhythmic inward currents, at a frequency similar to those observed with freshly isolated cells.11 The cell had been in culture 4 days and the recording was obtained at a membrane potential of −70 mV. The figure shows activity at 24 cycles/min.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Spontaneous rhythmic channel activity recorded with an electrode attached to a rhythmically contracting ICC. (A) Spontaneous inward currents recorded from an ICC in the cell-attached configuration. C, closed state; O, open state. The channel opened in a rhythmic manner at hyperpolarized patch potentials including the resting membrane potential of the cell. The frequency of the periods of activation was 18 cycles/min, similar to that of observed rhythmic whole-cell currents. Assuming a resting membrane potential of −65 mV, the reversal potential was −25 mV. Channels opened to a conductance level of 144 pS. Lower conductance levels were observed rarely. The second tracing shows a conductance level at half the dominant level, observed at a patch potential of −5 mV. The graph shows the relationship between current amplitude and voltage, the latter expressed as membrane potential across the patch (patch potential). (B) In another ICC, 5 dominant conductance levels were observed in a cell-attached patch; at a patch potential of −100 mV, unitary current amplitudes of 11 pA were obtained as shown in the current amplitude histogram. C, closed state; O, open state. The histogram indicates the relative time the channels were in the different open states. The closed state was determined by other sections of this recording (not shown). From current/voltage relationship (I/V) curves obtained at all conductance levels, the dominant conductance levels were 122, 244, 360, and 480 pS. Occasional current transitions of 5.5 pA (see inset at arrow) suggest a subconductance or existence of cochannels at a conductance of 67 pS (see Discussion section). The amplitude histogram was obtained from a 26-s recording and the major ticks represent 1000 events. The I/V curve expresses current amplitudes based on the first major conductance level obtained at the different holding potentials (expressed as membrane potential across the patch).
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Channel activity is not influenced by substitution of Na+ by Cs+ or NMDG+. A ramp protocol was applied to inside-out patches in which the patch potential was stepped from 0 to −60 mV for 100 ms followed by a ramp to +90 mV over a time period of 300 ms. Each figure shows two 300 ms sweeps. Currents are shown without additional filtering at the voltages between −60 and 90 mV. The extracellular (pipette) solution contained 140 mmol/L KCl. The intracellular (bath) solution contained 140 mmol/L NaCl (top figure). In the other graphs (2–4), 140 mmol/L NaCl was replaced by 140 mmol/L CsCl, 140 mmol/L NMDGCl, or 385 mmol/L sucrose. See the Methods section for complete description of solutions. When the patch potential was changed from 0 to −60 mV, channel activity was seen immediately at a conductance level of 140 pS (Na+), 140 pS (Cs+), and 134 pS (NMDG+). After 100 ms, the patch potential changed gradually from −60 to +90 mV as shown. In many sweeps, the channel(s) remained open for the duration of the ramp protocol. In the sweeps shown, cochannel closings can be seen at patch potentials between +50 mV and +90 mV. This type of experiment shows that the main conductance of the channel is 140 pS, maintained over a large voltage range. At more depolarized potentials a subconductance is revealed, or evidence of the existence of cochannels (see Discussion section). The reversal potentials were 2 mV (Na), 2 mV (Cs), and −1 mV (NMDG). In 5 such experiments, neither the conductances nor the reversal potentials were significantly different between results obtained with different cations. When NaCl was replaced by sucrose, no current went into the pipette. However, the open probability of the channel dramatically reduced such that currents observed leaving the pipette were rare.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Revealing subconductances or cochannels by ramp protocols. Channel activity was observed with NMDG-Cl on both sides of an inside-out patch. The tracing in the middle section of the figure shows channel activity obtained with a pulse protocol in which a holding potential corresponding to a patch potential of +20 mV was held for 13 seconds. Large current transitions were observed to conductance levels of 184 pS and 360 pS. The I/V curve shown represents the first conductance level. Three seconds after this experiment was completed, under identical conditions, a ramp protocol (see legend of Figure 5) was applied (bottom panel). Currents shown indicate a maximum channel conductance of 185 pS. At depolarizing potentials above 40 mV, currents decrease to subconductance levels of 126 and 62 pS, or alternatively it can be interpreted as showing single cochannel activity of 62 pS. Because most current transitions are to a level of 182 pS, the individual channels or cochannels show a high degree of cooperativity. The figure shows a composite of 3 sweeps.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Reduction in chloride concentration causes shift in reversal potential. In the inside-out configuration (similar to experiments shown in Figure 5), the bath solution contained 140 mmol/L KCl. Then the patch was superfused through a fast flow system with 140 mmol/L KCl replaced by 50 mmol/L KCl and 185 mmol/L sucrose (see Figure 1). In 140 mmol/L KCl, the dominant conductance level was 217 pS and the reversal potential was 4 mV. When part of the KCl was replaced by sucrose, the open probability decreased and the dominant conductance level was 64 pS, and the reversal potential shifted from +4 to −20 mV.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
Effect of DIDS on channel activity. A ramp protocol was applied in which the patch potential was gradually changed from −60 mV to +90 mV over a time period of 300 ms. Inside-out configuration with high K solution in bath and pipette. Top: control conditions. The figure shows a composite of 3 sweeps. Twenty sweeps showed channel activity at 182 pS, 130 pS, and 75 pS. In all sweeps, the 182 and 130 conductance levels were dominant. Middle: Two minutes after addition of DIDS (100 μmol/L) in the bath solution. The figure shows a composite of 3 sweeps. In 20 sweeps, the 130 pS and the 75 pS conductance levels were dominant. The channel was closed part of the time at hyperpolarizing and depolarizing potentials as shown. Bottom: Four minutes after addition of DIDS. The figure shows a composite of 2 sweeps. In all 20 sweeps, the channel was closed most of the time.
Gastroenterology  , DOI: ( /gast )
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10. Fig. 9
Periodic increase in amplitude and open probability reflect channel activity generating slow wave in membrane potential. Cell-attached configuration with 140 mmol/L K+ in the pipette and culture medium in the bath. The period frequency of the rhythmic changes was 19 cycles/min, similar to that of the rhythmic whole-cell inward currents described previously. The ICC that was patched in this experiment was connected to other ICCs close to the explant that was rhythmically contracting at 19 cycles/min, hence the ICC, but not smooth muscle cells, will generate rhythmic depolarizations as shown previously.11 (A) At a patch potential of 0 mV (−65 mV pipette potential), hence, at the expected resting membrane potential, the single-channel current was outward, corresponding to chloride entering the cell. The single-channel amplitude was seen to change in a rhythmic fashion but only when the channel was open. The bottom of the recording represents the closed state, no oscillation occurred here. In the expanded trace, the left part shows high amplitude channel activity whereas the right part shows relatively low amplitudes. The hypothesis is that the current amplitude fluctuates because of changes in cell membrane potential caused by slow wave activity, hence, cyclic changes in driving force for the ions occurs (see text). At 0 mV, when Cl− current flowed from the pipette into the cell, the driving force was highest when the membrane potential was in its depolarized phase (~−46 mV), hence, the area of highest current amplitudes, and it was lowest when the membrane potential was in its hyperpolarized phase (assumed resting membrane potential of −65 mV). It can be seen that the open probability is highest during the depolarized phase of the slow wave. The I/V curve (patch potential vs. current amplitude) was drawn from data at both the depolarized phase (○) and the hyperpolarized phase (▵). The reversal potential at the resting membrane potential was −20 mV, assuming a cell membrane potential of −65 mV. The graph at bottom right shows channel open probability vs. patch potentials. At all holding potentials, the open probability in the depolarized phase was higher compared with the open probability in the hyperpolarized phase. However, there was no relationship between open probability and holding potential, indicating that not depolarization but an intracellular event was driving the open probability and hence the generation of outward current. (B) At the resting membrane potential (with a holding potential of 0 mV pipette potential), the current was inward, corresponding to chloride leaving the cell. In this situation, the largest driving force was experienced during the most hyperpolarized phase, in which, hence, the current amplitude was highest. It is again seen that the open probability was the highest during the depolarized phase, that is, when the lowest amplitude currents were seen. The graphs show open probability of 53% during the hyperpolarized phase and 92% during the depolarized phase.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions