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Journal of Physiology (2002), 539.3, pp. 755-765
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013359
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ABSTRACT |
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K+ channels of isolated guinea-pig cardiomyocytes were studied using the patch-clamp technique. At transmembrane potentials between -120 and -220 mV we observed inward currents through an apparently novel channel. The novel channel was strongly rectifying, no outward currents could be recorded. Between -200 and -160 mV it had a slope conductance of 42.8 ± 3.0 pS (S.D.; n = 96). The open probability (Po) showed a sigmoid voltage dependence and reached a maximum of 0.93 at -200 mV, half-maximal activation was approximately -150 mV. The voltage dependence of Po was not affected by application of 50 µM isoproterenol. The open-time distribution could be described by a single exponential function, the mean open time ranged between 73.5 ms at -220 mV and 1.4 ms at -160 mV. At least two exponential components were required to fit the closed time distribution. Experiments with different external Na+, K+ and Cl- concentrations suggested that the novel channel is K+ selective. Extracellular Ba2+ ions gave rise to a voltage-dependent reduction in Po by inducing long closed states; Cs+ markedly reduced mean open time at -200 mV. In cell-attached recordings the novel channel frequently converted to a classical inward rectifier channel, and vice versa. This conversion was not voltage dependent. After excision of the patch, the novel channel always converted to a classical inward rectifier channel within 0-3 min. This conversion was not affected by intracellular Mg2+, phosphatidylinositol (4,5)-bisphosphate or spermine. Taken together, our findings suggest that the novel K+ channel represents a different 'mode' of the classical inward rectifier channel in which opening occurs only at very negative potentials.
(Resubmitted 3 October 2001; accepted after revision 3 December 2001)
Corresponding author J. Daut: Institut für Normale und Pathologische Physiologie, der Universität Marburg, Deutschhausstrasse 2, D-35037 Marburg, Germany. Email: daut{at}mailer.uni-marburg.de
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INTRODUCTION |
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Potassium channels (K+) are more diverse than any other type of ion channel expressed in cardiac myocytes (Coetzee et al. 1999). They play an important role in determining the shape of the cardiac action potential and in setting the level of the resting potential. The proper function of K+ channels under different conditions is essential for maintaining the normal electrical activity of the heart. Loss-of-function mutations of K+ channels or drug-induced inhibition of K+ currents can lead to ventricular arrhythmias and sudden cardiac death (Mitcheson et al. 2000; Tristani-Firouzi et al. 2001). K+ channels also provide for frequency-dependent changes in action potential duration and can be modulated by various hormones and intracellular second messengers; thus, they contribute to the adaptation of the heart to different functional states.
One of the most challenging questions in recent years has been the correlation between the K+ channels identified electrophysiologically in freshly isolated cells and the channels identified by molecular biological techniques (Coetzee et al. 1999; Nerbonne, 2000). In many cases the properties of the cloned channels expressed in mammalian cell lines or in Xenopus oocytes differ from the properties of the native channels. These discrepancies have led to the discovery of 
subunits or ancillary intracellular proteins associated with the channel (Coetzee et al. 1999; Tristani-Ferouzi et al. 2001). Nevertheless, the molecular structure of some K+ channels characterized electrophysiologically is still unknown, and, conversely, some of the K+ channels identified by RT-PCR or Northern blot analysis in the heart have not yet been correlated with native channels. Therefore a quantitative description of all channels detectable in cardiac muscle cells is needed.
In the present study we describe a novel K+ channel that conducts inward currents at transmembrane potentials more negative than -120 mV. The novel channel appears to be strongly rectifying because outward currents were not observed at potentials positive to -120 mV. As a first step towards molecular identification we have characterized the block by Ba2+ and Cs+ ions and studied the effects of patch excision on the properties of the novel channel. Our results suggest that the novel channel may represent a different mode of operation of one the classical inward rectifier channels of the Kir2 family.
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METHODS |
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The experiments were performed in accordance with the regional animal care committee guidelines (at the Regierungspräsidium Giessen, Germany). Guinea-pigs weighing 250-350 g were decapitated with a small-animal guillotine and the heart was rapidly excised. The aorta was attached to a cannula and the heart was perfused for 5-7 min with a nominally calcium-free solution containing (mM): 115 NaCl, 5.4 KCl, 1.5 MgCl2, 0.5 NaH2PO4, 16 taurine, 5 Hepes, 5 pyruvic acid, 15 NaHCO3, 5 glucose. The solution was bubbled with 95 % O2-5 % CO2 (pH, 7.36; temperature, 37 °C). The flow rate was kept constant at 6-9 ml min-1 (depending on the size of the heart) using a peristaltic pump. Subsequently, the heart was perfused for 4-6 min with a solution that in addition contained 0.05 % collagenase (Type I, Sigma), 40-60 µM CaCl2, and 0.1 % BSA. The heart was disconnected from the cannula and incubated for 10 min in 'recovery' solution containing (mM): 45 KCl, 70 potassium glutamate, 3 MgSO4, 15 KH2PO4, 16 taurine, 10 Hepes, 0.5 EGTA, 10 glucose (pH 7.38). In the same solution the heart was minced and the tissue dispersed with a glass pipette for 3-5 min. The suspension was filtered through a 200 µm nylon mesh, and kept at 15-25 °C for at least 1 h before switching to Eagle's minimum essential medium containing 1 mM Ca2+ (at 20-22 °C). Drops of the cell suspension were transferred to a small recording chamber mounted on the stage of an inverted microscope (Olympus IX 70). The chamber was perfused with physiological salt solution containing (mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 glucose, 5 Hepes (temperature, 20-22 °C; pH 7.4). The solution in the chamber could be exchanged completely within 10 s.
Single-channel currents were recorded using an Axon Instruments 200B amplifier. The pipette resistance was relatively high (8-12 M
); as a result, most patches contained only one or two channels. The pipette solution contained (mM): 145 KCl, 1 CaCl2, 1 MgCl2, 5 Hepes, pH 7.4. When needed, BaCl2 or CsCl was added to the pipette solution. For inside-out patches, the 'intracellular' solution contained (mM): 150 KCl, 1 K2-ATP, 10 EGTA and 10 Hepes (pH, 7.2). The data were acquired with pCLAMP 8 software and analysed with pCLAMP 8 software and a software developed in our laboratory (PC.DAQ 1.0) using LabView (National Instruments). Single-channel currents from cell-attached patches were sampled at 2.5 or 5 kHz and filtered with a -3 dB cut-off frequency of 0.5 or 1 kHz. The mean resting potential of the cardiomyocytes determined in the whole-cell mode (72.0 ± 1.3 mV; n = 92) was used to calculate the transmembrane potential of cell-attached patches. Open probability was determined using all-point-amplitude histograms (raw data) from recordings containing only one channel. The data were not corrected for the liquid junction potential, which was +2.8 mV, determined as described by Neher (1992). Data are given as means ± S.D.
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RESULTS |
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A novel channel activated at potentials negative to -120 mV
K+ channels in isolated guinea-pig cardiomyocytes were studied using the cell-attached mode of the patch-clamp technique. At negative potentials inward rectifier channels are the most frequently observed K+ channels (Sakmann & Trube, 1984a; Shioya et al. 1993; Liu et al. 2001). However, at potentials more negative than -120 mV we frequently observed an unknown channel (Fig. 1A), whose open probability and amplitude increased with hyperpolarization. Using patch electrodes with a low diameter we usually observed only one or two channels in cell-attached recordings. Occasionally, we observed a normal inward rectifier channel and one of the novel channels in the same patch (n = 46). Figure 1B illustrates that the novel channel and the classical inward rectifier channel differ substantially in their voltage dependence and in their kinetics. We found 221 of the novel channels and 715 classical inward rectifier channels under the same experimental conditions. Thus, the probability of finding one of the novel channels was about 31 % of the probability of finding a classical inward rectifier channel.
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Figure 1. A novel channel activated at negative membrane potentials A, typical cell-attached recording of the novel channel activated at -120 mV. No channel openings were seen at potentials between -100 and -120 mV. The pipette solution contained 150 mM K+, 1 mM Ca2+ and 1 mM Mg2+. Inward current is shown downwards; the transmembrane potentials are indicated to the right. The dashed line indicates the current level at which the channel is closed. B, one normal inward rectifier (Kir) channel and one novel channel in the same patch. The slope conductance between -200 and -160 mV was 40 pS for the novel channel and 35 pS for the classical inward rectifier channel. The dashed line labelled 'C' indicates the current level at which all channels are closed; the dashed line labelled 1 indicates the level at which the classical inward rectifier channel is open and the novel channel is closed. | ||
The current-voltage relations of the novel channel and of the classical inward rectifier channel are compared in Fig. 2A. The mean current amplitude of the novel channel (filled squares) was much lower than that of the inward rectifier channel (filled circles). However, the estimated slope conductance of the two channels in the range -200 to -160 mV was similar. In this voltage range the novel channel had a slope conductance of 42.8 ± 3.0 pS (n = 96); the classical inward rectifier channel (most likely Kir2.2, see Liu et al. 2001) had a slope conductance of 37.2 ± 3.7 pS (n = 64) between -120 and -80 mV. The extrapolated reversal potential of the novel channel was in the range -100 to -90 mV. However, as indicated by the dotted line, such an extrapolation might be misleading since the currents were not linearly dependent on membrane potential. No outward currents through the novel channel could be observed at any potential (tested up to +120 mV). Reduction of the K+ concentration in the pipette solution to 75 mM decreased the amplitude of the currents, consistent with a K+ selective channel.
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Figure 2. Comparison of the novel channel and the classical inward rectifier channel A, current-voltage relation of cell-attached single-channel recordings with 150 mM K+ ( Po = Pmax/(1 + e(V - V1/2)/k), where Pmax represents the maximum open probability observed in a patch, V the membrane potential, V1/2 the membrane potential at half-maximal activation and k the slope factor. C, typical plot of log(open time) vs. | ||
The open probability (Po) of the novel channel showed a sigmoidal voltage dependence and reached a maximum of 0.93 at -200 mV (Fig. 2B). The data were fitted with a Boltzmann function; half-maximal activation was around -150 mV, the slope factor was 7.3 mV. The open-time distribution at -200 mV was analysed using a plot of log(open time) vs. 
(number of events), as suggested by Sigworth & Sine (1987). A typical example is shown in Fig. 2C. At -200 mV mean open time (
o) showed only one peak at 35.8 ± 19.6 ms (n = 15). At -220, -180 and -160 mV o also showed only one peak. The mean open times measured ranged between 73.5 ms at -220 mV and 1.4 ms at -160 mV. Figure 2D shows that the mean value of o increased with hyperpolarization (filled squares), whereas in classical inward rectifier channels o decreased with hyperpolarization (filled circles) in the same potential range. The mean closed time was 5.4 ± 0.9 ms at -220 mV (n = 7). At this potential, the closed time distribution showed only one peak. However, at -200 and -180 mV at least two exponential components were required to fit the histogram (not illustrated).
The ion selectivity of the novel channel
Since the pipette solution contained mainly K+ and Cl- ions we first tested whether the novel channel was permeable to chloride ions. When the Cl- concentration in the pipette solution was reduced form 150 to 75 mM (replacing 75 mM KCl with 75 mM potassium glutamate) the conductance of the channel (measured between -200 and -160 mV) was 42.8 ± 1.3 pS (n = 3). This was not significantly different from the value measured under control conditions (42.8 ± 3.0 pS), which makes it unlikely that the channel is permeable to Cl-.
The next step was to test whether the channel was permeable to Na+ ions. When the pipette solution contained 150 mM K+ plus 150 mM Na+ (addition of 150 mM NaCl to the normal pipette solution) the conductance was virtually unchanged (43.0 ± 3.2 pS; n = 7). This finding makes it unlikely that the channels had a significant permeability for Na+ ions (and confirms the conclusion that the Cl- permeability of the channel was negligible). Furthermore, when the pipette contained 75 mM NaCl and 75 mM KCl (replacing 75 mM K+ by Na+) the conductance was reduced by 28 % (from 42.8 ± 3.0 to 31.1 ± 4.25 pS; n = 8). In comparison, the conductance of the classical inward rectifier channel was reduced by 33 % (from 37.2 to 24.8 pS; n = 22) under the same experimental conditions. These data suggest that the novel channel, like the classical inward rectifier channel, has a low permeability to Na+ ions.
The standard pipette solution also contained 1 mM Ca2+ and 1 mM Mg2+. In view of their low concentration it is unlikely that these ions carried a major part of the single-channel current. Nevertheless, we carried out control experiments without Ca2+ and without Mg2+ in the pipette solution (132 mM KCl, 5 mM EGTA, 5 mM Hepes, pH 7.2 adjusted with KOH, final K+ around 150 mM). Surprisingly, the conductance of the novel channel increased to 61.4 ± 2.8 pS (n = 7) under these conditions. Thus, removal of 1 mM Ca2+ and 1 mM Mg2+ from the pipette solution increased the single channel conductance by about 42 %. In comparison, removal of 1 mM Ca2+ and 1 mM Mg2+ from the pipette solution increased the single channel conductance of the classical inward rectifier channel by about 23 % (from 37.2 to 45.8 pS; n = 50), which is in reasonable agreement with the data obtained by Shioya et al. (1993) and Owen et al. (1999). These observations indicate that the conductance of the novel channel is reduced by Ca2+ and Mg2+ to a larger extent than is the case with the classical inward rectifier channel.
Taken together, our experiments suggest that the novel channel observed at membrane potentials negative to -120 mV is a K+ selective channel. Inspection of Fig. 2A shows that the single-channel current-voltage relation with symmetrical K+ is slightly curved (concave downward), as is the case with the classical inward rectifier channel (Liu et al. 2001) and, to a larger extent, with Kir7.1 (Döring et al. 1998). The novel channel has a very unusual voltage dependence, activating only at very negative potentials.
Block of the novel channel by Ba2+ and Cs+
Since the novel channel showed a number of similarities with the classical inward rectifier channel we studied the characteristics of channel block by Ba2+ and Cs+ ions. Cell-attached measurements were carried out with different Ba2+ or Cs+ concentrations in the pipette solution. Figure 3A shows that in the presence of Ba2+ additional long closed states were observed and that the duration of the closed states increased with hyperpolarization (upper three traces). At the same potential, the duration of the closed states increased with increasing Ba2+ concentration (lower three traces). For comparison, we measured the concentration dependence of Ba2+ block of the classical inward rectifier channels under the same experimental conditions (Fig. 3B). Figure 4A shows a plot of the normalized open probability of the novel channel at -200, -180 and -160 mV against log [Ba2+], and Fig. 4B shows the corresponding plot for Ba2+ block of classical inward rectifier channels. These data are summarized in the plot of membrane potential vs. logKd is shown in Fig. 4C. Although the voltage range in which Ba2+ block could be studied did not overlap between the two channel types, it is obvious that the novel channel is much less Ba2+ sensitive than the classical inward rectifier channels.
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Figure 3. Ba2+ block of novel channel and classical inward rectifier channel A, cell-attached recording of Ba2+ block of the novel channel. Upper traces, recorded at -160, -180 and -200 mV in the presence 5 µM Ba2+, illustrate the voltage dependence of Ba2+ block. The two lower traces, recorded at -200 mV, illustrate the concentration dependence of Ba2+ block. B, the effect of different concentrations of Ba2+ on a classical inward rectifier channel at -100 mV. Voltage and Ba2+ concentrations are indicated above the recordings. | ||
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Figure 4. Dependence of the open probability on Ba2+ concentration A, semi-logarithmic plot of the concentration dependence of Ba2+ block of the novel channels at -200 ( | ||
Cs+ block of the classical inward rectifier channels is characterized by short interruptions of individual channel openings resulting in an overall decrease of open probability (Sakmann & Trube, 1984b). The block is voltage dependent, increasing with hyperpolarization, and is attributable to a decrease in mean channel open time; closed times are unaffected by Cs+ (Klein et al. 1999). The block of the novel channel by Cs+ (Fig. 5A) was qualitatively similar to that seen in classical inward rectifier channels. At -200 mV, 1 µM Cs+ decreased Po from 0.93 to 0.87 and o from 35.8 to 32.1 ms (n = 4), 5 µM Cs+ decreased Po to 0.77 and o to 8.3 ms (n = 2) and 25 µM Cs+ decreased Po to 0.51 and o to 1.7 ms (n = 4). In the classical inward rectifier channel, 5 µM Cs+ decreased mean open time at -100 mV from 248 ms (control, n = 15) to 16 ms (n = 6; not illustrated).
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Figure 5. Effects of extracellular Cs+ on the novel channel A, concentration dependence of Cs+ block of the novel channel at -200 mV. With increasing Cs+ concentration in the pipette solution (indicated on the left) the openings were more frequently interrupted by short closures. B, voltage steps from -40 to -200 mV were applied in 20 mV steps in the presence of 25 µM Cs+. At -140 mV the inward rectifier channel was suddenly converted to a novel channel (arrow). At -180 and -200 mV, the mean open time was strongly decreased by Cs+ (compare Fig. 1A). | ||
The kinetics of open-channel block by Cs+ are difficult to analyse in the novel channel because its mean open time is relatively short and strongly voltage dependent (Fig. 2D). Figure 5B shows a typical experiment in which 25 µM Cs+ was added to the pipette solution and the membrane potential was varied between -40 and -200 mV. In this experiment the channel switched from a classical inward rectifier channel to a novel channel at the arrow (this is explained in detail below). Comparison between Fig. 1A and Fig. 5B (lower traces) clearly shows that in the novel channel Cs+ markedly decreased mean open time and that the effect of Cs+ on open probability was much more conspicuous at more negative voltages. In conclusion, the analysis of Ba2+ and Cs+ block revealed a number of similarities between the novel channel and the classical inward rectifier channel in cardiac muscle, but the sensitivity to Ba2+ block is lower in the novel channel.
Lack of sensitivity to isoproterenol
Recently, a hyperpolarization-activated current has been described in cardiac ventricular muscle cells (Yu et al. 1993, 1995; Cerbai et al. 1996; Hoppe et al. 1998) which shows some similarities with the pacemaker current found in sino-atrial node and in the cardiac conduction system (DiFrancesco, 1993; Vassalle et al. 1995). The sino-atrial pacemaker current is due to hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels which are permeable to both Na+ and K+ ions (Ludwig et al. 1999). One of its hallmarks is the shift of the activation curve to more positive voltages in response to cAMP and cAMP derivatives (DiFrancesco & Mangoni, 1994; Bois et al. 1997), and this was also found with the pacemaker-like currents in canine, rat and human cardiac ventricular muscle cells (Yu et al. 1995; Hoppe et al. 1998; Cerbai et al. 1999). Therefore, we tested the effects of isoproterenol on the novel K+ channel. The relation between membrane potential and Po was unchanged after application of 50 µM isoproterenol (n = 3). These findings and the low permeability to Na+ suggest that the novel channel described here is not related to the pacemaker-like current described previously in cardiac ventricular muscle.
Switching of channel characteristics between two different modes
The novel K+ channel was blocked by Ba2+ and Cs+ with characteristics similar to those found in classical inward rectifier channels (Sakmann & Trube, 1984b; Klein et al. 1999; Liu et al. 2001). When we studied the novel channel in more detail we noticed that it was quite frequently converted to a classical inward rectifier channel (n = 27 of 221 recordings) and vice versa (n = 109 of 715 recordings). In all cases one form of the channel disappeared and the other form appeared instantaneously. Figure 6A shows a typical example of a switch of channel properties observed during a series of voltage steps. Between -40 and -120 mV a normal inward rectifier channel was seen, between -140 and -180 mV a typical 'novel' channel was recorded, and at -200 mV the channel suddenly switched back to the classical inward rectifier pattern. It is very unlikely that in these recordings of channel conversion there were two different channels in the same patch because there was no superposition of a classical inward rectifier channel with a novel channel (as was the case, for example, in Fig. 1B).
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Figure 6. Switching between novel channel and classical inward rectifier channel A, series of voltage steps between -40 and -200 mV. A classical inward rectifier channel switched to a novel channel at -120 mV and switched back to a normal inward rectifier channel at -200 mV. B, histogram of the number of conversions from normal inward rectifier channel to novel channel at different potentials. The protocol illustrated in A was used and only the channel conversions occurring during the voltage steps were counted. C, histogram of the number of conversions from novel channel to normal inward rectifier channel at different potentials. At potentials positive to -100 mV (where the novel channel is closed) conversions could still be reliably observed because the mean closed time of the normal channel was very short compared to the duration of the sweep (3 s). | ||
Such switching processes could occur 'spontaneously' at any potential. The voltage dependence of the switching events observed during voltage steps is shown in Fig. 6B and C; B shows a histogram of the conversions from the novel to the classical inward rectifier channel and C shows a histogram the reverse conversions. The data show some scatter, but there was no obvious dependence of the switching process on membrane potential. Subsequently we carried out long-lasting experiments in which the membrane potential alternated between -80 and -180 mV. Figure 7 shows a continuous record in which the channel switched back and forth three times. First, it switched from a normal inward rectifier channel to a novel channel at -80 mV and then back again at -180 mV (a); then it switched briefly to a novel channel at -180 mV (b), and finally it switched again to a novel channel at -180 mV (c). We applied this experimental protocol to 45 patches containing one inward rectifier channel or one novel channel (total recording time, 4.5 h). In 8 of 28 patches initially displaying a classical inward rectifier channel we observed a switch to the novel channel (2.1 switches h-1 at -80 mV and 4.3 switches h-1 at -180 mV). In 4 of 17 recordings initially displaying a novel channel we observed a switch to a classical inward rectifier channel (2.1 switches h-1 at -80 mV and 3.7 switches h-1 at -180 mV). Although the number of switching processes was somewhat higher at -180 mV these data provide no evidence for a statistically significant potential dependence.
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Figure 7. The switching of channel mode during long-lasting recordings Continuous recordings were carried out in which the membrane potentials alternated between -80 and -180 mV. In the upper trace three reversible channel conversions from a normal inward rectifier channel to a novel channel are shown, indicated by the dotted lines marked a, b and c. The transitions are shown at a higher time resolution in the lower traces. | ||
We speculated that the conversion from the normal to the novel inward rectifier channel might be related to detachment from the cytoskeleton. Therefore we tried to disrupt the cytoskeleton by adding cytochalasin B to the bathing solution while a classical inward rectifier channel was recorded in the cell-attached configuration. However, application of 20 µM cytochalasin B for 15 min had no measurable effect on the inward rectifier channels (n = 11).
Conversion of the novel channel to an inward rectifier channel in inside-out patches
To get more information on the mechanisms underlying the transition between the two channel types we excised patches containing the novel channel. After excision a very similar channel was observed in inside-out patches. It also activated at transmembrane potentials negative to -120 mV, no single-channel currents were seen between -100 and +120 mV. Interestingly, the novel channel was always converted to a classical inward rectifier channel within 0-3 min, as illustrated in Fig. 8A. In the experiment shown in Fig. 8B a classical inward rectifier and a 'novel' channel were recorded in the cell-attached mode; after excision two classical inward rectifier channels were observed. The mean delay between patch excision and switching of channel properties was 75 ± 60 s (n = 18). In the inside-out configuration the classical inward rectifier channels had a slope conductance of 33.9 ± 2.6 pS between -120 and -80 mV (n = 23), whereas inward rectifier channels that were converted from a novel channel had a slope conductance of 34.8 ± 2.5 pS (n = 17).
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Figure 8. Conversion of channel mode after patch excision Two typical examples of the conversion of a novel channel to a classical inward rectifier channel after excision of the patch. The transmembrane potentials are indicated to the left. A, a single novel channel recorded in a cell-attached recording converted to a classical inward rectifier channel after excision of the patch. B, a small channel and a larger channel were observed in the same cell-attached patch; a series of recordings at different voltages confirmed that the small channel was a typical 'novel channel'. After excision, two classical inward rectifier channels were observed in the same patch. | ||
These experiments suggest that the novel channel described here may represent a different functional state or a different 'mode' of the classical inward rectifier channel of cardiac muscle cells. It is tempting to speculate that some membrane-bound substance needs to detach from the channel for the switching to occur. Therefore we tried to prevent the transition in single-channel properties by excising the patches into 'intracellular' solutions to which various substances were added. None of the substances tested, Mg2+ (0.1-1 mM), Ca2+ (0.1-10 µM), spermine (10-100 µM), phosphatidylinositol (4,5)-bisphosphate (PIP2, 5-10 µM) prevented or delayed the conversion of the novel channel to a classical inward rectifier channel.
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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By using very small pipettes that allow recording of only one or two channels per patch in guinea-pig cardiomyocytes we were able to identify and characterize a novel channel that was activated at membrane potentials negative to -120 mV. Several lines of evidence suggest that this channel is mainly permeable to K+ ions: (1) addition of 150 mM NaCl to the pipette solution did not change single-channel conductance; (2) reduction of Cl- in the pipette solution did not change single-channel conductance; (3) reduction of K+ in the pipette solution by 50 % (KCl replaced NaCl) decreased single-channel conductance from 43 to 31 pS. (4) Omission of Ca2+ and Mg2+ from the pipette solution increased single-channel conductance to about 61 pS. The channel described here is not related to the hyperpolarization-activated Cl- current (Duan et al. 2000) or to the Ba2+ insensitive pacemaker-like current (Yu et al. 1993, 1995) described previously in cardiac ventricular muscle.
In the presence of external Ba2+ ions long closures of the novel channel were observed, and in the presence of Cs+ the openings were interrupted by short closures, as is the case in inward rectifier channels. However, the half-maximal inhibitory Ba2+ concentration of the novel channel was lower that that of the cardiac inward rectifier channels. The most striking observation was that in the cell-attached configutation the novel channel could spontaneously convert to a classical inward rectifier channel and vice versa. This conversion showed no obvious voltage dependence (Fig. 6 and Fig. 7). After excision of the patch the novel channel always converted to an inward rectifier channel within 0-3 min. These findings suggest that the novel channel represents a different conformational state of the inward rectifier channels, which we denote the 'sleepy' mode because in this mode the open probability is extremely low in the physiological range of potentials.
The delay between excison of the patch and the conversion to a classical inward rectifier channel may be related to the loss of a membrane constituent that is normally present. Our attempts to identify such a component were not successful. Addition of Ca2+, Mg2+, spermine or PIP2 to the intracellular solution had no effect. Further studies are required to identify possible intracellular ligands of cardiac inward rectifier channels. Another possibility is that the switch in channel conformation was due to interaction with a component of the cytoskeleton. The intracellular binding of PDZ domains, for example, can substantially change conductance and kinetics of inward rectifier channels (Nehring et al. 2000). However, application of 20 µM cytochalasin B, which is supposed to disrupt the cytoskeleton, has no measurable effect on channel amplitude or kinetics.
Our results suggest that a considerable fraction of the inward rectifier channels, about one-third, may be in a 'sleepy' mode, in which the channels can be activated only by strong hyperpolarization. Long-lasting periods without any inward rectifier channel activity have been noted previously in cell attached recordings (Trube & Hescheler, 1984; Balser et al. 1991), but it is not known whether this was associated with a conversion of the channels to the sleepy mode described here. We do not know whether the fraction of sleepy inward rectifier channels can be modulated. Any increase in the sleepy fraction would result in a decrease of the resting potential and a prolongation of the action potential. Interestingly, in patients with severe heart failure (Beuckelmann et al. 1993) and in patients with idiopathic dilated cardiomyopathy (Koumi et al. 1995) the density of the inward rectifier current measured in cardiomyocytes was found to be reduced by about 40 %. The associated prolongation of the action potential has been postulated to be the major reason for the altered diastolic relaxation in the patients with heart failure. Thus, it appears possible that a conversion of inward rectifier channels from the normal mode to the sleepy mode may be relevant under pathophysiological conditions.
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Acknowledgements
We thank Robert Graf, Kersten Schneider, Lothar Krapp and Susanne Bamerny for excellent technical and secretarial help and Günter Schlichthörl and Allen Bassaly for support in programming and data evaluation. Our work was supported by the 'Ernst und Berta Grimmke-Stiftung' and the Deutsche Forschungsgemeinschaft (grant Da 177/7-3).
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, novel channels, n = 17; 
, classical inward rectifier channels, n = 64) and with 75 mM K+ (
, novel channels, n = 9; 
, classical inward rectifier channels, n = 12) in the pipette solution. Error bars indicate S.D. The dotted line represents an arbitrary extrapolation. B, plot of open probability vs. membrane potential. The data were fitted with a Boltzmann function:
). B, Ba2+ block of the classical inward rectifier channels at -120 (