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Nicardipine-sensitive Na⁺-mediated single channel currents in guinea-pig sinoatrial node pacemaker cells

Tamotsu Mitsuiye, Jiquing Guo and Akinori Noma

MS 9113 Received 4 January 1999; accepted after revision 10 September 1999.

	ABSTRACT

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1. The Na⁺-dependent inward currents underlying slow diastolic depolarization of sinoatrial (SA) node cells were examined. Using a Na⁺-rich, Ca²⁺-free pipette solution a novel single channel current was recorded in addition to the conventional Na⁺ and L-type Ca²⁺ currents. The current (termed i_st, as it reflects the whole-cell sustained inward current, I_st) does not show obvious inactivation during a 700 ms depolarization and is unique in having a smaller amplitude (1·1 ± 0·18 pA at -60 mV, n = 12) than the Na⁺ current through conventional Na⁺ (~3·3 pA) and Ca²⁺ channels (9·6 ± 0·32 pA at -60 mV, n = 8). The mean unitary conductance of i_st channels was 13·3 pS.

2. The recording of i_st was infrequent, was observed only in spontaneously beating SA node cells, and was facilitated by adding Bay-K 8644 to the pipette solution. Overlapping of i_st events was observed and i_st was abolished by bath application of nicardipine.

3. In the ensemble average, the activation of i_st was evident by depolarization beyond -70 mV, and the dynamic voltage range of activation (-70 to -30 mV) encompassed the extent of the slow diastolic depolarization. The current density of i_st was 0·33 pA pF^-1 at -60 mV, as estimated from the number of channels per membrane patch, the open probability and the unitary amplitude.

4. Cumulative histograms for both open and closed times were fitted with a sum of two exponential components. The slow time constants decreased with depolarization, while the fast time constants and the fraction of the fast component were voltage independent. The number of bursts per sweep increased with depolarization. The time constant of the first latency histogram was about two orders of magnitude larger than those in cardiac L-type Ca²⁺ channels and decreased with depolarization.

5. It is suggested that the i_st channels might be responsible for the whole-cell I_st.

	INTRODUCTION

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Voltage-dependent activation of inward current systems is essential for driving the slow diastolic depolarization in cardiac pacemaker cells. The identity of the ionic channels which are responsible for the pacemaker depolarization in the sinoatrial (SA) node cells is still a matter of debate (Irisawa et al. 1993). Activation of the L-type Ca²⁺ channel occurs at potentials positive to -40 mV (Hagiwara et al. 1988), which are less negative than the maximum diastolic potential of -60 to -70 mV, although studies have suggested that these channels can be activated at potentials of between -60 and -40 mV (Verheijck et al. 1999). The hyperpolarization-activated cation current (I_f) is partly responsible (DiFrancesco et al. 1986a,b, 1994; DiFrancesco, 1991), but the pacemaker depolarization has been reported as being hardly affected (Noma et al. 1983; Denyer & Brown, 1990; Van Ginneken & Giles, 1991; Sohn & Vassale, 1995) or only slightly (< 30 %) affected (Nikmaram et al. 1997) by the suppression of I_f. Recently an inward current component was found to be activated by depolarization to potentials in the range of the slow diastolic depolarization in sinoatrial (SA) node cells of rabbit and guinea-pig (Guo et al. 1995, 1996a,b; also atrioventricular (AV) node, Guo & Noma, 1997) and rat (Shinagawa et al. 1997). Since the current was suppressed by removing extracellular Na⁺, but remained in Ca²⁺-free solution, the carrier ion was suggested to be Na⁺. This current was not affected by a high concentration of TTX, but was blocked by conventional Ca²⁺ antagonists. Since the current was also unique in not showing marked inactivation during a depolarizing pulse, a novel sustained inward current (I_st) was proposed. However, identification of the channel responsible for I_st is still not unequivocal, because the whole-cell current was hardly separated from the L-type Ca²⁺ current.

Here we report a single channel sustained inward current (i_st) which is activated by depolarization to the diastolic potential range of SA node cells. Although the channel is blocked by nicardipine, the current is carried by Na⁺ in the absence of Ca²⁺. The conductance and gating kinetics of this current are clearly different from those of the L-type Ca²⁺ or the Na⁺ channel currents. Here the characteristics of the novel single channel current are compared with those of the whole-cell current I_st.

	METHODS

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Single cell preparation

SA node cells were dissociated from guinea-pig heart as described previously (Guo et al. 1996a). Briefly, adult guinea-pigs weighing 200-300 g were given an overdose of sodium pentobarbitone (40 mg kg^-1). All animal treatments were performed according to the guidelines of the Declaration of Helsinki, and the experimental protocols used were approved by the animal use and care committee of Kyoto University. Under artificial respiration the chest cavity was opened and the aorta was cannulated in situ to start perfusion of coronary vessels with the control Tyrode solution. The heart was then excised and mounted on a Langendorff-type apparatus. The perfusate was switched from control to Ca²⁺-free Tyrode solution, and then to the same solution containing 0·04 % collagenase (200 units mg^-1, Wako, Tokyo, Japan). After 10-15 min of the collagenase treatment, the SA node region bordered by crista terminalis, atrial septum and the cranial and caudal vena cava was excised and cut into small strips of 0·5-1 mm in width. The strips were incubated in Ca²⁺-free Tyrode solution containing 0·1 % collagenase and 0·01 % elastase (Boehringer, Germany) for 5-10 min. After the enzyme treatment, myocytes were gently and mechanically dissociated in high-K⁺, low-Cl^- Kraft-Brühe (KB) solution, and stored at 4°C for later experiments on the same day.

Atrial cells were also dissociated from the digested right atrial wall after the Langendorff perfusion of whole heart with collagenase.

Solutions

The control Tyrode solution contained (mM): NaCl 140·0, KCl 5·4, NaH₂PO₄ 0·33, CaCl₂ 1·8, MgCl₂ 0·5, glucose 5·5 and N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulphonic acid) (Hepes) 5·0, and pH was adjusted to 7·4 with NaOH. The content of CaCl₂ was decreased to 0 mM (nominally Ca²⁺-free solution) where appropriate. The modified KB solution, (high-K⁺, low-Cl^- solution) contained (mM): glutamic acid 70·0, KCl 25·0, KH₂PO₄ 10·0, 2-aminoethane sulphonic acid (taurine) 10·0, glucose 11·0, O,O'-bis(2-aminoethyl) ethylene-glycol-N,N,N',N'-tetraacetic acid (EGTA) 0·5 and Hepes 10·0, and pH was adjusted to 7·2 with KOH. The standard Na⁺-rich pipette solution (Ca²⁺-free) contained (mM): NaCl 140, MgCl₂ 0·5 and EGTA 0·1, and pH was adjusted to 7·3 with 10 mM Hepes-NaOH. The Ca²⁺ channel blocker, nicardipine and the agonist Bay-K 8644 were used. When Bay-K 8644 was added to the pipette solution, the pH was adjusted to 8·0 to facilitate the drug effect (Lansman et al. 1986). Nicardipine was added to the bath solution.

Cell-attached patch single channel recordings

Before experiments, a drop of the cell suspension was added to the recording chamber filled with the control Tyrode solution. Spontaneously beating spindle-shaped SA node cells, called pacemaker cells in the present study, were few in number. The cells were 5-10 µm wide and 50-80 µm in length, with an input capacitance of 40 pF according to our whole-cell current analysis (for example, see Fig. 1 in Guo et al. 1997). The remainder of the cells were typically of atrial or intermediate shape, and were either quiescent or showed irregular beating, probably due to transient depolarizations caused by the spontaneous release of Ca²⁺ from the sarcoplasmic reticulum. Undamaged quiescent cells, as determined by whole-cell current recordings (see also Guo et al. 1995), were used for comparison with spontaneously beating cells. After identification of pacemaker cells, the external solution was switched to a solution in which 30 mM K⁺ was substituted for equimolar Na⁺ to stop the spontaneous beating and to facilitate the tight-seal formation with the patch electrode. The resting potential of the pacemaker cells in the 30 mM K⁺ solution was measured in separate experiments, and was -41·1 ± 0·6 mV (n = 7), which agrees well with the value estimated from the Nernst equation (-40·5 mV). The holding potential was set to -100 mV, assuming a resting potential of -41 mV in the 30 mM K⁺ solution in the sinoatrial node cells, except during the recording of rapid TTX-sensitive Na⁺ currents when the holding potential was -140 mV. For recording single channels from atrial cells, resting potential was nullified by superfusing the cell with a high K⁺ external solution (140 mM KCl, 2 mM EGTA and 10 mM glucose adjusted to pH 7·3 with KOH). The test pulse was applied at intervals of 5 s, except during the Na⁺ channel recording when intervals were much shorter (0·1-0·5 s). All experiments were conducted at 35°C.

The tips of the patch pipettes were silicone coated with a drop of N,N-dimethyltrimethylsilylamin to decrease the stray capacitance (Inoue et al. 1991). In some experiments, the pipette solution was changed during experiments using the pipette perfusion technique (Soejima & Noma, 1984).

The output of the amplifier (List EPC-7) was low-pass filtered (4 or 2 kHz) with a Bessel filter and stored on computer hard disk via an A/D converter. The sampling frequency was 5 kHz, except for rapid Na⁺ channel recording when it was 50 kHz. Leak and capacitive current traces were obtained by averaging traces with no openings, and were used for subtraction. Amplitudes of the open events were determined by constructing a current level histogram. The latency for the first opening of the channel on depolarization, and life times of the open and closed events were determined by defining a threshold level in the middle of the open-closed transitions. Life time distribution histograms were first constructed (not shown), then the values of sequential bins were summed to construct the cumulative histograms (see Figs 7-10). Finally the values were normalized by the total number of events. All computer programs were custom-written by T.M. Statistical values are given as means ± S.D.

	RESULTS

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Three types of Na⁺-mediated single channel currents in SA node cells

To record single channel currents carried selectively by Na⁺, a Na⁺-rich, Ca²⁺-free pipette solution was used to which 0·5 mM Mg²⁺ was added to inhibit the Na⁺ conductance of L-type Ca²⁺ channels. Three types of voltage-dependent current were recorded from pacemaker cells as shown in Fig. 1A-C. The records in Fig. 1A were obtained by repetitive depolarizing pulses to -60 mV from a holding potential of -140 mV. Activation of the inward current occurred within a few milliseconds of depolarization, followed by complete inactivation within the 40 ms pulse. These gating kinetics are typical for the TTX-sensitive Na⁺ channel. If a Na⁺ equilibrium potential of +50 mV is assumed, a unitary amplitude of 3·3 pA at -60 mV gives a conductance of 30 pS at 35°C. This value agrees well with the estimate extrapolated from 20 pS (Kunze et al. 1985; Kimitsuki et al. 1990b) obtained at room temperature using a Q₁₀ of 1·5 (Correa et al. 1991). The activity of this type of channel was progressively abolished when a less negative holding potential of -100 mV was used as previously described (the time-dependent voltage shift of the steady-state inactivation curve towards more negative potentials in the cell-attached patch; Kunze et al. 1985; Kimitsuki et al. 1990a). This type of channel was observed in 32 % of 43 patches tested, but mostly only one channel appeared in a given patch. It seems that the density of distribution of Na⁺ channels in SA node cells is less than in ventricular cells, where most of the patches showed overlaps of more than five open events (Kimitsuki et al. 1990b).

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Figure 1. Three types of single channel current recorded in standard Na+-rich pipette solution Depolarizing pulses to -60 mV were applied from a holding potential of either -140 (A) or -100 mV (B and C). A, the rapid Na+ channel current, sampled at 50 kHz through a low-pass filter at 4 kHz. Pulse interval, 500 ms. Note the faster time scale of records in A compared with those in B and C. B and C, currents recorded by applying the test pulse every 7 s with 10 µM Bay-K 8644 present in the pipette solution, sampled at 5 kHz through a low-pass filter at 2 kHz. The dashed lines to the right of the uppermost trace in C indicate the multiple open levels. A, B and C were obtained from different cells.

Two other types of channel activity were recorded using the same pipette solution, but containing Bay-K 8644 to increase the rate of observing channels (Fig. 1B and C). The channel events in Fig. 1B were unique in showing extremely short life times, which resulted in variable amplitudes of individual events (1-3 pA at -60 mV) due to limited time resolution of the recording system (using the low-pass filter of 2 kHz). On the other hand, those in Fig. 1C showed much longer open events or bursts with clearly smaller amplitudes. The type of channel activity shown in Fig. 1B was observed in 21 % of patches obtained from pacemaker SA node cells (n = 53), and 22 and 17 % of patches from quiescent SA node cells (n = 125) and atrial cells (n = 86), respectively. The third type of channel activity (Fig. 1C) was observed in 32 % of patches from pacemaker SA node cells (n = 53). Overlapping of open events was frequently observed with this channel type. The number of channels was estimated from the maximum number of overlapping events (excluding patches from quiescent cells) 2·8 ± 0·3 channels per patch were determined in the pacemaker cells (n = 17). The channel activity illustrated in Fig. 1C was never observed in 125 patches from non-beating SA node cells and 86 patches from atrial cells. The unit amplitude of open events determined by eye was -1·1 ± 0·18 pA at -60 mV (n = 12).

Identification of the L-type Ca²⁺ channel

The current shown in Fig. 1B was scarcely observed in the absence of Bay-K 8644. To determine whether this was the Na⁺ current passing through L-type Ca²⁺ channels partially blocked by Mg²⁺, we first recorded the single channel current using Mg²⁺-free pipette solution containing 10 µM Bay-K 8644. Without the Mg²⁺ blockade, the unit amplitude of Na⁺ current was well resolved, as shown in Fig. 2Ba, and measured 9·6 ± 0·32 pA at -60 mV, (n = 8). Compared with Ca²⁺-mediated current through the L-type Ca²⁺ channel, activation of Na⁺-mediated current was observed at more negative potentials with much larger unit conductance, and with much less time-dependent inactivation (Matsuda, 1986; Hess et al. 1986). Furthermore, the channel activity was largely abolished 3 min after adding 5 µM nicardipine to the bath solution (Fig. 2Ab).

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Figure 2. Examination of the Na+ current passing through L-type Ca2+ channels A, Na+-mediated single channel current recorded using Mg2+-free pipette solution before (a) and 5 min after (b) adding 5 µM nicardipine to the bath solution. B, the Na+ current was recorded using Mg2+-free pipette solution for control recordings (a), the pipette solution was then switched to solution containing 0·5 mM MgCl2. Sequential traces recorded 2 (b) and 10 min (c) after the start of perfusion of the pipette are shown. Note the different time scales for b and c. Bay-K 8644 (10 µM) was present in the pipette solution. The membrane potential during the pulse was -50 (A) or -60 mV (B).

After recording the channel activity in Fig. 2Ba, the pipette solution was switched to solution containing 0·5 mM Mg²⁺. The current traces in Fig. 2Bb and c were obtained 2 and 10 min after starting the pipette perfusion, respectively. It is obvious that the inward current was partially blocked by the external Mg²⁺, and indeed the channel events became quite similar to those demonstrated in Fig. 1B. Thus, we concluded that the current shown in Fig. 1B was due to the Mg²⁺ blockade of Na⁺ influx through L-type Ca²⁺ channels.

Nicardipine blockade of the novel single channel Na⁺ current

Since the channel events shown in Fig. 1C have not previously been described, their characteristics were studied. We first examined the channel's sensitivity to a Ca²⁺ agonist and antagonist. Although overlap of open events was frequently observed in the presence of Bay-K 8644 (see above), the overlap was not observed in the absence of Bay-K 8644. The frequency of observation of the channel type was 36 % (78 patches); that is, the density of available channels was 0·36 per patch. Furthermore, the channel was blocked by nicardipine. In Fig. 3A, five sequential pulses to -60 mV from -100 mV, applied every 10 s, are demonstrated in the control (a), 9 min after applying nicardipine to the bath solution (b), and 12 min after washing out the drug (c). It is evident that the channel activity was abolished in a reversible manner. To demonstrate the time course of the blocking effect, the open probability (Fig. 3B) or the availability (Fig. 3C) of the channel was summed over the sequential pulses to construct the cumulative P_o and availability graphs (Fig. 3B and C, respectively). The availability of the channel was defined as 1 when at least one opening event was observed during the depolarizing pulse. In both graphs, the slope of the cumulative histogram was clearly decreased by nicardipine, and a delayed wash-out is obvious. Abolishment of the channel activity was consistently observed in another five patches within 5 min after the application of nicardipine. In the following section, we call this sustained inward current i_st.

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Figure 3. Blockade of the ist channel by nicardipine The pipette solution contained no Bay-K 8644. A, original cell-attached recordings in control solution (a), 9 min after the bath application of 5 µM nicardipine (b), and 12 min after washing out the drug (c). B, open probability (Po) of the channel during individual depolarizing pulses summed throughout the experiment and plotted against the pulse number. In C, availability of the channel was defined as 1 when open channel events were observed during a pulse, and 0 for a blank sweep. The values for individual sweeps were summed throughout the same experiment as in B. The depolarizing pulse was to -60 mV and applied every 5 s.

Single channel conductance

To avoid modification of the channel gating by Bay-K 8644, the following experiments (Figs 4-10) were carried out without Bay-K 8644 in the standard Na⁺-rich pipette solution. It should be noted that the presence of L-type Ca²⁺ channel activity is negligible in the absence of this Ca²⁺ channel agonist. Figure 4 shows records of i_st evoked by sequential pulses to different potentials (a, -80 mV; b, -60 mV; c, -40 mV; d, -20 mV). Bursting and non-bursting openings are evident with infrequent blank sweeps. It seems that the bursting events became more frequent with larger depolarizations at the expense of the isolated short openings. Accordingly, the tail openings on repolarization to the holding potential (-100 mV) were observed as the preceding test pulse became more positive (see events marked with dots in Fig. 4D). The channel showed no obvious time-dependent inactivation, as evident from the repetitive openings all through the pulse duration at these test potentials.

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Figure 4. ist at different test pulses ist evoked by sequential pulses to -80 (A), -60 (B), -40 (C) and -20 mV (D). Standard Na+-rich pipette solution containing no Bay-K 8644 was used here and in subsequent figures. Bay-K 8644 was not used to avoid L-type Ca2+ channel openings. Note that the amplitude of the current becomes smaller with larger depolarization. Tail openings (···) were observed in D.

The unitary amplitude of i_st was determined by fitting a Gaussian distribution to the amplitude histogram obtained for each of the test potentials as shown in the left panels of Fig. 5A. Figure 5B indicates the mean value of the current amplitudes (n = 6-9). The slope of the regression line over the linear part of the relationship was 13·3 pS. This value is smaller than the single channel conductance of the Na⁺ channel (30 pS). The mechanisms underlying the deviation of the data points at -80 and -100 mV were not examined in the present study. The reversal potential was +13·2 mV. These data were obtained in the absence of Bay-K 8644, but as expected the amplitude of i_st at -60 mV (0·98 ± 0·19 pA; n = 12) was not significantly different from that obtained in the presence of Bay-K 8644 (1·1 ± 0·18 pA, n = 12).

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Figure 5. The current-voltage relationship of the ist channel A, current level histograms during pulses to the various potentials indicated. The smooth superimposed curves are best fits of a sum of two Gaussian distributions. The amplitudes of open events were determined in six or seven experiments in different cells and their mean values at different test potentials are plotted in B. B, bars indicate standard deviation. The slope of the fitted line is 13·3 pS. Data are from the same experiment as shown in Fig. 4.

Voltage dependence of i_st activation

The traces shown in Fig. 6A are ensemble averages of the open probability calculated from 70-120 recordings of i_st evoked at various potentials. Channel activation was observed at potentials positive to -80 mV and open probability increased with more positive depolarizations. The sustained channel activity is obvious during the 700 ms pulse, with rapid deactivation occurring on switching off the test pulses. For comparison with the whole-cell current, the voltage dependence of activation of the channel was evaluated by calculating the mean open probability during the 700 ms test pulse in four experiments. It is obvious that the activation range of the mean i_st is between -70 and -30 mV with a tendency for saturation at more positive potentials. This potential range overlaps well with the voltage range of the slow diastolic depolarization in the SA node cells.

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Figure 6. Voltage-dependent activation of ist A, the individual open events in the original recordings were idealized with a square pulse of a constant amplitude (1·0), but of a duration equal to the open event. These traces, thus determined from 70-120 sequential sweeps, were averaged. B, the mean open probability during the test pulse was determined at different test potentials in four cells, and was plotted in B against the test potential. The arrows indicate the pulse offset. The smooth curves in B are fitted by eye. Standard Na+-rich pipette solution was used.

Voltage dependence of the life times for open and closed states

In the present study, stable recordings of i_st which were long enough to allow measurement of many channel events were difficult to achieve because of run-down of channel activity and determination of channel gating in terms of state-transition model was also difficult. However, analysis of i_st clearly demonstrated some unique characteristics of the ion channel responsible. Only those experiments which showed no overlapping of i_st channel events were used. Contamination of the Na⁺ current through the L-type Ca²⁺ channel was indicated in only a few experiments by the variable amplitudes of the open events due to Mg²⁺ block (Fig. 2). These experiments were also rejected from the i_st life time analysis. Traces in Fig. 7A indicate the cumulative open-time histogram obtained from a continuous recording at different test potentials. The histogram at -80 mV could only be fitted by single exponential, whereas those at different potentials were fitted with a sum of two exponentials. The histogram determined from the tail openings at -100 mV after depolarization to -20 mV was also fitted with a sum of two exponentials. These findings may suggest the presence of more than one open state. Voltage-dependent changes were not observed in the fast time constant or the fractional weight of the fast component (Fig. 7Bb and c). Only the slow time constant decreased with depolarization (Fig. 7Aa).

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Figure 7. Analysis of open time A, cumulative histograms of open times at various potentials (a, -80 mV; b, -60 mV; c, -40 mV; d, -100 mV). d was obtained by analysing tail openings. The superimposed exponential curves indicate the slow component and the sum of fast and slow components in each graph. Time constants for the two components are indicated. N is the total number of events. Arrows indicate the intersection of the slow exponential curve with the ordinate, these values indicate the fraction of the fast component. In a, only a single component was observed. B, two time constants (a and b) of the open time distribution and the fractional amplitude of the fast component (c). Filled symbols indicate events obtained from the tail current, and the open symbols were from events during the test pulse. Lines connect data points obtained in a given patch. Standard Na+-rich pipette solution was used.

The cumulative closed time histograms in Fig. 8 were determined at different test potentials. All these histograms were fitted with a sum of two exponentials. The slow and fast time constants were plotted against the test potential in Fig. 8Ba and b, respectively, and its fractional amplitude of the fast component in Fig. 8Bc. Only the slow time constant decreased with depolarization (Fig. 8Ba) while no voltage dependence was found in the fast time constant (Fig. 8Bb) or in the fractional amplitude of the fast component (Fig. 8Bc).

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Figure 8. Analysis of the closed time A, cumulative histograms of closed times at various potentials (a, -80 mV; b, -60 mV; c, -40 mV). In a, the right panel shows the fast component on an expanded time scale. The superimposed exponential curves indicate the slow component and the sum of fast and slow components in each graph. Time constants for the two components are indicated. N is the total number of events. Arrows indicate the intersection of the slow exponential curve with the ordinate, thereby indicating the fraction of the fast component. B, two time constants (a and b) of the open time distribution and the fractional amplitude of the fast component (c). Lines connect data points obtained in a given patch. The standard Na+-rich pipette solution was used.

It seemed that the increase in open probability with depolarization was mainly determined by the more frequent bursts with larger depolarization, as suggested in Fig. 4. To test this view we tentatively assumed that a burst was a train of more than two openings interrupted by gaps of less than 5 ms. According to the closed time analysis, the fast (Fig. 8Bb) and slow (Fig. 8Ba) time constants differed by about two orders of magnitude, and therefore we chose a critical gap duration of 5 ms, which is about two times the fast time constant. The histogram was fitted with a sum of two exponentials at all potentials tested (Fig. 9A). The fast exponential component consisted of trains of a few short openings, and may have small weight in terms of the open probability of the channel. Therefore the fast component was neglected. The number of bursts per 700 ms pulse was plotted in Fig. 9Ba. Depolarization induced more frequent bursts. Unexpectedly the mean burst duration thus determined was decreased with larger depolarization (Fig. 9Bb).

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Figure 9. Burst opening kinetics The cumulative burst duration in A (a, -60 mV; b, -40 mV) was obtained assuming that open events interposed with closed events shorter than 5 ms were within a burst. The number of bursts per 700 ms pulse was plotted in Ba, and the time constant of the slow component in Bb. At potentials more negative than -70 mV, bursts were not obvious. Different symbols indicate different cells. The standard Na+-rich pipette solution was used.

The cumulative first latency histograms in Fig. 10 were fitted with single exponentials. The time constant for the first openings was voltage dependent, and was about two orders of magnitude larger than those for cardiac L-type Ca²⁺ channels. At physiological temperature, the mean first latency is 2-3 ms (-45 to -20 mV) for L-type Ca²⁺ channel (Isenberg & Han, 1994). The lack of overlapping of the open events in records from a given patch does not necessarily indicate that the patch contained only one channel, because the open probability of the channels was very low. Therefore, the mean closed time and the first latency of i_st channel might be underestimated by a factor of real number of channels within a patch. However, values obtained in different patches did not vary by more than twofold, suggesting that the number was not different among different patches.

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Figure 10. Analysis of the first latency A, examples of cumulative distribution of the time to first opening (a, -80 mV; b, -60 mV; c, -40 mV). B, mean first latency plotted against test potentials. Different symbols indicate different experiments.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

A novel single channel current

The present study revealed a new kind of single channel current, i_st, which is activated by depolarization to the potential range of the slow diastolic depolarization in pacemaker SA node cells (-70 to -30 mV). Although Cl^- and Na⁺ are the major components of the pipette solution, the reversal potential of +13·2 mV (Fig. 5) strongly suggests that i_st is not a Cl^- current. The Cl^- equilibrium potential should be negative in the SA node, similar to ventricular myocytes, in which recordings of single CFTR-type Cl^- channels under the cell attached patch mode revealed a Cl^- reversal potential between -40 and -30 mV (Ehara & Ishihara, 1990; see also Matsuoka et al. 1990). The reversal potential of the i_st channel may be explained by assuming a permeability ratio (P_Na/P_K) of 0·6 for a non-selective cation channel. Although the inward i_st is carried by Na⁺, the single channel conductance and the gating kinetics are totally different from the conventional Na⁺ channel. The single channel conductance of 13·3 pS, as revealed by the clear open channel current levels is also different from that of the Na⁺ current through L-type Ca²⁺ channels as demonstrated in Figs 1 and 2. No corresponding single channel current has been described, to our knowledge, in the cardiac myocytes. The single channel conductance of 13 pS may be similar to the Ca²⁺-activated non-selective cation current described in the ventricular myocyte (15 pS, Ehara et al. 1988), or in primary-cultured rat heart cells (Colquhoun et al. 1981). Although the ion selectivity of the i_st channel was not systematically studied in the present study, the voltage-dependent activation of the i_st channel is clearly different from the virtually voltage-independent kinetics of the Ca²⁺-activated non-selective cation current.

The nicardipine sensitivity and the voltage-dependent kinetics of bursting and non-bursting openings of i_st are similar to those described in cardiac L-type Ca²⁺ channels (Cavalie et al. 1983; Lansman et al. 1986; Dirksen & Beam, 1996). However, the latency to first opening was more than one order of magnitude longer in i_st than in cardiac L-type Ca²⁺ channels. The i_st channel did not show voltage-dependent inactivation during depolarization. However, this does not necessarily exclude the possibility of the Ca²⁺-mediated inactivation of i_st channel, since the carrier ion is always Na⁺. The increase in intracellular [Ca²⁺], through the activation of the L-type Ca²⁺ channel and/or the Ca²⁺ release from the sarcoplasmic reticulum, may possibly inactivate the i_st channels. It is speculated that i_st channel may have a similar molecular structure to the ₁ subunit of the L-type Ca²⁺ channel with unique voltage sensors in the S4 segment (Hille, 1992; Chin et al. 1992). However, the charged residues in the region within or near to the selectivity filter (Parent & Gopalakrishnan, 1995; Bahinski et al. 1997) may be different from those of L-type Ca²⁺ channels.

Comparison with the whole-cell current, I_st

The gating kinetics of i_st, as well as the activation by Bay-K 8644 or inhibition by nicardipine, compare well with the characteristics of the whole-cell current, I_st. The dynamic range of I_st activation was from -70 to -40 mV in SA node cells of rabbits and guinea-pigs (Guo et al. 1995, 1996a,b; Guo & Noma, 1997) or in rat (Shinagawa et al. 1997). It should be noted that the time constant of the first latency histogram ( > 150 ms) does not directly correspond to the rising phase of the whole-cell I_st activation. The fast components in both the open and closed time histograms have much larger weight than the slow component in determining the activation of whole-cell current. I_st was depressed by removing external Na⁺, but not by removing Ca²⁺, and yet showed pharmacological characteristics similar to those of the L-type Ca²⁺ channels. The current density may also be compared between i_st and I_st. At -60 mV (without Bay-K 8644), the channel open probability was 0·0047 and the unit amplitude was 0·98 pA. If the membrane area covered by the patch electrode tip of 0·8 µm in diameter is assumed to be 0·5 µm², the number of channels (0·36 in the absence of Bay-K 8644) per patch gives a current density of 3·32 × 10^-3 pA µm^-2, or 0·332 pA pF^-1 based on the unit membrane capacitance of 1 µF cm^-2. This value is within the cell-to-cell variation of I_st (0·1-1 pA pF^-1). Considering the tendency of channel run down in the single channel recording, it may be concluded that the current size is also comparable between i_st and I_st. Thus, the i_st channel discovered in the present study may underlie the whole-cell I_st.

Proof of the existence of a unique ion channel i_st in SA node pacemaker cells further supports the view of the ionic mechanisms underlying the slow diastolic depolarization. Namely, the voltage-dependent activation of I_st provided by the i_st channels may play a pivotal role in accelerating the depolarization, which is basically dependent on the voltage-dependent deactivation of the delayed rectifier K⁺ channels (I_Kr, HERG and/or I_Ks, K_VLQT channels). Activation of I_Ca,L may also occur during the diastolic depolarization as suggested by Verheijck et al. (1999). It should also be noted that the enhancement of I_st, as well as I_Ca,L, also supports the positive chronotropic effect of -adrenergic stimulation on the cardiac pacemaker.

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Acknowledgements

The authors thank Dr T. Powell for reading the manuscript and Kanako Fujita for her secretarial work. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture.

Corresponding author

T. Mitsuiye: Department of Physiology, Faculty of Medicine, Kyoto University, Sakyo-ku, Yoshida-Konoe, Kyoto 606-8501, Japan.

Email: tamo3{at}card.med.kyoto-u.ac.jp

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