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MS 0166 Received 28 September 1999; accepted after revision 23 December 1999.
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ABSTRACT |
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INTRODUCTION |
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Slow diastolic depolarization underlies the automaticity of the sino-atrial node (SAN) cells. Until recently, most ionic currents contributing to the slow diastolic depolarization have been identified and analysed extensively in both multicellular and single cell preparations (for review see Irisawa et al. 1993). The currents include the delayed rectifier K+ current (IK), the Na+-K+ pump current (Sakai et al. 1996), current due to the spontaneous opening of muscarinic K+ channels (Ito et al. 1994), L- and T-type Ca2+ currents (Hagiwara et al. 1988), hyperpolarization-activated cation current (If or Ih; DiFrancesco et al. 1986), background cation current (Hagiwara et al. 1992) and a dihydropyridine-sensitive sustained inward current (Guo et al. 1995). Using the conductance and gating parameters of individual current components, it is possible to reproduce the pacemaker activity in computer simulations (Yanagihara et al. 1980; Noble & Noble, 1984; Wilders et al. 1991). It should be noted, however, that most studies were carried out using rabbit hearts, and that the simulated action potentials only represent automaticity of the rabbit heart, the rate of which is approximately 200 beats min-1. It appears that there are substantial differences in current components among different species. For example, IK is largely attributed to the rapidly activating component of IK (IK,r) in rabbit SAN cells (Ito & Ono, 1995; Verheijck et al. 1995), whereas the slowly activating component of IK (IK,s) is a major component in guinea-pig SAN cells (Anumonwo et al. 1992). Since the conductance and gating properties of IK,r and IK,s are quite different (Sanguinetti & Jurkiewicz, 1991), their role in forming the spontaneous action potentials may differ depending on the species and heart rate.
In the present study, we isolated single pacemaker cells from porcine hearts. The porcine heart rate, ranging from 60 to 80 beats min-1 in resting conditions, is much slower than that of rabbit. We show that the major component of IK in porcine heart is IK,s. The possible contribution of IK,s to the action potentials of porcine SAN cells will be discussed.
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METHODS |
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Preparation
Porcine heart was obtained from a slaughterhouse near the laboratory. Six-month-old pigs weighing 90-100 kg were killed by electrical shock and exsanguination, and their intra-thoracic and abdominal organs were quickly removed. The heart was also quickly excised, and the right coronary artery was cannulated to enable perfusion with Ca2+-free Tyrode solution. Ventricular branches of the coronary artery were clamped in order to facilitate perfusion towards the right atrium including the SAN region. The heart was first perfused with 150 ml Ca2+-free Tyrode solution (approximately 30°C) using a disposable syringe and was cooled by subsequent perfusion with 250-300 ml cardioplegic solution (see below) at 4°C. Thereafter, the right atrial region enclosed laterally by the crista-terminalis and the interatrial septum was dissected out, and stored in cardioplegic solution at 4°C for transfer to the laboratory. Approximately 1 h elapsed before the following isolation procedure was commenced in the laboratory.
A piece of SAN tissue measuring approximately 20 mm × 5 mm was cut perpendicular to the crista-terminalis into about 20 strips. In some preparations, the SAN artery of 200-300 µm in diameter could be clearly identified under the stereo-microscope. In such cases, strips were made so as to include the SAN artery at their centre. The pieces of nodal tissue were incubated in Ca2+-free Tyrode solution for 5 min and then in the same solution containing 480 U ml-1 collagenase (Wako, Osaka, Japan), 0·1 mg ml-1 elastase (Boehringer, Germany), 10 U ml-1 protease (Type X; Sigma, St Louis, USA) and 3·5 U ml-1 hyaluronidase (Wako) for 45-50 min at 34°C. After the enzyme treatment, strips of SAN tissue were placed into high-K+, low-Cl- medium (modified KB medium) and gently teased apart using forceps and a glass pipette in a Petri dish. The cell suspension was stored in modified KB medium at 4°C for later use.
The protocols for isolating SAN cells described in this paper were previously approved by the Animal Committee, Akita University School of Medicine; the 'Guidelines for Animal Experimentation' of the University were completely adhered to in all subsequent experiments.
Solutions
Normal Tyrode solution contained (mM): NaCl, 136·9; KCl, 5·4; MgCl2, 0·53; CaCl2, 1·8; NaH2PO4, 0·33; glucose, 5·5; Hepes, 5·0 (pH = 7·4 with NaOH). Ca2+-free Tyrode solution was made by simply omitting CaCl2 from the above normal Tyrode solution. To block L-type Ca2+ current, 0·3 µM nisoldipine was added to the normal Tyrode solution. The cardioplegic solution contained (mM): NaCl, 82; KCl, 20; MgCl2, 0·5; glucose, 111·1; taurine, 20; Hepes, 10 (pH = 7·4 with NaOH), and was supplemented with 20 units l-1 bovine insulin (Wako). Modified KB medium contained (mM): potassium glutamate, 70; KCl, 20; KH2PO4, 10; MgCl2, 1·0; taurine, 20; glucose, 10; EGTA, 0·3 (pH = 7·2 with KOH).
The pipette solution for conventional whole-cell clamp experiments contained (mM): KOH, 150; HCl, 30; NaCl, 10; CaCl2, 2; EGTA, 5; Na2ATP, 5; Na2GTP, 0·1; MgCl2, 5; Hepes, 5·0 (pH = 7·2 with aspartic acid). The free concentrations of Ca2+ and Mg2+ were calculated to be 0·1 µM and 0·55 mM, respectively (Fabiato & Fabiato, 1979). In the perforated patch recording (Fig. 1), the pipette solution contained (mM): KCl, 30; potassium gluconate, 120; NaCl, 10; Hepes, 5·0 (pH = 7·2 with KOH). Nystatin (Sigma) was dissolved in methanol as a 10 mg ml-1 stock solution, and added to the pipette solution to give a final concentration of 200 µg ml-1 (Akaike & Harata, 1994).
E4031 was kindly donated by Eisai Pharmaceutical Co. and dissolved directly in the external solution. Chromanol 293B, a chromanol derivative, was supplied by Hoechst Co. (Frankfurt, Germany) and dissolved in dimethylsulfoxide as a 10 mM stock solution. Before the experiment, the stock solution was diluted in external solution to obtain the concentrations described in the text.
Electrophysiological experiments
Whole-cell recordings were performed using the technique originally described by Hamill et al. (1981). Patch electrodes were made of glass capillaries of 1·5 mm o.d. and resistance in the range 3-5 M
when filled with the internal solution. The currents were recorded with a patch-clamp amplifier (List EPC-7, Darmstadt, Germany). An agar-Tyrode-Ag-AgCl bridge was used as an indifferent electrode. Current and voltage signals were stored on digital audiotape (TEAC RD-101T, Tokyo, Japan) for subsequent computer analysis (NEC PC98Ap, Tokyo, Japan). All experiments were carried out at 35-37°C.
Results are expressed as means ± S.E.M. (n, number of cells). Comparisons between two groups were performed using Student's paired or unpaired t test and values of P < 0·05 were considered statistically significant.
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RESULTS |
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Action potentials of porcine SAN cells
Figure 1 shows a typical record of spontaneous activity recorded using the nystatin perforated patch-clamp technique. The overall electrical activity of porcine SAN cells was qualitatively similar to that recorded in rabbit preparations (Irisawa et al. 1993). Quantitatively, however, the action potential of the porcine SAN cells was characterized by a much slower spontaneous rate, pacemaker depolarization and upstroke, and by a longer duration, compared with that of the rabbit preparation. This is clearly demonstrated in Fig. 1B, where typical action potentials of rabbit SAN cells are shown superimposed on the same time scale. The spontaneous rate, measured in 11 cells, ranged between 47·8 and 118·3 min-1. The action potential parameters are summarized in Table 1. For comparison, the action potential parameters of rabbit SAN cells are also shown (Ono & Ito, 1995).
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A, top: original current trace on the chart recorder. The action potentials were recorded under the nystatin perforated clamp mode. The dashed line indicates the zero current level. For comparison, typical action potentials of rabbit SAN cells are shown in the bottom panel (data from Ono & Ito, 1995). The spontaneous rate was 80·9 min-1 for pig and 202·0 min-1 for rabbit. B, spontaneous action potentials shown on an expanded time scale. | ||
Table 1. Action potential parameters of the isolated porcine SAN cells
| Parameters | Pig | Rabbit |
| Cycle length (ms) | 781·7 ± 58·7 | 294·3 |
| Spontaneous rate (min-1) | 80·5 ± 5·4 | 203·9 |
| Amplitude (mV) | 75·1 ± 2·4 | 95·8 |
| Vmax (V s-1) | 1·57 ± 0·15 | 10·3 |
| APD50 (ms) | 196·6 ± 19·9 | n.d. |
| MDP (mV) | -58·4 ± 1·9 | -70·7 |
| DDR (mV s-1) | 34·4 ± 2·4 | 68·7 |
| Reference | Present study | Ono & Ito (1995) |
Under conventional whole-cell voltage-clamp conditions using the standard internal solution and Tyrode solution, square pulses of 500 ms duration were applied from a holding potential of -50 mV (Fig. 2). Upon depolarization a transient inward current due to L-type Ca2+ current was followed by a slowly activating delayed rectifier K+ current (IK). The activation of IK is evident as a decaying tail current on repolarization to -50 mV. Hyperpolarization activated a slowly activating inward current, If. The current-voltage (I-V) relationships measured at the initial peak (
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) of the pulses are shown in Fig. 2B. The configuration of the I-V curve for the initial current was in rough agreement with that reported in rabbit SAN cells (Irisawa et al. 1993; Ito & Ono, 1995). For the late current, however, there was a difference between the two species: the I-V curve of rabbit SAN cells showed upward curvature between -30 and +20 mV (Irisawa et al. 1993; Ito et al. 1994; Ono & Ito, 1995) whereas the late current in porcine SAN cells increased monotonically as the test potential was increased (Fig. 2B). These findings indicate that the properties of IK may differ between rabbit and porcine SAN cells.
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A, whole-cell current of a SAN cell in normal Tyrode solution. Traces shown were obtained by applying 500 ms depolarizing (upper traces) or hyperpolarizing (lower traces) pulses from a holding potential of -50 mV in 10 mV increments. Dashed lines indicate the zero current level. B, I-V relationships for the initial current ( | ||
To examine the properties of IK, long depolarizing pulses were applied during block of the L-type Ca2+ current by 0·3 µM nisoldipine (Fig. 3A). In response to a 3 s depolarizing pulse, IK was activated gradually with little saturation during the pulse. In Fig. 3B, the current densities of the initial () and late () current measured in eight cells are plotted against the test pulses (Vt). In Fig. 3C, the amplitude of the tail current is plotted against Vt. The relationship was well fitted by the Boltzmann equation:
where V½ is the half-activation potential and S is a slope factor. The values of V½and S were determined by a least-squares fit; they were 20·5 ± 2·1 mV and 16·4 ± 1·2 mV, respectively (n = 8).
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A, whole-cell current in external solution containing 0·3 µM nisoldipine. The holding potential (Vh) was -40 mV and 3 s depolarizing pulses were applied in 10 mV increments. Vt, test potential. B, I-V relationships for the initial current ( | ||
Envelope test
The time course of development of the tail current compared with that of the activated outward IK provides a clue to the relative contribution of IK,r and IK,s to net IK (Sanguinetti & Jurkiewicz, 1990). In the experiment shown in Fig. 4A, the duration of the test pulse at +60 mV (upper traces) and +10 mV (lower traces) was varied. As the duration of the test pulse was prolonged, the amplitude of the tail current increased in proportion to that of the time-dependent outward current activated during depolarization. Data obtained from five cells for +60 mV and six cells for +10 mV are summarized in Fig. 4B, where the tail amplitude (Itail) is plotted against the activated outward current upon depolarization (Iout). Iout was measured as the time-dependent current during the step depolarization. A linear relationship is evident between Itail and Iout during the entire course of the pulses in each cell examined. The slope of the linear regression line was 0·50 ± 0·05 for +60 mV and 0·56 ± 0·02 for +10 mV. These findings indicate a single population of IK in porcine SAN cells.
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A, tail currents at -40 mV were recorded by changing the duration of the test depolarization to +60 mV (upper traces) and +10 mV (lower traces). B, relationship between the amplitude of the tail current (Itail) and the activated current (Iout) during the depolarizing pulses. Different symbols indicate different cells. | ||
Pharmacological properties of porcine SAN cell IK
In rabbit SAN cells, IK was markedly inhibited by E4031, a selective IK,r blocker (Ito & Ono, 1995; Ono & Ito, 1995; Verheijck et al. 1995). By contrast, the drug hardly affected the IK of porcine SAN cells. To monitor the amplitudes of both IK,r and IK,s, a pair of 3 s depolarizing pulses to +10 and +60 mV were applied every 60 s. After observation of the control IK, 2 µM E4031 was applied. Little or no change in the current configuration was observed at both +10 and +60 mV (Fig. 5A). In fact, when the E4031-sensitive current was calculated by subtracting the current in the presence of E4031 from the control current, there was little or no current during the depolarization step or upon repolarization to -40 mV (Fig. 4B). In five cells, the mean amplitude of the activated outward current was 101·0 ± 7·8 pA at +10 mV and 696·2 ± 70·1 pA at +60 mV in the control, and 94·3 ± 7·8 pA at +10 mV and 665·6 ± 70·6 pA at +60 mV in the presence of 2-5 µM E4031. The amplitude of the tail current was 47·5 ± 3·9 pA at +10 mV and 273·6 ± 24·7 pA at +60 mV in the control, and 45·8 ± 4·0 pA at +10 mV and 262·0 ± 24·7 pA at +60 mV in the presence of 2-5 µM E4031. These differences were not statistically significant.
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A, superimposed currents in response to 3 s test pulses to +60 and +10 mV recorded in the absence and presence of 2 µM E4031. The holding potential was -40 mV and L-type Ca2+ current was blocked by 0·3 µM nisoldipine. B, E4031-sensitive current obtained by subtracting the currents in the presence of E4031 from control currents. In A and B, the dashed line indicates the zero current level. | ||
It has been shown that chromanol 293B, a chromanol derivative, selectively inhibits IK,s in guinea-pig ventricular cells without affecting IK,r (Busch et al. 1996). In the experiment shown in Fig. 6, whole-cell outward currents were compared before (Fig. 6A) and during (Fig. 6B) application of 30 µM chromanol 293B. In the presence of 30 µM chromanol 293B, no obvious time-dependent current was recorded over the potential range -30 to +60 mV, and IK tail current was almost completely blocked. These findings may support the view that IK,r is negligibly small in this preparation. The chromanol 293B-sensitive current was obtained by digitally subtracting the current under the effect of chromanol 293B from the control current at each potential (Fig. 6C). The amplitude became larger as the depolarizing pulse was increased, and rectification was not detected over the voltage range examined.
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Whole-cell current in the control external solution (A) and in the presence of 30 µM chromanol 293B (B). The holding potential was -40 mV and 3 s depolarizing pulses were applied in 10 mV increments. C, chromanol 293B-sensitive current obtained by subtracting the currents in the presence of chromanol 293B from control currents. D, relationship between the amplitude of the tail current and the concentration of chromanol 293B. The amplitude of the tail current was normalized to that recorded in the absence of chromanol 293B. The smooth curve is the best fit with the Hill equation: I = 1/(1 + ([Drug]/IC50)nH), where IC50 and nH are the half-inhibitory concentration and the Hill coefficient, respectively. The values of IC50 and nH were 8·79 µM and 1·22, respectively. In A-C, the dashed line indicates the zero current level. | ||
The concentration-response relationship was examined in 18 cells and the results are shown in Fig. 6D. The relationship was fitted by the Hill equation:
where IC50 and nH are the half-inhibitory concentration and the Hill coefficient, respectively. The values of IC50 and nH were 8·79 µM and 1·22, respectively. The IC50 value was slightly larger than that reported in guinea-pig ventricular cells (IC50 = 2·1 µM; Busch et al. 1996).
Ion selectivity of IK
The ion selectivity of IK was examined by measuring the reversal potential (Vrev) of the tail current. In the experiment shown in Fig. 7A, the membrane was first depolarized to +60 mV for 3 s and then repolarized to various test voltages. The initial () and late () currents recorded during the second pulse were plotted against membrane potential, except at potentials more negative than -70 mV where the currents before If activation were plotted as the late current. Vrev, determined as the intersection of these relationships, was -79 mV in this experiment. The mean Vrev, determined in four cells in standard external solution (5·4 mM [K+]o), was -75·2 ± 2·3 mV. In Fig. 7B, summarized results of Vrev measured at variable [K+]o are shown. The linear regression line of the Vrev-log[K+]o relationship had a slope of 52·0 mV per tenfold change in [K+]o. The value was obviously smaller than that for a K+-selective electrode expected from the Nernst equation (dotted line in Fig. 7B), indicating that IK in porcine SAN cells is sensitive to, but not completely selective for, K+.
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A, left: current traces obtained using the double pulse protocol shown at the top. The dotted line indicates the zero current level. Right: I-V relationships for the initial ( | ||
Time constants
The kinetics of chromanol 293B-sensitive IK were analysed. The time course of activation in response to depolarizing pulses was fitted with either a single or double exponential function. The data were better fitted by the sum of two exponential functions:
,f(1 - exp(-t/
f)) + I,s(1 - exp(-t/s)) + I0,
where I is a free parameter, I0 is the instantaneous current, and f and s are the fast and slow activation time constants, respectively. It should be noted that, in previous studies, the onset kinetics of the cardiac delayed rectifier K+ current exhibited significant sigmoidicity (Matsuura et al. 1987; Balser et al. 1990). However, such sigmoidicity was not obvious in the chromanol 293B-sensitive current of porcine SAN cells, as illustrated in Fig. 6C.
The deactivating tail current was obtained using the double pulse protocol (see Fig. 7). The tail current was also fitted with the sum of two exponential functions, except at potentials more negative than -60 mV where a single exponential function was sufficient. Data obtained from five cells are summarized in Fig. 8. Both the slow and fast time constants were clearly dependent on the membrane voltage and, when plotted against membrane potential, showed a bell-shaped curve with a maximum around -10 mV. The slope was much steeper at negative potentials whereas both time constants showed only weak voltage dependency at potentials more positive than +10 mV.
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A, time constants for slow (filled symbols) and fast (open symbols) components plotted against membrane potential. Activation phases (circles) were recorded during depolarization for 3 s from -40 mV to various potentials, while deactivation phases (squares) were recorded by repolarization from +60 mV. Each phase was fitted with the sum of two exponentials. Data are means ± S.E.M. of 5 experiments. Inset, original current traces. The dashed line indicates the zero current level. B, relative amplitude of the fast component (F/(F + S)) plotted against membrane potential. S, slow component. Data obtained from activation ( | ||
Effect of chromanol 293B on the spontaneous action potentials
Complete block of IK,s by chromanol 293B abolished the spontaneous activity of porcine SAN cells. In the experiment shown in Fig. 9, the action potential was recorded using the nystatin-perforated patch-clamp method. After confirming regular action potentials, 10 and 30 µM chromanol 293B was cumulatively applied, causing complete cessation of the action potential in a reversible manner. Essentially similar results were obtained in three other cells.
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A, after obtaining a stable firing of action potentials in a nystatin-perforated patch, 10 and 30 µM chromanol 293B was superfused into the bath. The bar indicates the period of drug application. B, traces at the times indicated in A shown on an expanded time scale. | ||
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DISCUSSION |
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IK,s is characterized as a slowly activating IK; the activation threshold is around -20 mV and V½ is about +10 mV. The time course of activation and deactivation requires several seconds and the fully activated I-V relationship shows little rectification. By contrast, IK,r is activated at more negative potentials (V½ is around -25 mV) and shows strong inward rectification (Sanguinetti & Jurkiewicz, 1990; Ono & Ito, 1995). These currents can also be distinguished pharmacologically. IK,r is specifically blocked by class III antiarrhythmic agents (Sanguinetti & Jurkiewicz, 1990), whereas IK,s is inhibited by chromanol 293B (Busch et al. 1996). In the present study, we have demonstrated that IK of porcine SAN cells possesses electrophysiological and pharmacological properties quite similar to those of IK,s. The reversal potential of -75 mV, measured with 5·4 mM external and 150 mM internal K+, is 
10 mV positive to the equilibrium potential expected for a K+-selective electrode from the Nernst equation. This is consistent with reports that IK,s is sensitive to, but not completely selective for, K+ (Matsuura et al. 1987; Sanguinetti & Jurkiewicz, 1990; Anumonwo et al. 1992). Furthermore, block of IK,s inhibited the spontaneous action potentials of porcine SAN cells. All these findings indicate that not only is IK,s the dominant outward K+ current, but also that it plays a functional role in the pacemaker activity of porcine SAN cells.
The isolated porcine SAN cells exhibited spontaneous activity of which the spontaneous rate was similar to the native heart rate of pigs, indicating that the mechanism underlying different heart rates among various species can be attributed to intrinsic properties of isolated SAN cells in individual species. The action potentials of porcine SAN cells showed a relatively slower upstroke, longer duration and slower diastolic depolarization compared with those of rabbit SAN cells (Table 1). The slow upstroke indicates that the density of the Ca2+ current might be low, or that the property of the Ca2+ current might be different from that of rabbit SAN cells which usually show the maximal upstroke, around 10 V s-1 (see Table 1; Wilders et al. 1991; Ono & Ito, 1995). On the other hand, the relatively longer duration of the action potential and slower diastolic depolarization of porcine SAN cells than those of rabbits may be brought about, at least in part, by the difference in IK kinetics between the two species. The functional role of time-dependent outward current, IK, in the pacemaker potential has long been suggested (Irisawa et al. 1993). Namely, the activation of IK during the preceding action potential drives the membrane towards repolarization, and IK deactivation triggers the slow diastolic depolarization after the maximum diastolic potential (Irisawa et al. 1993). In the case of rabbit SAN cells, IK,r has been shown to play this active role (Ono & Ito, 1995; Verheijck et al. 1995). Application of chromanol 293B to the rabbit SAN cells did not affect spontaneous activity (Lei et al. 1997). This finding indicates that IK,s, which has been reported to exist in rabbit SAN cells (Ono & Ito, 1995; Lei & Brown, 1996), cannot participate in regulating the spontaneous action potentials of rabbit SAN cells, probably due to its slower gating and relatively higher threshold compared with IK,r. On the other hand, the present study has demonstrated that chromanol 293B inhibited the spontaneous action potentials in porcine SAN cells in a reversible manner. This effect is not likely to be due to possible block of L-type Ca2+ current by the drug, since chromanol 293B selectively inhibited IK,s in human and guinea-pig ventricular cells without affecting L-type Ca2+ current and Na+ current (Bosch et al. 1998). Also, If of porcine SAN cells was not affected by chromanol 293B (K. Ono & T. Iijima, unpublished data). Thus our results indicate that IK,s is required for generating pacemaker activity in porcine SAN cells. Furthermore, IK,s is the only time-dependent outward current that can drive the membrane towards the maximum diastolic depolarization after the peak of the action potential. The longer action potential duration of pigs may enable IK,s to replace the role of rabbit IK,r in porcine SAN cells, thereby causing a slower heart rate compared with that of rabbits.
Although we failed to record IK,r in porcine SAN cells which showed little striation and were spindle shaped, IK,r did exist in large, rod-shaped quiescent cells in the same batches of preparation (authors' unpublished data). IK,r and IK,s may be distributed heterogeneously in the SAN region depending on the cell shape. Heterogeneous distribution of IK,r or IK,s has been suggested in various species. Recent molecular studies have revealed that the minK channel (also termed IsK), which is considered to form IK,s channels along with another K+ channel subunit, KvLQT1 (Barhanin et al. 1996; Sanguinetti et al. 1996a), is expressed in the SAN region of the guinea-pig (Freeman & Kass, 1993a), ferret (Brahmajothi et al. 1996) and mouse (Kupershmidt et al. 1999). On the other hand, mRNA of ERG, which forms the IK,r channel (Sanguinetti et al. 1996b) together with another subunit, MiRP1 (Abbott et al. 1999), is substantially expressed in the SAN region of the rabbit heart (Wymore et al. 1997) and in the crista-terminalis of ferret heart including the SAN region (Brahmajothi et al. 1997). Thus, the distribution of IK,s and IK,r channels varies depending on the species, and could be potentially related to the different spontaneous rate of SAN cells in various species. In this respect, it would be interesting to determine the expression and distribution of various K+ channels including ERG, minK, MiRP1 and KvLQT1 in the SAN region of porcine hearts.
Apart from the molecular analysis described above, direct measurement of membrane currents from mammalian SAN cells has been performed in rabbits (for review see Irisawa et al. 1993), guinea-pigs (Anumonwo et al. 1992; Guo et al. 1997) and pigs (present study). The different kinetics of IK,s and IK,r may, in part, account for the difference between the heart rate of rabbits (approximately 200 beats min-1) and pigs (80 beats min-1). It is interesting to note that IK of guinea-pig SAN cells is also derived from IK,s, not IK,r, despite a marked difference in the spontaneous rate compared with that of porcine SAN cells (Anumonwo et al. 1992; Guo et al. 1997). It should be noted that the properties of IK,s appear to be different, depending on the mammalian species. The half-activation voltage was +32 mV in guinea-pig (Anumonwo et al. 1992) and +20 mV in porcine SAN cells (Fig. 3). The relationship between fast and slow time constants and the membrane potential showed a clear bell-shaped curve peaking near 0 to +20 mV in guinea-pig SAN cells. By contrast, the voltage dependence of porcine IK,s was not obvious at potentials more positive to +10 mV (Fig. 8A). Furthermore, the values for fast and slow time constants are generally slower for porcine IK,s (present study) than for IK,s of guinea-pig SAN cells (Anumonwo et al. 1992). These properties might be beneficial for forming a longer action potential duration in porcine SAN cells, compared with that in guinea-pig. In addition to the different gating properties of IK,s, the current density may also provide an important variable for determining the contribution of IK,s to the action potential in various species. The current density of IK,s is only 8 pA pF-1 at +60 mV in porcine SAN (Fig. 3), whereas Anumonwo et al. (1992) reported a current size of approximately 1 nA at +80 mV in guinea-pig SAN cells which have cell capacitance of less than 10 pF. Such a large current density might be required for IK,s to play a functional role in the spontaneous activity of guinea-pig SAN cells.
It should also be noted that the properties of IK,s do not only depend on the species but also on the cell type in the same heart. Anumonwo et al. (1992) reported that the time constants for activation and deactivation of IK,s were faster in guinea-pig SAN cells than those in ventricles. Freeman & Kass (1993b) reported that regulation of IK,s in guinea-pig SAN cells is not temperature dependent, in contrast to ventricular IK,s in which regulation by kinases A and C was diminished by lowering the temperature (Walsh & Kass, 1988). Non-stationary fluctuation analysis predicted a single channel conductance of 6 pS in the SAN IK,s channel, which is larger than that estimated for ventricular IK,s channels of guinea-pigs (Freeman & Kass, 1993b).
Possible contributing factors that could explain the differences in IK,s kinetics among various species and cell types described so far remain to be elucidated. One possibility is that primary structure (KvLQT1 and/or minK) and the resulting kinetic properties of the IK,s channel may differ depending on the species. A second possibility may be related to previous observations that coexpression of minK with KvLQT1 slowed activation kinetics without much alteration in the deactivation process, decreased the single channel amplitude of KvLQT1 (Romey et al. 1997), and slowed the blocking rate of KvLQT1 by chromanol 293B (Loussouarn et al. 1997). Romey et al. (1997) proposed that a tetramer of KvLQT1 can bind one to four minK subunits depending on the concentration of minK in the membrane, thereby producing the complex kinetic behaviour of IK,s channels. It might be speculated that the stoichiometry of minK in the IK,s channel complex is different in various regions of the heart. Finally, there may be KvLQT1 isoforms that have different gating properties or serve various functions, like that reported in human heart (Jiang et al. 1997).
Not only IK but also various other ionic channels are important in controlling the rate of diastolic depolarization of SAN cells. In rabbit SAN cells, the currents involved include T- and L-type Ca2+ current (Hagiwara et al. 1988), background cation current (Hagiwara et al. 1992), If (DiFrancesco et al. 1986), sustained inward current (Guo et al. 1995), Na+-K+ pump current (Sakai et al. 1996) and ionic current due to spontaneous opening of muscarinic K+ channels (Ito et al. 1994). Shibata et al. (1999) have demonstrated recently that the activation kinetics of If in porcine SAN cells are quite similar to those of rabbit. As for other current systems, however, little is known of whether gating property and/or current density of these currents are different between rabbits and pigs. Further studies are clearly necessary to elucidate the mechanism underlying the pacemaker activity in porcine SAN cells.
In conclusion, we have recorded for the first time the spontaneous action potentials of porcine SAN cells, and demonstrated that IK,s plays a major role in the membrane repolarization of these cells. The difference in heart rate between various species, which might be caused by differences in current components generating the pacemaker potential and/or the current density of various ionic channels in SAN cells, should be given attention for understanding the pacemaker mechanism of the mammalian heart, including that of humans.
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REFERENCES |
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We thank Dr A. Shinbo for the initial work of this study and Dr K. Yazawa for reading the manuscript. This work was supported by grants from the Ministry of Education, Science Sports and Culture of Japan, and in part from the Naitoh Memorial Foundation.
Corresponding author
T. Iijima: Department of Pharmacology, Akita University School of Medicine, Hondoh 1-1-1, Akita 010-8543, Japan.
Email: tiijima{at}med.akita-u.ac.jp
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