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Pacemaker currents in mammalian nodal cells
j Mol Cell Cardiol 16, 777-781 (1984)
EDITORIAL REVIEW Pacemaker
Currents in Mammalian
In recent years, there has been a growing interest in the mechanisms underlying the pacemaker activity of cardiac muscle. At least four types of rhythmic activities m a y be defined according to their ionic mechanisms in various parts of the m a m m a l i a n heart. These are as follows.
Type 1: Spontaneous rhythms observed in sinoatrial (S-A) and atrioventricular (A-V) membrane potential node cells (low oscillation). W h e n the atrial cells are depolarized with a constant current, the cells also show repetitive action potentials E4]. In these cells, the major ionic currents responsible for the phase 4 depolarization are the decaying potassium currents (i~) and the increasing slow inward calcium currents (ica) [4, 11, 14"]. Type 2: Spontaneous rhythms observed in sheep Purkinje fibres (high m e m b r a n e potential oscillation). The diastolic depolarization of this cell has long been attributed to the decaying outward current (iK2), which is now reinterpreted as the increasing inward current (if) [7]. Type 3: Triggered activity observed under the condition of digitalis intoxication in ventricular and Purkinje cells. The origin of this type of rhythmic activity is due largely to the transient depolarization T D or the transient inward current TI. T h e intracellular accumulation of Ca m a y activate the non-selective cation channel [6, 16]. Type 4: Spontaneous activity induced by various pharmacological agents in ventricular, atrial and Purkinje cells. The current responsible in this type of r h y t h m is still not totally clear [5]. Due to the limited scope of this editorial review, we will discuss only the pacemaker mechanism of the primary pacemaker in the S-A and A-V node. 0022--~2828/84/090777+ 05 $03.00/0
Nodal Cells
The spontaneous change of the m e m b r a n e potential derives primarily from the time- and voltage-dependent nature of the conductance of the current systems. An increase in the conductance for the inward current system tends to depolarize the cell membrane, while a decrease in it hyperpola-rizes the membrane, and vice versa in the case of the conductance changes for the outward current system. In the nodal pacemaker cells, four current systems are known to have such a time- and voltage-dependent nature : the sodium current, iNa , the slow inward calcium current, ic, , the hyperpolarization activated current, ih, and the potassium current, i K . Because of the insensitivity of the pacemaker depolarization to T T X , i~a is not discussed in the present review.
Participation of the hyperpolarization-activated current (ih) W h e n the m e m b r a n e potential of the S-A node cell was clamped for the first tinae using the sucrose gap technique [12], a timedependent inward-going current change was recorded over the pacemaker potential range. Later, detailed analysis of this current change revealed two components, i.e. the decaying outward current system (i•) and the activating inward current system (ih) [2, 3, 9, 18, 39]. Meanwhile, the pacemaker current system iK2 observed in Purkinje fibres [21] was reinterpreted as the inward current system if [7]. The similarity of i h in the nodal cell to the pacemaker current if in the Purkinje fibre p r o m p t e d several investigators to examine the extent to which i h contributed to the pacemaker activity in the nodal cells [2, 3, 20]. Brown et al. [2] found an increase in i h during 9 1984 Academic Press Inc. (London) Limited
778
H. I r i s a w a a n d A. N o m a
the positive chronotropic effect induced by epinephrine, and proposed the hypothesis that the time-dependent activation of i h is an important mechanism of the pacemaker potential. T h e kinetic analysis of i h reported by Yanagihara and Irisawa [38], however, suggested that i h is not playing an essential role in the normal pacemaker activity of the sinus node. The reasons are as follows. T h e activation potential range is too negative (more negative than -- 60 to -- 70 mV) for the pacemaker potential. T h e time course of activation (r = 1 to 2 s) is too slow for the higher frequency of the primary pacemaker cell. Recently, Cs was shown to block i h selectively [9, 20] and the effect of Cs on the diastolic depolarization was examined. T h e spontaneous activity remains almost intact after blocking ih with Cs [17, 28]. The spontaneous activity was also recorded in cells which lacked the presence of i h [18, 253. Furthermore, Noma, M o r a d and Irisawa [28] blocked i h with Cs and yet were able to demonstrate the positive chronotropic effect of adrenaline. These observations all favour the notion that i h is not essential for the pacemaker activity of the nodal cell. The above arguments in nodal cells do not exclude the possibility that in Purkinje fibres, where the m a x i m u m diastolic potential is more negative than -- 70 mV, activation ofi e could cause the pacemaker activity.
Potassium current as a source of the slow diastolic depolarization
Trautwein and Ka~sebaum [34] showed in the frog sinus venosus that slow deactivation of the potassium conductance represented the major cause of the diastolic depolarization. The delayed K current was described first in 1976 in a small S-A node specimen [24] and later analysed more quantitatively [8, 39, 40]. Steady state activation ofi K (described by the kinetic variable p) suggests that Po0 activated at about - 5 0 m V becomes fully activated at - 1 0 mV. T h e time constant for the faster component of i K was longest (0.33 s) at --40 mV. W h e n these kinetics are incorporated into the Hodgkin-Huxley type model of pacemaker activity, the i K is activated by half during the action potential and causes the m e m b r a n e to hyperpolarize to the m a x i m u m
diastolic potential. After the m a x i m u m diastolic potential, p falls continuously to a m i n i m u m value of about 0.15 : thereby generating a decaying outward current, which contributes to the diastolic depolarization.
The slow inward
current
(ica)
W h e n iK was blocked using 5 m M B a , the m e m b r a n e depolarized to around - 1 0 m V and the spontaneous activity was stopped [39]. Following application of a constant hyperpolarizing current, rhythmic action potentials resumed. Later in a simulation study, N o m a et al. [27] reproduced this experiment well. Since the spontaneous activity can be reproduced after perfusing the S-A node specimen with both Ba and Cs, participation of i h in this spontaneous activity during hyperpolarization pulse could be neglected. T h e remaining time- and voltage-dependent current system under these conditions is ica. Thus, the kinetic change in ica appears to share with i K the essential role for the pacemaker depolarization. The question thus arises as to what extent ica contributes to the slow diastolic depolarization. If the above hypothesis is correct, the conductance for ica should increase in a timedependent m a n n e r during the diastolic period. In their mathematical model, Yanagihara et al. [40] attributed this conductance increase to the slow recovery of ica from its inactivation, which occurred during the preceding action potential (time-dependent increase of the kinetic variable f). Actually, the recovery time course Ofica proceeds with a time constant of 40 to 150 ms over the potential range of the diastolic depolarization [25]. T h e conductance of ica may be proportional to the product of the activation variables and the inactivation variables. Thus, the increase of the inactivation variable during diastole can cause an increase in the conductance provided that the activation variable is not zero [1, 10, 14]. I n order to examine at which potential the activation curve began to increase, the threshold potential of ica was examined employing a holding potential of --70 to --60 inV. The significant amplitude of the slow inward current was recorded with a depolarization to --55 to --60 m V [253 . T h e y concluded that the activation variable
Editorial Review began to increase at about --60 mV, and so ic, can contribute to the diastolic depolarization positive to --60 mV. However, these experiments were performed on multicellular preparations, and determination of the threshold potential in conventional preparations is severely limited by a possible nonhomogeneity of the potential distribution within the preparation, or by an incomplete suppression ofiNa with T T X . It is essential to evaluate this point in single cell voltage clamp experiments as well as patch clamp experiments. Recently, it has been strongly suggested that the inactivation of ic~ is at least partially mediated through Ca ions which pass through the channel [17, 35]. This finding raises the possibility that an intracellular mechanism is involved in determining the rate of the diastolic depolarization. It m a y be concluded that the pacemaker potential is not the result of activation or deactivation of only one ionic current system, but is attributable to both a decrease in the conductance for iK and an increase in the conductance for iCa. The mathematical model demonstrated that the amplitude of iK is almost constant or only slightly decreases during the diastolic period. This is because the driving force for i K increases during the diastolic depolarization and counterbalances the decrease in the conductance. T h e increase o f the conductance for ica is also partially neutralized by the decrease of the driving force. T h e amplitude of the net m e m b r a n e current is inward during the diastolic depolarization and is almost constant or only slightly increases with time as examined from the first differential of the m e m b r a n e potential change. T h e significant contribution of the leakage current cannot be ignored. However, the mechanisms underlying this background current are entirely speculative. The N a - K p u m p current, Ca p u m p i n g as well as N a - C a exchange mechanisms are all included as the background current in our model, but the precise amplitude and kinetics of these mechanisms remain unknown in nodal cells.
L o w r e s t i n g p o t e n t i a l in the n o d a l cell O n e of the c o m m o n phenomena of the pacemaker tissues is that they show a low mere-
779
brane potential. T h e resting potential can be seen when the nodal ceil becomes quiescent as a result of low temperature, depletion of Na § from the bathing solution, recovery from the acetylcholine effect, etc. A more accurate method for measuring the resting potential is to voltage clamp the potential and to determine the holding potential where no net current is observed in the steady state, i.e. zero current level. A value of around --40 m V has been obtained. Concerning the cause of this low resting potential, a high p N a : p K ratio in the nodal cell has been postulated [11]. Indeed, nodal cells reveal a remarkably constant resting potential even with a large variation in the external K concentration [23]. Recently, as one of the mechanisms for this low resting potential, the absence of an inward rectifier K channel in the nodal cell has been proposed [25]. I n the ventricular cell, the current voltage relationship shows a marked inward-going rectification (iK1) due to the presence of this channel, whereas in the nodal cell, inward-going rectification in the initial current is less pronounced. Another mechanism for the low m e m b r a n e potential is the presence of the inward current system, i,, which activates with strong hyperpolarization and causes the steady state I-V relation to bend in an inward direction negative to - 5 0 inV. Thus, whenever the membrane is hyperpolarized for a period which is sufficiently long to activate ih, i h drives back the m e m b r a n e potential to less negative potentials necessary for spontaneous activity [38]. Such a mechanism m a y be operating at the junctional region between the S-A node and the atrium or between the A-V node and the His bundle. Without an i h system in the nodal ceils, the m e m b r a n e of the pacemaker cell will be hyperpolarized due to the physiologically higher resting potential of the electrically coupled atrial or His bundle cells. When the m e m b r a n e potential is hyperpolarized by acetylcholine applied artificially or through vagal activities, the activation ofi h m a y play a role in buffering the ACh action.
N e w era in p a c e m a k e r s t u d i e s As studies on single isolated cardiac cells have developed, they have opened a new field of research in electrophysiology. Single cell
780
H. I r i s a w a and A. N o m a
studies offer a m o r e a c c u r a t e analysis o f the ionic c u r r e n t systems, since the c o n v e n t i o n a l s p e c i m e n had g e o m e t r i c a l c o m p l e x i t i e s a n d p r e s e n t e d l i m i t a t i o n s for analysis. S i n g l e cell studies also p r o v i d e a n o p p o r t u n i t y for the i n t r a c e l l u l a r m i l i e u to be c h a n g e d by i n j e c t i n g c e r t a i n c h e m i c a l substances [19] or by dialysis w i t h g i v e n solutions [13]. T h u s , t h e r e are several n e w findings w h i c h i n d i c a t e t h a t ic~ is s u p p o r t e d by the i n t r a c e l l u l a r m e t a b o l i c activities. I n c r e a s e in the i n t r a c e l l u l a r C a concentration reduces ic~ and causes C a m e d i a t e d i n a c t i v a t i o n . I n j e c t i o n of c a t a l y t i c s u b u n i t increases ic~ [30]. A T P also affects the iCa f r o m the i n t e r i o r of the cell [13]. T h e i n t r a c e l l u l a r m e c h a n i s m is therefore n o w b e l i e v e d to h a v e a close r e l a t i o n to the p a c e m a k e r m e c h a n i s m . F u r t h e r m o r e , using single cells,
the details of the single ionic c h a n n e l h a v e b e e n e x p l o r e d . W e are n o w able to discuss the p r o b a b i l i t y of o p e n i n g a n d closing o f the single C a c h a n n e l , a n d b y s u m m i n g up all events we c a n r e c o n s t r u c t the net C a c u r r e n t . T h e single c a l c i u m c h a n n e l has b e e n investig a t e d i n d e p e n d e n t l y in several l a b o r a t o r i e s [29, 30, 31, 37]. As r e g a r d s the K c h a n n e l , several K c h a n n e l s h a v e b e e n d e s c r i b e d such as the t i m e - a n d v o l t a g e - d e p e n d e n t K c h a n n e l , A T P sensitive K c h a n n e l w h i c h m a y be responsible at the t i m e of h y p o x i a [22, 33], A C h sensitive c h a n n e l [32], a n d i n w a r d rectifier K c h a n n e l [15, 36]. I t still r e m a i n s u n c e r tain w h i c h is the single c h a n n e l responsible for i K . N e v e r t h e l e s s , in a few years we c a n e x p e c t to e n t e r a n e w era in o u r u n d e r s t a n d i n g o f the pacemaker mechanism.
H. I r i s a w a a n d A. N o m a
National Institute for Physiological Sciences, Okazaka, 444 Japan
References 1 BEELER,G. W., REUTER, H. Reconstruction of action potential of ventricular myocardiac fibres. J Physiol 268, 177 210 (1977). 2 BROWN,H. F., DIFRANCESCO,D., NOBLE, S. J. How does adrenaline accelerate the heart? Nature [Lond] 280, 235 236 (1979). 3 BROWN,H. F., DIFRANCESCO,D. Voltage-clamp investigation of membrane currents underlying pace-maker activity in rabbit sino-atrial node. J Physio1308, 331 351 (1980). 4 BRow~, H. F. Electrophysiology of the sinoatrial node. Physiol Rev 62,505 530 (1982). 5 CARMF.LIET,E., VAN DER HEYDEN, G., VEREECKE. Spontaneous activity in cardiac ventricular cells. Proc Int Union Physiol Sci [Sydney] 15, 213, 03 (1983). 6 COLOQUHOUN,D., NEHER, E., REVTER,H., STEVENS,C. F. Inward current channels activated by intracellular Ca in cultured cardiac cells. Nature [Lond] 29't, 752 754 (1981). 7 DIFRANCESCO,D., NOBLE,D. Implications of the re-interpretation ofika for the modelling of the electrical activity of pacemaker tissues in the heart. In: (Eds) Bouman, L. N. and Jongsma, H.J. (Eds) CardiacRateand Rhythm,pp. 93-128 The Hague, Martinus NijhoffPublishers (1982). 8 DIFRANCESCO,D., NOMA,A., TRAU'rWEIN,W. Kinetics and magnitude of the time-dependent K current in the rabbit S-A node : effect of external potassium. Pfluegers Archiv 381, 271 279 (1979). 9 DIFRANCESCO,D., OJEDA, C. Properties of the 'Pacemaker' current, if, in the sinoatrial node of rabbit: a comparison with the current iK2 in Purkinje fibres, J Physiol 301), 353 367 (1980). 10 GINNEK~g,VAN A. C. G., JONGSMA,H. J. Slow inward current in aggregates of neonatal rat heart cells and its contribution to the steady state current-voltage relationship. Pfluegers Archiv 397, 265 271 (1983), 11 IRISAWA,H. Comparative physiology of the cardiac pacemaker mechanism. Physiol Rev 58, 461-498 (1978). 12 ImSAWA,H. Electrical activity of rabbit sino-atrial node as studied by the double sucrose gap method. In: Rijlant, P. (Ed) Brussels: Presses Academiques Europ~ennes (1972). 13 IRISAWA,H., KOKU~UN,S. Modulation by intracellular ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of the guinea-pig.J Physiol 3:38, 321-337 (1983). 14 IRISAWA,H., NOMA,A. Pacemaker mechanisms of tlae rabbit sinoatrial node cells. In: Bouman, L. N., Jongsma, H.J. (Eds) CardiacRateandRhythm,pp. 35-51 The Hague, Martinus Nijhoff. 15 KAME,ZAMA,M., KIYOSUF.,T., SOEJ1MA,M. Single channel analysis of the inward rectifier K current in the rabbit ventricular cells.JpnJ Physio133, 1039 1056 (1983). 16 KASS,R. S., TSlEN, R. W., WEINCART,R. Ionic basis of transient inward current induced by strophanthidin in cardiac Purkinje fibres. J Physio1281,209 226 (1978). 17 KIMURA,J. Electrophysiological studies of the sino-atrial node of the rabbit. Thesis, Oxford University (1982). 18 KOKU1aUN,S., NISHIMURA,M., NOMA,A., IRISAWA,H. Membrane currents in the rabbit atrioventricular node cell. Pfluegers Archiv. 393, 15 22 (1982).
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20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
38 39
40
78 l
KURACm, Y. The effects of intracellular protons on the electrical activity of single ventricular cells. Pfluegers Archly 394, 264 270 (1982). MAYLIE,J., MORAD, M., WEISS,J. A study of pace-maker potential in rabbit sino-atrial node: Measurement of potassium activity under voltage-clamp conditions.J Physio1311,161-178 (1981). NOBLE,D. The Initiation of the Heart Beat. Oxford, Clarendon Press (1975). NOMA,A. ATP-regulated K + channels in cardiac muscle. Nature 305, 147 148 (1983). NOMA,A., IRlSAWA,H. Effects ofNa + and K + on the resting membrane potential of the rabbit sinoatrial node cell. J p n J Physiol 25, 287 302 (1975). NOMA, A., IRISAWA, H. Time- and voltage-dependent potassium current in the rabbit sinoatrial node cell. Pfluegers Archly 366, 251-258 (1976). NOMA, A., IRISAWA, H. Ionic current in the isolated single S-A and A-V node cells. Proc Int Union Physiol Sci [Sydney] 15, 167, 08 (1983). NOMA, A., KOTAKI~, H., IRISAWA, H. Slow inward current and its role mediating the chronotropic effect of epinephrine in the rabbit sinoatrial node. Pfluegers Archiv. 388, 1 9 (1980). NOMA,A., KOTAKE, H., KOKUBUN,S. Kinetics and rectification of the slow inward current in the rabbit sinoatrial node cell.JpnJ Physio131, 491500 (1981). NOMA, A., MORAD, M., IRISAWA, H. Does the 'pacemaker current' generate the diastolic depolarization in the rabbit SA node cell. Pfluegers Archiv 397, 190-194 (1983). Ocm, R., I-hNo, N. Ca and Ba currents through single Ca channels in isolated guinea-pig ventricular cells. Proc Int Union Physiol Sci 15,486,06 (1983). OSTERRIDER,W., BRUM, G., HESCHELER,J., TRAUTWEIN, W., FLOCKERZEI,V., HOFMANN, F. Injection ofsubunits of cyclic AMP-dependent proteinkinase into cardiac myocytes modulates Ca 2+ current. Nature 298, 576 578 (1982). RE.UTER,H. Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301,569 574 (1983). SAKMANN,B., NOMA,A., TRAUTWE1N,W. Acetylcholine activation of single muscarinic K channels in isolated pacemaker cells of the mammalian heart. Nature 303, 250 -253 (1983). TANW,UCHI, J., NO~IA, A., IRISAWA, H. Modification of the cardiac action potential by intracellular injection of ATP and related substances in guinea-pig single ventricular cells. Circ Res 53, 131-139 (1983). TRAUTWEIN,W., KASSEBAUM,D. G. On the mechanism of spontaneous impulse generation in the pacemaker of the heart.J Gen Physio145, 317 330 (1961). TRAUTWEIN,W., TANtaUCHI,J., NOMA, A. The effect ofintracellular cyclic nucleotides and calcium on the action potential and acetylcholine response of isolated cardiac cells. Pfluegers Archiv 392, 307-314 (1982). TRUBE, G., HESCHELER,J. Potassium channels in isolated patches of cardiac cell membrane. Pfluegers Archly (Suppl) 396, R64 (1983). TSlEN, R. W., HEss, P,, BEAN, B. P., NOWYCK'z, M. C., LEE, K. S. Single Ca channels in ventricular heart cells: channel density, gating and unitary current as influenced by permeant ions blockers and adrenergic agonists. Proc Intern Union PhysioI Sci 15, 167,04 (1983). YANAGIHARA,K., IRISAWA,H. Inward current activated during hyperpolarizations in the rabbit sinoatrial node cell. Pfluegers Archly 385, 11 19 (1980). YANAGIHARA,K., IRISAWA,H. Potassium current during the pacemaker depolarization in rabbit sinoatrial node cell. Pfluegers Archiv 388, 255 260 (1980). YANAGIHARA,K., NOMA, A., IRISAWA,H. Reconstruction of sinoatrial node pacemaker potential based on the voltage clamp experiments.JpnJ Physio130, 841 857 (1980).
EDITORIAL REVIEW Pacemaker
Currents in Mammalian
In recent years, there has been a growing interest in the mechanisms underlying the pacemaker activity of cardiac muscle. At least four types of rhythmic activities m a y be defined according to their ionic mechanisms in various parts of the m a m m a l i a n heart. These are as follows.
Type 1: Spontaneous rhythms observed in sinoatrial (S-A) and atrioventricular (A-V) membrane potential node cells (low oscillation). W h e n the atrial cells are depolarized with a constant current, the cells also show repetitive action potentials E4]. In these cells, the major ionic currents responsible for the phase 4 depolarization are the decaying potassium currents (i~) and the increasing slow inward calcium currents (ica) [4, 11, 14"]. Type 2: Spontaneous rhythms observed in sheep Purkinje fibres (high m e m b r a n e potential oscillation). The diastolic depolarization of this cell has long been attributed to the decaying outward current (iK2), which is now reinterpreted as the increasing inward current (if) [7]. Type 3: Triggered activity observed under the condition of digitalis intoxication in ventricular and Purkinje cells. The origin of this type of rhythmic activity is due largely to the transient depolarization T D or the transient inward current TI. T h e intracellular accumulation of Ca m a y activate the non-selective cation channel [6, 16]. Type 4: Spontaneous activity induced by various pharmacological agents in ventricular, atrial and Purkinje cells. The current responsible in this type of r h y t h m is still not totally clear [5]. Due to the limited scope of this editorial review, we will discuss only the pacemaker mechanism of the primary pacemaker in the S-A and A-V node. 0022--~2828/84/090777+ 05 $03.00/0
Nodal Cells
The spontaneous change of the m e m b r a n e potential derives primarily from the time- and voltage-dependent nature of the conductance of the current systems. An increase in the conductance for the inward current system tends to depolarize the cell membrane, while a decrease in it hyperpola-rizes the membrane, and vice versa in the case of the conductance changes for the outward current system. In the nodal pacemaker cells, four current systems are known to have such a time- and voltage-dependent nature : the sodium current, iNa , the slow inward calcium current, ic, , the hyperpolarization activated current, ih, and the potassium current, i K . Because of the insensitivity of the pacemaker depolarization to T T X , i~a is not discussed in the present review.
Participation of the hyperpolarization-activated current (ih) W h e n the m e m b r a n e potential of the S-A node cell was clamped for the first tinae using the sucrose gap technique [12], a timedependent inward-going current change was recorded over the pacemaker potential range. Later, detailed analysis of this current change revealed two components, i.e. the decaying outward current system (i•) and the activating inward current system (ih) [2, 3, 9, 18, 39]. Meanwhile, the pacemaker current system iK2 observed in Purkinje fibres [21] was reinterpreted as the inward current system if [7]. The similarity of i h in the nodal cell to the pacemaker current if in the Purkinje fibre p r o m p t e d several investigators to examine the extent to which i h contributed to the pacemaker activity in the nodal cells [2, 3, 20]. Brown et al. [2] found an increase in i h during 9 1984 Academic Press Inc. (London) Limited
778
H. I r i s a w a a n d A. N o m a
the positive chronotropic effect induced by epinephrine, and proposed the hypothesis that the time-dependent activation of i h is an important mechanism of the pacemaker potential. T h e kinetic analysis of i h reported by Yanagihara and Irisawa [38], however, suggested that i h is not playing an essential role in the normal pacemaker activity of the sinus node. The reasons are as follows. T h e activation potential range is too negative (more negative than -- 60 to -- 70 mV) for the pacemaker potential. T h e time course of activation (r = 1 to 2 s) is too slow for the higher frequency of the primary pacemaker cell. Recently, Cs was shown to block i h selectively [9, 20] and the effect of Cs on the diastolic depolarization was examined. T h e spontaneous activity remains almost intact after blocking ih with Cs [17, 28]. The spontaneous activity was also recorded in cells which lacked the presence of i h [18, 253. Furthermore, Noma, M o r a d and Irisawa [28] blocked i h with Cs and yet were able to demonstrate the positive chronotropic effect of adrenaline. These observations all favour the notion that i h is not essential for the pacemaker activity of the nodal cell. The above arguments in nodal cells do not exclude the possibility that in Purkinje fibres, where the m a x i m u m diastolic potential is more negative than -- 70 mV, activation ofi e could cause the pacemaker activity.
Potassium current as a source of the slow diastolic depolarization
Trautwein and Ka~sebaum [34] showed in the frog sinus venosus that slow deactivation of the potassium conductance represented the major cause of the diastolic depolarization. The delayed K current was described first in 1976 in a small S-A node specimen [24] and later analysed more quantitatively [8, 39, 40]. Steady state activation ofi K (described by the kinetic variable p) suggests that Po0 activated at about - 5 0 m V becomes fully activated at - 1 0 mV. T h e time constant for the faster component of i K was longest (0.33 s) at --40 mV. W h e n these kinetics are incorporated into the Hodgkin-Huxley type model of pacemaker activity, the i K is activated by half during the action potential and causes the m e m b r a n e to hyperpolarize to the m a x i m u m
diastolic potential. After the m a x i m u m diastolic potential, p falls continuously to a m i n i m u m value of about 0.15 : thereby generating a decaying outward current, which contributes to the diastolic depolarization.
The slow inward
current
(ica)
W h e n iK was blocked using 5 m M B a , the m e m b r a n e depolarized to around - 1 0 m V and the spontaneous activity was stopped [39]. Following application of a constant hyperpolarizing current, rhythmic action potentials resumed. Later in a simulation study, N o m a et al. [27] reproduced this experiment well. Since the spontaneous activity can be reproduced after perfusing the S-A node specimen with both Ba and Cs, participation of i h in this spontaneous activity during hyperpolarization pulse could be neglected. T h e remaining time- and voltage-dependent current system under these conditions is ica. Thus, the kinetic change in ica appears to share with i K the essential role for the pacemaker depolarization. The question thus arises as to what extent ica contributes to the slow diastolic depolarization. If the above hypothesis is correct, the conductance for ica should increase in a timedependent m a n n e r during the diastolic period. In their mathematical model, Yanagihara et al. [40] attributed this conductance increase to the slow recovery of ica from its inactivation, which occurred during the preceding action potential (time-dependent increase of the kinetic variable f). Actually, the recovery time course Ofica proceeds with a time constant of 40 to 150 ms over the potential range of the diastolic depolarization [25]. T h e conductance of ica may be proportional to the product of the activation variables and the inactivation variables. Thus, the increase of the inactivation variable during diastole can cause an increase in the conductance provided that the activation variable is not zero [1, 10, 14]. I n order to examine at which potential the activation curve began to increase, the threshold potential of ica was examined employing a holding potential of --70 to --60 inV. The significant amplitude of the slow inward current was recorded with a depolarization to --55 to --60 m V [253 . T h e y concluded that the activation variable
Editorial Review began to increase at about --60 mV, and so ic, can contribute to the diastolic depolarization positive to --60 mV. However, these experiments were performed on multicellular preparations, and determination of the threshold potential in conventional preparations is severely limited by a possible nonhomogeneity of the potential distribution within the preparation, or by an incomplete suppression ofiNa with T T X . It is essential to evaluate this point in single cell voltage clamp experiments as well as patch clamp experiments. Recently, it has been strongly suggested that the inactivation of ic~ is at least partially mediated through Ca ions which pass through the channel [17, 35]. This finding raises the possibility that an intracellular mechanism is involved in determining the rate of the diastolic depolarization. It m a y be concluded that the pacemaker potential is not the result of activation or deactivation of only one ionic current system, but is attributable to both a decrease in the conductance for iK and an increase in the conductance for iCa. The mathematical model demonstrated that the amplitude of iK is almost constant or only slightly decreases during the diastolic period. This is because the driving force for i K increases during the diastolic depolarization and counterbalances the decrease in the conductance. T h e increase o f the conductance for ica is also partially neutralized by the decrease of the driving force. T h e amplitude of the net m e m b r a n e current is inward during the diastolic depolarization and is almost constant or only slightly increases with time as examined from the first differential of the m e m b r a n e potential change. T h e significant contribution of the leakage current cannot be ignored. However, the mechanisms underlying this background current are entirely speculative. The N a - K p u m p current, Ca p u m p i n g as well as N a - C a exchange mechanisms are all included as the background current in our model, but the precise amplitude and kinetics of these mechanisms remain unknown in nodal cells.
L o w r e s t i n g p o t e n t i a l in the n o d a l cell O n e of the c o m m o n phenomena of the pacemaker tissues is that they show a low mere-
779
brane potential. T h e resting potential can be seen when the nodal ceil becomes quiescent as a result of low temperature, depletion of Na § from the bathing solution, recovery from the acetylcholine effect, etc. A more accurate method for measuring the resting potential is to voltage clamp the potential and to determine the holding potential where no net current is observed in the steady state, i.e. zero current level. A value of around --40 m V has been obtained. Concerning the cause of this low resting potential, a high p N a : p K ratio in the nodal cell has been postulated [11]. Indeed, nodal cells reveal a remarkably constant resting potential even with a large variation in the external K concentration [23]. Recently, as one of the mechanisms for this low resting potential, the absence of an inward rectifier K channel in the nodal cell has been proposed [25]. I n the ventricular cell, the current voltage relationship shows a marked inward-going rectification (iK1) due to the presence of this channel, whereas in the nodal cell, inward-going rectification in the initial current is less pronounced. Another mechanism for the low m e m b r a n e potential is the presence of the inward current system, i,, which activates with strong hyperpolarization and causes the steady state I-V relation to bend in an inward direction negative to - 5 0 inV. Thus, whenever the membrane is hyperpolarized for a period which is sufficiently long to activate ih, i h drives back the m e m b r a n e potential to less negative potentials necessary for spontaneous activity [38]. Such a mechanism m a y be operating at the junctional region between the S-A node and the atrium or between the A-V node and the His bundle. Without an i h system in the nodal ceils, the m e m b r a n e of the pacemaker cell will be hyperpolarized due to the physiologically higher resting potential of the electrically coupled atrial or His bundle cells. When the m e m b r a n e potential is hyperpolarized by acetylcholine applied artificially or through vagal activities, the activation ofi h m a y play a role in buffering the ACh action.
N e w era in p a c e m a k e r s t u d i e s As studies on single isolated cardiac cells have developed, they have opened a new field of research in electrophysiology. Single cell
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H. I r i s a w a and A. N o m a
studies offer a m o r e a c c u r a t e analysis o f the ionic c u r r e n t systems, since the c o n v e n t i o n a l s p e c i m e n had g e o m e t r i c a l c o m p l e x i t i e s a n d p r e s e n t e d l i m i t a t i o n s for analysis. S i n g l e cell studies also p r o v i d e a n o p p o r t u n i t y for the i n t r a c e l l u l a r m i l i e u to be c h a n g e d by i n j e c t i n g c e r t a i n c h e m i c a l substances [19] or by dialysis w i t h g i v e n solutions [13]. T h u s , t h e r e are several n e w findings w h i c h i n d i c a t e t h a t ic~ is s u p p o r t e d by the i n t r a c e l l u l a r m e t a b o l i c activities. I n c r e a s e in the i n t r a c e l l u l a r C a concentration reduces ic~ and causes C a m e d i a t e d i n a c t i v a t i o n . I n j e c t i o n of c a t a l y t i c s u b u n i t increases ic~ [30]. A T P also affects the iCa f r o m the i n t e r i o r of the cell [13]. T h e i n t r a c e l l u l a r m e c h a n i s m is therefore n o w b e l i e v e d to h a v e a close r e l a t i o n to the p a c e m a k e r m e c h a n i s m . F u r t h e r m o r e , using single cells,
the details of the single ionic c h a n n e l h a v e b e e n e x p l o r e d . W e are n o w able to discuss the p r o b a b i l i t y of o p e n i n g a n d closing o f the single C a c h a n n e l , a n d b y s u m m i n g up all events we c a n r e c o n s t r u c t the net C a c u r r e n t . T h e single c a l c i u m c h a n n e l has b e e n investig a t e d i n d e p e n d e n t l y in several l a b o r a t o r i e s [29, 30, 31, 37]. As r e g a r d s the K c h a n n e l , several K c h a n n e l s h a v e b e e n d e s c r i b e d such as the t i m e - a n d v o l t a g e - d e p e n d e n t K c h a n n e l , A T P sensitive K c h a n n e l w h i c h m a y be responsible at the t i m e of h y p o x i a [22, 33], A C h sensitive c h a n n e l [32], a n d i n w a r d rectifier K c h a n n e l [15, 36]. I t still r e m a i n s u n c e r tain w h i c h is the single c h a n n e l responsible for i K . N e v e r t h e l e s s , in a few years we c a n e x p e c t to e n t e r a n e w era in o u r u n d e r s t a n d i n g o f the pacemaker mechanism.
H. I r i s a w a a n d A. N o m a
National Institute for Physiological Sciences, Okazaka, 444 Japan
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