Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility.

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

Modulation of delayed rectifier potassium current, iK, by isoprenaline in rabbit isolated pacemaker cells.

Permeabilized patch whole-cell voltage clamp methods were used to investigate the effects of isoprenaline (ISO) on total delayed rectifier potassium current, iK, in rabbit sino-atrial (SA) node pacemaker cells; total iK is composed of the rapidly activating iKr and the slowly activating iKs, but predominantly iKr in this species. ISO (20 nM) increased the amplitude of total iK and caused a negative shift of approximately 10 mV in the activation curve for iK, both in the absence and in the presence of 300 nM nisoldipine to block the L-type Ca2+ current, iCa,L. The same concentration (20 nM) of ISO increased the spontaneous pacemaker rate of SA node pacemaker cells by 16%. In addition to increasing the amplitude of iK, ISO (20-50 nM) also increased the rate of deactivation of this current. The stimulation of iK by ISO was reversed by 10 microM H-89, a selective protein kinase A inhibitor, but not by 200 nM bisindolymaleimide I, a selective protein kinase C inhibitor. It therefore appears that the mechanisms by which -adrenoceptor agonists increase pacemaking rate in sinoatrial node pacemaker cells include an increase in the rate of deactivation of iK in addition to the well-documented augmentation of iCa,L and the positive shift of the activation curve for the hyperpolarization-activated inward current, if. The observations are also consistent with a role for protein kinase A in the stimulation of iK by ISO in SA node cells.

Inhibition by Compound II, a sotalol analogue, of delayed rectifier current (iK) in rabbit isolated sino-atrial node cells.

The effects of Compound II, a sotalol analogue, on spontaneous electrical activity and on three membrane currents (the delayed rectifier current, iK, the long-lasting inward calcium current, i(Ca,L) and hyperpolarization activated inward current, i(f)) were investigated in rabbit isolated sino-atrial node cells by whole cell clamp with amphotericin-permeabilised patches. A submaximal concentration of Compound II (50 nM) had a significant effect on the time and voltage dependent activation of iK and caused a positive shift of the iK activation curve. As well as blocking i(Kr), it caused some degree of block of i(Ks). Block of iK by Compound II was found to be concentration dependent with an IC50 of approximately 40 nM. 1 microM Compound II nearly completely blocked iK without significantly affecting the peak current or I/V relationships of i(Ca,L) or i(f). 50 nM Compound II caused a significant prolongation of APD100 and of cycle length. It also decreased diastolic depolarization rate without significantly affecting MDP and action potential amplitude. It is concluded that Compound II, a sotalol analogue, slows spontaneous activity of isolated rabbit SA node cells through a selective inhibition of iK.

Nitric oxide can increase heart rate by stimulating the hyperpolarization-activated inward current, I(f).

We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n = 8) or 3-morpholinosydnonimine (SIN-1, n = 6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n = 6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 mumol/L, n = 6) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 mumol/L, n = 6), and (3) mimicked by 8-bromo-cGMP (n = 6) with no additional positive chronotropic effect of SIN-1 (n = 5). The response to 10 mumol/L SNP (n = 28) or 50 mumol/L SIN-1 (n = 16) was unaffected by IcaL antagonism with nifedipine (0.2 mumol/L) but was abolished after blockade of the hyperpolarization-activated inward current (I(f)) by Cs+ (2 mmol/L) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 mumol/L). The effect on I(f) was further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n = 21), where we found that 5 mumol/L SNP or SIN-1 caused a reversible Cs(+)-sensitive increase in this current (+130% at -70 mV and +250% at -100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of I(f) via the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO.

Two components of the delayed rectifier potassium current, IK, in rabbit sino-atrial node cells.

The delayed rectifier current was studied in rabbit isolated sino-atrial (SA) node cells using the whole-cell voltage clamp technique with amphotericin-permeabilized patches. The envelope of tails test indicated that in SA node cells the decay of IK (IK.tall) comprises two distinct current components similar to the specific fast and slow components of IK (IKr and JKs) that have been found in atrial and ventricular myocytes. Dofetilide, a Class III antiarrhythmic agent and a selective blocker of IKr. separated the delayed rectifier current into drug-sensitive current (IKr) and drug-insensitive current (IKs). The dofetilide-sensitive current activated rapidly and it showed two components of deactivation, the larger of which was very slow, while the dofetilide-insensitive current activated more slowly and deactivated quickly. The effects of propofol, an IKs blocker, on the delayed rectifier current were also investigated. The results show that IKs contributes to IK.tall and that the more positive the membrane potential, the more the contribution of IKs. The ratio of IKs to IKr in tail currents at -40 mV after a 1 s clamp pulse to +40 mV was 0.3-0.4:1. Dofetilide slowed spontaneous activity, suggesting that IKr contributes to the pacemaker activity of the SA node cell.

Voltage- and time-dependent block of delayed rectifier K+ current in rabbit sino-atrial node cells by external Ca2+ and Mg2+.

1. The properties of the delayed rectifier K+ current (IK) of rabbit isolated sino-atrial node cells were investigated in high (140 mM) [K+]o using the whole-cell-clamp technique. 2. Hyperpolarizing clamp pulses from 0 mV induced an instantaneous current jump (I-V relation linear) followed by a time-dependent increase in inward current to a peak, whereas depolarizing clamp pulses induced little outward current. The peak I-V relation showed a strong inward rectification. The inwardly rectifying current was blocked by E-4031. 3. The inward K+ current induced by hyperpolarizing clamp pulses from 0 mV relaxed after reaching its peak. The rate of the relaxation increased as the membrane potential became more negative and concentrations of external Ca2+ or Mg2+ were increased. The steady-state current was smaller as the relaxation of the current accelerated on increasing [Ca2+]o or [Mg2+]o. 4. Depolarizing clamp pulses from -80 mV induced an increase in inward current, reaching a steady state. The amplitude of the steady-state current became smaller and the rate of current increase became slower as [Ca2+]o or [Mg2+]o was increased. 5. The effects of Ca2+ and Mg2+ are well explained by a time- and voltage-dependent blockade of the K+ channel by these ions. The fractional electrical distance of the binding site calculated from the voltage dependence of the blocking rate constant is 0.69 for Ca2+ and 0.88 for Mg2+. The blocking rate constant at 0 mV for Ca2+ is about 15 times faster than that for Mg2+, indicating stronger effects of Ca2+. 6. A re-interpretation of IK in sino-atrial node cells is proposed: there are two independent gates (an activation gate which opens on hyperpolarization and an inactivation gate which closes on hyperpolarization) and a binding site for Ca2+ and Mg2+ inside the channel. Binding of these ions, which is facilitated by hyperpolarization, causes channel blockade, resulting in the observed voltage dependence of IK in physiological concentrations of Ca2+ and Mg2+.

Effect of raised extracellular calcium on characteristics of the guinea-pig ventricular action potential.

The whole-cell patch-clamp method was used to investigate the role of Na/Ca exchange current (INa,Ca) in the shortening action of raised extracellular calcium on the ventricular action potential. Experiments were performed either using BAPTA to buffer intracellular calcium, or by replacing extracellular Na+ with Li- to abolish INa,Ca. A blocker of Ik, compound II. was used to investigate whether changes in this current may also play a role. Raising extracellular calcium from 1.8 mM to 5.0 mM increased the amplitude of the action potential by 8% and decreased its duration (at 90% repolarization, APD90) by 23%. Compound II increased APD90 by 40% and resulted in the appearance of early afterdepolarizations but did not affect the response to raised extracellular calcium. Intracellular BAPTA (20 mM) did not prevent the calcium-induced shortening but did abolish the initial rapid phase of repolarization and increase the inactivation time constant of Icsl recorded under voltage clamp. Replacement of extracellular Na+ with Li+ dramatically shortened the action potential and under these conditions raising extracellular calcium lengthened the action potential. Using a voltage-clamp protocol to mimic an action potential, whole-cell current in the absence and in the presence of Li(+)-replacement was recorded in normal and raised extracellular calcium. The lithium-sensitive current was inward during the "plateau" and was reduced by raising extracellular calcium. The results do not support a role for Ik in mediating action potential shortening in raised extracellular calcium. It is suggested that a decrease in inward INa,Ca may be largely responsible, with a role for increased Icsl inactivation in the early part of the action potential.

Effects of high potassium and the bradycardic agents ZD7288 and cesium on heart rate of rabbits and guinea pigs.

The effects of 2 mM cesium (Cs+) and a novel selective bradycardic agent ZD7288 (0.64 microM) on sinoatrial Node (SAN) pacing rate were investigated in an isolated guinea pig SAN/atrial preparation, rabbit SAN preparation, and isolated working rabbit heart preparation. The effect of Cs+ and ZD7288 on the response of the preparations to increased extracellular potassium concentration ([K+]o) was also studied. Cs+ reduced beating frequency by 24% in isolated rabbit SAN (n = 16, p < 0.01) and by 21% in isolated working rabbit heart (n = 9, p < 0.01). ZD7288 decreased beating rate by 53% in guinea pig SAN (n = 7, p < 0.01) and by 38% in isolated working rabbit heart (n = 6, p < 0.01). In all three preparations, increased [K+]o significantly decreased the rate (p < 0.01) in normal Tyrode's solution but had no effect in the presence of Cs+ and caused tachycardia (p < 0.01) in the presence of ZD7288. We conclude that Cs+ and ZD7288 decrease pacing rate in rabbits and guinea pigs, possibly through modulation of the hyperpolarization-activated current (I(f)). ZD7288 is a more effective bradycardic agent than Cs+.

High selectivity of the i(f) channel to Na+ and K+ in rabbit isolated sinoatrial node cells.

The ionic selectivity of the hyperpolarization-activated inward current (i(f)) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K+ pipette, replacement of 90% external Na+ by Li+ caused a -24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs+ pipette, the i(f) current decreased almost proportionally to the decrease in external [Na+]o as Li+ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG+) substitution, suggesting that the relative permeability of Li+ compared with Na+ for the i(f) channel is as low as that of NMG+. When Cs+ or Rb+ was substituted for internal K+, the fully activated I/V relationship for i(f) showed strong inward rectification with a positive reversal potential, indicating low permeability of the i(f) channel for Cs+ and Rb+. These results show that the i(f) channel is highly selective for Na+ and K+ and will not pass the similar ions Li+ and Rb+. Such a high degree of selectivity is unique and may imply that the structure of the i(f) channel differs greatly from that of other Na+ and K+ conducting channels.

Internal K ions modulate the action of external cations on hyperpolarization-activated inward current in rabbit isolated sinoatrial node cells.

We have investigated the effect of change in external Na+ concentration on the hyperpolarization-activated inward current (I(f)) in the presence of different internal cations. Rabbit single isolated sinoatrial node cells were studied using the whole-cell patch-clamp technique. With 140 mM K+ pipettes, lowering [Na+]o causes the fully activated I/V curve for I(f) to shift in a negative direction without a significant decrease of the slope conductance. The PNa/PK ratio, as defined by the Goldman-Hodgkin-Katz equation, is concentration-dependent: the lower the [Na+]o, the higher PNa/PK. The conductance/concentration relationship for I(f) shows saturation at low [Na+]o or [K+]o, indicating that the channel has a strong affinity for external cations. With 140 mM Cs+ pipettes, the I/V curve shows strong inward rectification and inward I(f) current decreases almost proportionally to the decrease in [Na+]o; the conductance/concentration relationship for I(f) shifts to the right suggesting that the binding affinity of the external binding site is reduced. These results suggest that the I(f) channel is a multi-ion channel with a high-affinity external binding site, the affinity of which is modulated by internal cations.

Effect of catecholamines on the ventricular myocyte action potential in raised extracellular potassium.

We describe the relationship between catecholamines and raised extracellular potassium ([K+]o) on action potential parameters and calcium currents in isolated ventricular myocytes of the guinea-pig and relate these findings to the problem of understanding how the heart is protected from exercise-induced hyperkalaemia ([K+]a up to 8.5 mM). Action potential duration (APD90), amplitude and upstroke velocity were recorded in stimulated (2Hz) guinea-pig ventricular myocytes using whole-cell patch electrode recordings (37 degrees C). Cells were superfused with normal K+ Tyrode and with raised K+ Tyrode in the presence of either noradrenaline, adrenaline or raised calcium. Inward calcium current was measured using voltage clamp. Raised K+ (8, 12, 16 mM K+ Tyrode) caused a significant (P < 0.01) depolarisation, shortened the APD90 and decreased the action potential amplitude and upstroke velocity. In raised K+ Tyrode addition of noradrenaline (0.08-0.1 microM) or adrenaline (0.1-0.2 microM) increased action potential amplitude (P < 0.01), APD90 (P < 0.01) and upstroke velocity (P < 0.01) (measured only in 16 mM K+ Tyrode). In 12 mM K+ Tyrode raised Ca2+ (5-6 mM) increased action potential amplitude (P < 0.05) and shortened APD90 (P < 0.05). Addition of NA (0.08-0.1 microM) increased the inward Ca2+ current. All effects were fully reversible. In raised [K+]o increases in catecholamines and [Ca2+]o cause changes in action potential parameters that would be expected to maintain propagation of the cardiac action potential in the whole heart. Thus, in the ventricular myocyte the increase in conductance to Ca2+ caused by catecholamines may be one factor that is important in minimising the potentially adverse effects of exercise-induced hyperkalaemia.

Reciprocal role of the inward currents ib, Na and i(f) in controlling and stabilizing pacemaker frequency of rabbit sino-atrial node cells.

Experiments and computations were done to clarify the role of the various inward currents in generating and modulating pacemaker frequency. Ionic currents in rabbit single isolated sino-atrial (SA) node cells were measured using the nystatin-permeabilized patch-clamp technique. The results were used to refine the Noble-DiFrancesco-Denyer model of spontaneous pacemaker activity of the SA node. This model was then used to show that the pacemaker frequency is relatively insensitive to the magnitude of the sodium-dependent inward background current ib, Na. This is because reducing ib, Na hyperpolarizes the cell and so activates more hyperpolarizing-activated current, i(f), whereas the converse occurs when ib, Na is increased. The result is that i(f) and ib, Na replace one another and so stabilize nodal pacemaker frequency.

Effects of catecholamines and potassium on cardiovascular performance in the rabbit.

Resting subjects risk cardiac arrest if plasma potassium ([K+]p) is raised rapidly to 7-9 mM, but brief bouts of exhaustive exercise in healthy subjects can give similar [K+]p without causing cardiac problems. We investigated the effects of [K+]p and catecholamines on systolic blood pressure (SBP) and mean aortic flow (MAF) in anesthetized rabbits and on maximum output pressure (MOP) in isolated working rabbit hearts. In six rabbits, hyperkalemia (11.4 +/- 0.4 mM) caused a fall in SBP from 116 +/- 6 to 49 +/- 6 mmHg and in MAF from 373 +/- 30 to 181 +/- 53 ml/min (P < 0.01). Raising [K+]p (11.6 +/- 0.3 mM) with norepinephrine (NE) (1.3 micrograms.kg-1.min-1 iv), however, increased SBP from 108 +/- 7 to 150 +/- 6 mmHg (P < 0.01) and MAF from 347 +/- 42 to 434 +/- 35 ml/min (P < 0.01). In 19 isolated working hearts, perfusion with 8 mM K+ Tyrode and then 12 mM K+ Tyrode reduced MOP from 87 +/- 3 (control 4 mM K+) to 67 +/- 3 (8 mM K+) and 51 +/- 2 cmH2O (12 mM K+) (P < 0.01); 12 mM K+ Tyrode with 0.08 microM NE or epinephrine, however, increased MOP from 67 +/- 6 (in 8 mM K+) to 85 +/- 6 cmH2O (NE) and from 58 +/- 2 to 76 +/- 5 cmH2O (epinephrine) (P < 0.01). Catecholamines may therefore play a key role in protecting the heart from exercise-induced hyperkalemia.

Pacemaking in rabbit isolated sino-atrial node cells during Cs+ block of the hyperpolarization-activated current if.

1. Experiments have been carried out using the whole-cell patch clamp technique to investigate how the spontaneous pacemaker activity of rabbit isolated sino-atrial (SA) node cells is affected by the block of the hyperpolarization-activated current if by 1 and 2 mM-caesium. 2. Two millimolar caesium reduced the amount of if activated at -90 mV to less than 10% of the control value. In the pacemaking range of SA node cells, if was often completely blocked by this concentration of Cs+. 3. Two millimolar caesium slowed but did not arrest spontaneous pacemaking in isolated SA node cells. In freely beating non-patched cells, 2 mM-CsCl caused a 30% reduction in rate of beating, indicating that in all cells observed if was normally contributing to pacemaking. 4. No increase in instantaneous inward current was seen in response to hyperpolarizing voltage clamp pulses from a holding potential of -40 mV when 1 or 2 mM-CsCl was applied to SA node cells. These concentrations of Cs+ do not therefore induce an 'extra' inward current. 5. Neither the inward calcium current (iCa) nor the outward potassium current (iK) showed changes which could be attributed to Cs+ application. 6. Since spontaneous pacemaking continues during Cs+ block of if while other membrane currents show no Cs(+)-induced changes which could account for this, these experiments provide strong, though indirect, evidence for the presence in SA node of a time-independent background current, ib, which will contribute to a mode of pacemaking controlled by the decay of potassium conductance (gK). The nature of this inward current has yet to be clarified. 7. The results strongly suggest that the hyperpolarization-activated current if normally makes an important contribution to the pacemaker depolarization of all SA node cells.

Rabbit sino-atrial node cells: isolation and electrophysiological properties.

1. A method has been developed for isolating calcium-tolerant, single rabbit sinoatrial node cells which maintain their natural shape following isolation. The majority of viable, spontaneously active cells were elongated and measured about 100 microns in length. 2. Staining fixed cells with Haematoxylin-Eosin revealed that a 'cell' with projections was usually an aggregate of more than one cell. 3. Single, elongated, spontaneously active cells were current and voltage clamped using the whole-cell configuration of the patch-clamp recording technique. The spontaneous activity and time-dependent currents recorded were similar to those reported previously in multicellular nodal preparations and in single cells. 4. An assessment was made of the time course of L-type calcium current run-down: a stable period of between 10 and 20 min followed by a rapid run-down (over about 2 min) was typically observed. 5. In most cells, a fast, TTX-sensitive Na+ current component was seen. A few cells showed a transient outward K+ current (iA). 6. The activation range for the hyperpolarization-activated current, if, varied from cell to cell. In the majority of actively beating cells, the threshold for if was near the maximum diastolic potential (about -65 mV in most cells) but in other cells, no if could be recorded within the pacemaker range. 7. Millimolar concentrations of MnCl2 caused a marked increase in if, but only when the pipette solution did not contain EGTA. Inclusion of EGTA (to buffer Ca2+ to about pCa 8) significantly reduced the effect of Mn2+ which therefore probably occurs through inhibition of Na(+)-Ca2+ exchange and consequent rise in intracellular Ca2+ concentration.

The relationship between the transient inward current (TI) and other components of slow inward current in mammalian cardiac muscle.

When rabbit sino-atrial node preparations and isolated guinea-pig ventricular cells are subjected to Na-K pump blockade (either by reducing external K+ by a factor of 10: sinus node; or by the presence of 10(-7) M ouabain: ventricular cells) they develop oscillatory transient inward currents of the kind already recorded in Purkinje fibres and ventricular muscle strands. The time course of these transient currents, generally known as TI's, closely resembles that of the slow component of second inward current (isi,2) previously reported by us as occurring in rabbit sinus node when recorded near its threshold (-40 mV). Moreover, we have found that, under voltage clamp conditions, the 'envelope' of isi currents activated by depolarization from negative membrane potentials matches the outline of the iTI which develops during the initial hyperpolarization. In the sinus node, oscillations of iTI become smaller near O mV but are never flat and there is no clear cut reversal potential, whilst in ventricular cells oscillations and contractions cease at very positive membrane potentials (+35 mV) without the TI current ever becoming net outward. Replacing 75% of the external Na+ with Li+ reduces isi and iTI in the node by about the same proportion strongly suggesting that both are carried by a Na-Ca exchange mechanism. This idea is supported by reproducing the conditions of Na-K pump block in a computer model of the sinus node activity++, when oscillatory currents are generated by variations in activity of the Na-Ca exchange mechanism triggered by fluctuating levels of intracellular calcium. The same model when used to test the hypothesis that isi,2 might be carried by a non-specific ion channel showed that considerable distortion of the action potential would then occur. From the experimental and computed results it is concluded that the majority of isi,2 and iTI currents are both mediated by Na-Ca exchange.

Relationship between the transient inward current and slow inward currents in the sino-atrial node of the rabbit.

In low K+ (0.3 mM) solutions rabbit sinus node preparations show the oscillatory transient inward current, iTI, already recorded in these conditions in Purkinje and ventricular preparations. The time course of iTI closely resembles that of the slow component of the slow inward current (isi) previously reported by us (Brown, Kimura, Noble, Noble & Taupignon, 1984a) in rabbit sinus node, when recorded near its threshold (-40 mV). When the duration of voltage-clamp steps is varied there is a strong correlation between the 'envelope' of isi amplitudes on depolarization and the time course of iTI on hyperpolarization. Although oscillations of iTI become smaller near 0 mV, there is no potential at which the current records are completely flat, suggesting that there is no simple reversal potential. 75% substitution of Na+ by Li+ greatly reduces both iTI and slow isi in about the same proportion. Reducing the activity of the Na-K exchange pump by the amount expected in 0.3 mM-K+ solutions is sufficient to induce oscillatory iTI in a computer model of the sino-atrial node (Noble & Noble, 1984). The model reproduces the current as variations in the Na-Ca exchange current dependent on intracellular Ca2+ concentration ([ Ca]i). The model was also used to test the alternative hypothesis that the slow inward currents might be generated by [Ca]i-activated non-specific cation channels. It is shown that this would distort the shape of the repolarization phase of the action potential. It is concluded that the experiments and computations are consistent with the hypothesis that a large fraction of iTI and the slow component of isi could both be generated by Na-Ca exchange and that only a relatively small fraction might be generated by non-specific channels.

The slow inward current, isi, in the rabbit sino-atrial node investigated by voltage clamp and computer simulation.

The properties of the slow inward current, isi, in the sino-atrial (s.a.) node of the rabbit have been investigated using two microelectrodes to apply voltage clamp to small, spontaneously beating, preparations. Many of the experimental results can be closely simulated using the computer model of s.a. node electrical activity (Noble & Noble 1984) which has been developed from models of Purkinje fibre activity (Noble 1962; DiFrancesco & Noble 1984). Comparison of the computed reconstructions with experimental results provides a test of the validity of the modelling. Experiments using paired depolarizing clamp pulses show that inactivation of isi is calcium-entry dependent although, unlike the inactivation of Ca2+ currents in some other systems, it also shows some voltage-dependence. Re-availability (recovery from inactivation) of isi in s.a. node is much slower than inactivation at the same potential, showing that isi is not controlled by a single first order process. This very slow recovery from inactivation of isi in the s.a. node and the slow time course of its activation and inactivation at voltages near threshold (-40 to -50 mV) can be closely modelled by assuming that there are two components of 'total isi': a fast inward current, iCa,f' representing the 'gated' fraction and a second, slower, inward current component, iNaCa which, we propose, is caused by the sodium-calcium exchange that ensues when the initial Ca2+ -entry triggers the release of stored intracellular Ca2+. When repetitive trains of clamp pulses are given, a 'staircase' of isi magnitude is seen which can be increasing ('positive') or decreasing ('negative') according to the potential level and frequency of the pulse train given. When computer reconstructions of such staircases are made, it is found that the positive staircases (which, in contrast to negative staircases, imply that more complex processes than simple inactivation are present) can be closely simulated by a model which incorporates slower processes (suggested Na-Ca exchange current) in the total isi in addition to the gated current component.(ABSTRACT TRUNCATED AT 400 WORDS)

The ionic currents underlying pacemaker activity in rabbit sino-atrial node: experimental results and computer simulations.

The membrane currents underlying the pacemaker depolarization have been investigated in rabbit s.a. node preparations using the two-microelectrode voltage clamp technique. Many of the experimental results have been simulated using a computer model of s.a. node electrical activity. Changes of three time-dependent membrane currents which could contribute to pacemaker depolarization are found to occur in the relevant potential range: decay of the potassium current, iK, and activation of the inward current, if, and of the slow inward current, isi. The contribution of if activation to the pacemaker depolarization ranges from nil to an appreciable part depending on the preparation; when Cs (1 mM) blocks if, it nevertheless does not prevent pacemaking. In the model, holding the if activation variable at zero slows but does not stop pacemaking; doubling if conductance and shifting its activation curve by 15 mV in the positive direction causes a 15% faster rate of pacemaking. The slow time course of re-availability of isi must be allowed for when determining the isi threshold. A voltage clamp protocol designed to mimic as closely as possible an action potential followed by a pacemaker depolarization gives an estimate of isi threshold at the potential level of the last third of the pacemaker depolarization. This has been confirmed in experiments in which the voltage clamp was switched on at different points in the pacemaker depolarization. In the computer simulation, 'blocking' isi depolarizes the membrane to the zero current level (close to the potential reached at the end of a pacemaker depolarization) and stops the generation of action potentials. The decay of iK contributes to the pacemaker depolarization; with both our own model and that of K. Yanagihara, A. Noma and H. Irisawa, Jap. J. Physiol. 30, 841-857 (1980) 'blocking' iK decay abolishes pacemaker activity. Computations of extracellular K+ concentration changes compared with iK decay in a cylindrical model allow re-assessment of the interpretation of K+ concentration measurements during pacemaking made by J. Maylie, M. Morad and J. Weiss, J. Physiol., Lond. 311, 167-178 (1981).(ABSTRACT TRUNCATED AT 400 WORDS)