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Cardiovascular Research 2002 53(1):68-79; doi:10.1016/S0008-6363(01)00459-X
© 2002 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Role of the 293b-sensitive, slowly activating delayed rectifier potassium current, iKs, in pacemaker activity of rabbit isolated sino-atrial node cells

Ming Lei*, Patricia J Cooper, Patrizia Camelliti and Peter Kohl

Department of Physiology, University of Oxford, Oxford OX1 3PT, UK

ming.lei{at}physiol.ox.ac.uk

* Corresponding author. Fax: +44-1865-272-554

Received 11 October 2000; accepted 27 August 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: (i) to characterize the electrophysiological properties of the slowly activating delayed rectifier potassium current, iKs, defined as the 293b-sensitive current, during the action potential (AP) of rabbit sino-atrial node (SAN) pacemaker cells; (ii) to evaluate the contribution of iKs to the pacemaker AP under physiological conditions and during β-adrenergic stimulation. Methods: Rabbit SAN pacemaker cells were studied using the perforated patch clamp technique in voltage–, AP– and current–clamp modes. Results: Voltage–clamp findings. Block of iKs by 293b is dose-dependent, with an IC50 (half block) in rabbit SAN cells of 1.35 µM and an IC80 (sub-maximal block) of 5 µM. Sub-maximal concentrations of 293b have no significant effects on long-lasting and transient inward calcium currents, iCa,L and iCa,T, inward hyperpolarization activated current, if, and transient outward current, ito. AP–clamp experiments. The 293b-sensitive current activates near the peak of the SAN pacemaker action potential, reaches a mean maximal current density of 1.0±0.3 pA/pF (n=8, cell capacitances 27 to 62 pF, mean 35±4.0 pF) during late repolarization, and inactivates towards the end of repolarization. Additionally, in two smaller cells (cell capacitances 15 and 23 pF), no discernible 293b-sensitive current component was detected. Current–clamp data. In spontaneously beating SAN cells under control conditions, sub-maximal block of iKs by 5 µM 293b has negligible effects on action potential characteristics and does not change average cycle length (n=11). In contrast, after pre-treatment with 10 nM isoprenaline to mimic β-adrenergic stimulation, cells showed a 293b-induced depolarization of maximum diastolic potential by 2.2±1%, a decrease in diastolic depolarization rate by 9.9±4%, and a slowing of late action potential repolarization by 28.7±10.2%, resulting in a prolongation of spontaneous cycle length by 9.8±3.0% (P<0.05, n=10; for all parameters). Conclusion: Our findings suggest that in rabbit SAN: (i) iKs is activated during the normal pacemaker AP; (ii) the contribution of iKs to beating rate is small under control conditions; and (iii) iKs contributes significantly to spontaneous pacemaker rate during β-adrenergic stimulation.

KEYWORDS Adrenergic (ant)agonists; K-channel; Sinus node


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The delayed rectifier potassium current, iK, plays important roles in cardiac action potential (AP) repolarization and, possibly, pacemaking [1–3]. Previous studies have shown that iK can be separated into at least two kinetically distinct components: a rapidly activating (iKr) and a slowly activating (iKs) component [4]. Molecular biology studies have demonstrated that the channels underlying these two currents are composed of proteins encoded by separate genes: HERG, a human homologue of the ‘ether-a-go-go’ gene, encodes a protein that produces iKr; while the minK protein (encoded by minK gene) acts as the β-subunit that co-assembles with an {alpha}-subunit KvLQT1 (encoded by KvLQT1 gene) to form iKs channels [5,6]. The presence of mRNA for HERG, and of the minK protein, have been confirmed in rabbit sino-atrial node (SAN) cells, suggesting that both channel sub-types are expressed [7,8]. Furthermore, functional iKr and iKs have been identified electrophysiologically in these cells, and their activating kinetic properties are similar to those in guinea-pig cardiac myocytes [9,10]. In contrast to guinea pig, iK in the rabbit SAN appears to consist mainly of iKr, whose role in pacemaker activity has been extensively studied [2,9]. The relative contribution of iKs to rabbit SAN pacemaking, however, remained unclear, originally due to the lack of a selective blocker.

In the mid-1990s, a new selective blocker of iKs (Chromanol 293b, Hoechst) became available. This blocker was first shown to inhibit a cAMP-regulated K+ channel in rat colon crypts [11]. A subsequent study showed that the IsK channel may underlie the cAMP-regulated K+ channel and that 293b also blocks the rat IsK channel, expressed in Xenopus oocytes [12]. IsK is also known to be an iKs channel subunit. In the same study, 293b did not block the inward rectifying K+ channels, Kv1.1 and Kir2.1 [12]. 293b selectively inhibited native iKs in guinea-pig ventricular cells and the cloned guinea pig IsK channel, expressed in Xenopus oocytes, with a similar 50%-effective inhibitory concentration, IC50 (2–6 µM). Significantly higher concentrations of 293b (30 µM) had only a negligible effect on iKr in guinea-pig ventricular cells and none on the HERG channel expressed in Xenopus oocytes, the inward Ca2+ current, iCa, in guinea-pig ventricular cells, or the cardiac iCa channel expressed in Xenopus oocytes [12], suggesting a reasonably specific action of the drug on iKs.

In a more recent study, Bosch et al. [13] extensively characterized the inhibition of iK by 293b in ventricular myocytes. They showed that the properties of 293b-resistant and -sensitive currents correspond closely to those of iKr and iKs, respectively, as identified by the pharmacological probes E-4031 and dofetilide. Their data indicated that 293b is highly selective for iKs (IC50 1.02 µM) over iKr, with no effects on inward rectifier K+ current, iK1, Na+ current, iNa, L-type Ca2+ current, iCa,L, or the transient outward current, ito, at concentrations that were sufficient to completely inhibit iKs (50 µM 293b).

In the present study, we investigate the contribution of iKs, defined as the 293b-sensitive current, to rabbit SAN pacemaking in the absence and presence of β-adrenergic stimulation (10 nM isoprenaline), using the perforated patch technique in voltage–, current– and AP–clamp modes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell isolation
Cells were isolated as previously described by Lei and Brown [9]. Briefly, hearts were excised from 600–1000 g New Zealand White rabbits after cervical dislocation, and washed by coronary perfusion, using HEPES-buffered oxygenated normal Tyrode solution, before the right atrium was dissected (all solutions were at 37°C, unless otherwise stated). The SAN was located between Crista terminalis, atrial septum, and venae cavae. Thin strips of SAN tissue (approx. 3.0x1.0 mm) were dissected perpendicularly to the Crista terminalis and allowed to regain their spontaneous activity in Ca2+-containing Tyrode solution. Beating strips were then placed in Ca2+-free Tyrode solution for 5 min, incubated for 30–40 min in Ca2+-free Tyrode solution containing 230 units/ml collagenase (Type I, Sigma Chemical Co., UK) and 15 units/ml elastase (Type IIA, Sigma), and finally washed and stored in KB medium at 4°C for at least 1 h. For electrophysiological studies, single cells were released from the strips by glass pipette suction. During experiments, the cells were superfused with Tyrode solution at a rate of about 1 ml/min at 35°C in a laminar flow chamber of 400 µl volume (Warner Instrument Corp., Hamden, CT, USA).

2.2 Solutions
Solutions were prepared according to the following recipes, and checked for pH and osmolality. Normal Tyrode solution (mM): 140 NaCl; 5.4 KCl; 1.8 CaCl2; 1 MgCl2; 10 glucose; 5 HEPES; titrated with NaOH to pH 7.4. Ca2+-free solution: normal Tyrode but without CaCl2. KB solution (mM): 25 KCl; 80 L-glutamic acid; 20 taurine; 10 KH2PO4, 3 MgCl2; 10 glucose; 10 HEPES; 0.5 EGTA; titrated to pH 7.4 with KOH. Pipette solution (mM): 140 KCl; 1.8 MgSO4; 5 HEPES; 1 EGTA; titrated to pH 7.3 with KOH; amphotericin (200 µg/ml) was added to the pipette solution before the beginning of experiments.

2.3 Whole cell current and voltage clamp
Cells were studied using the amphotericin-permeabilized patch technique to limit disturbance of the intracellular environment. Electrodes were made from glass capillaries of 0.75 mm inner and 1.0 mm outer diameter (Clark Electromedical Instruments, Pangbourne, UK) using a Narishige electrode puller (PP-83, Narashige, Japan). Electrode tip diameters were approximately 1–2 µm, yielding resistances of 3–8 M{Omega}. The liquid junction potential between electrode and superfusate was corrected once the electrode made contact with the bath solution. An Axopatch-200B patch clamp amplifier (Axon Instruments Inc., Foster City, CA, USA) was used for current–, voltage– and AP–clamping. Electrical signals were displayed during the experiments on an oscilloscope (PM-3335, Philips, Watford, UK), simultaneously digitised at 5 kHz by a DigiData 1200 AD converter (Axon Instruments Inc., Foster City, CA, USA), and stored on computer hard disk for later analysis using Axon software.

The following parameters were individually analysed for 10 consecutive APs during steady state conditions (summarized in Table 1): maximum diastolic potential (MDP); maximum systolic potential (MSP); threshold potential (THP, voltage at the intersection of tangents to the membrane potential during diastolic depolarization and during AP upstroke); time to threshold potential (TTT, time required for the membrane potential to move from MDP to THP); average diastolic depolarization rate (DDR, THP minus MDP divided by TTT); maximum upstroke velocity (MUV, maximum of the first derivative of voltage changes during AP upstroke); AP duration at 50 and 90% repolarization (APD50, APD90, calculated as the time lag between THP-timing and corresponding levels of AP repolarization); late repolarization duration (LRD, APD90 minus APD50); and cycle length (CL, peak-to-peak duration of MSPs).


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  		Table 1 Action potential parameters of spontaneously active sino-atrial node cells during intrinsic (left) and β-adrenergically stimulated pacemaking (right) in the absence and presence of 5 µM 293ba

 
2.4 Action potential clamp
AP–clamp experiments were carried out using the technique originally described by Doerr et al. [14], with some modifications. In current–clamp mode, spontaneous SAN APs were recorded for 30–60 s after a stable perforated whole-cell configuration was formed. For each cell, the last five APs were digitised, and this signal was then reapplied (as a continuous loop) via the command voltage in voltage–clamp mode (AP–clamp). Since the command voltage mimicked the spontaneous AP of that cell, the compensation currents were near zero (with the exception of the AP upstroke, where a ‘current artefact’ indicated slight inter-cycle lag in several cells).

Experimental interventions that affect a particular ion current system (e.g. application of a selective blocker) reveal themselves in the form of an observable change in the compensation current that is required to maintain AP–clamp. This compensation current has the opposite polarity compared to the affected membrane current that gave rise to it [15]. This has to be taken into account when interpreting signals. In order to identify the time-course of iKs during the AP, we applied 5 µM 293b (a concentration that is sufficient to cause sub-maximal block of iKs without affecting other pacemaker current systems; see first two chapters of Results). The 293b-sensitive current revealed itself as a change in compensation current (see Fig. 3C). In order to reduce any possible contribution by imperfect AP–clamp (in particular as the membrane resistance of SAN cells is high), and to account for the inverse polarity of the recorded signal, we identified the 293b-sensitive membrane current as the difference current between compensation currents recorded in control conditions and during superfusion with the drug (see Fig. 3D).


Figure 3
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  		Fig. 3 Action potential clamp recording in rabbit SAN cell. Pre-recorded spontaneous action potentials (A) were reapplied as the command pulse (continuous loop) to the same cell, and compensation currents were recorded before (B) and during (C) exposure to 5 µM 293b (note that compensation currents have the opposite polarity compared to underlying membrane currents, for detail see text). The 293b-sensitive membrane current (D) was obtained by digitally subtracting the compensation current in presence of drug (C) from that under control conditions (B). Inset: time course of 293b-sensitive current relative to SAN action potential.

 
2.5 Drugs
Chromanol 293b (trans-6-cyano-4-4(N-ethysulphonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chromane, Hoechst AG, Frankfurt, Germany) was dissolved in 50% ethanol at 500 µM. Batches of stock solution were stored at room temperature for 5–10 days. Nisoldipine (Bayer plc., Newbury, England), Nickel (Sigma Chemical Co., UK) and E-4031 ((1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonylaminobenzoyl))piperidine, Eisai Pharmaceuticals, Tokyo, Japan) were dissolved in distilled water to make 10 mM stock solutions, and stored at 4°C. Isoprenaline ((–)-isoproterenol, (+)-bitartrate salt, Sigma–Aldrich, Poole, UK) was dissolved in distilled water to make 1 mM stock solution and kept in the dark on ice until application at final concentrations of 10 nM (note that use of the (+) or (±) forms of isoproterenol may necessitate higher final concentrations to achieve similar effects).

2.6 Data analysis
All results are presented as mean±S.E.M. Statistical significance was determined by Student's t-test for paired observations. P<0.05 was considered to indicate a significant difference between means. Statistical analysis was performed using SigmaStat version 4.0 (Jandel Scientific, San Rafael, CA, USA), and GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).

This study was carried out in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Concentration-dependent block of iKs by 293b
Voltage clamp experiments quantified iKs by measuring tail currents at –40 mV after 1 s depolarizing clamp pulses from –40 to +40 mV in the presence of 300 nM nisoldipine and 5 µM E-4031, to block iCa,L and iKr, respectively (see Fig. 1A). Mean data for the dose-dependent effect of 293b on iKs are summarised in Fig. 1B. These data have been fitted with a typical dose-response relation using a Hill coefficient of 1. Block of iKs by 293b occurs in a concentration-dependent manner, with an IC50 of 1.35 µM. At a concentration of 5 µM, 293b blocks 80% of iKs. This concentration has been used for sub-maximal block of iKs in all following experiments and is indicated by the light traces in all figures.


Figure 1
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  		Fig. 1 Dose-response data for iKs block by 293b. A: iKs tail currents at –40 mV after 1 s clamp pulses from –40 to +40 mV (see inset for overall pulse and tail current responses, not to scale) in the presence of different concentrations of 293b (from top to bottom): 0, 1, 5 and 50 µM. Light traces indicate 5 µM 293b, the concentration at which sub-maximal block of iKs occurred, and which was used in all subsequent experiments. All experiments were performed in the presence of 300 nM nisoldipine and 5 µM E-4031 to block iCa,L and iKr, respectively. B: Concentration-dependent block of iKs by 293b (mean data for four or five cells at each drug concentration, as indicated).

 
3.2 Lack of effect of 293b on iCa,L, iCa,T, if, and ito
The selectivity of 293b has thus far been assessed in guinea-pig ventricular cells and cloned guinea pig and rat IsK channels, expressed in Xenopus oocytes. Its effect on key membrane currents of rabbit SAN cells (like iCa,L, iCa,T, if and ito) has not previously been examined. In this series, we investigate the effect of 5 µM 293b on these currents in the presence of 30 µM TTX to block any potential contamination by iNa.

The effects of 5 µM 293b on iCa,L (n=7) and iCa,T (n=7) were investigated in the presence of 5 µM E-4031 to block iKr (Fig. 2A and B), using protocols of depolarizing clamp pulses to various test potentials for at least 500 ms. Both the complete time-course and an enlarged view of the first 100 ms of the depolarizing pulse are presented for better resolution of iCa,L and iCa,T. It is inherent to the nature of these experiments, that currents such as iCaL, iCaT, if and ito were not blocked. Currents recorded upon early depolarization do not, therefore, follow the ‘typical’ iKs activation shape. Furthermore, iKs activates slowly, so that the effects of 293b on pulse current amplitude become prominent only towards the end of depolarizing steps.


Figure 2
Figure 2
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  		Fig. 2 Lack of effect of 5 µM 293b on iCa,L, iCa,T, if and ito. All experiments were carried out in the presence of 30 µM TTX to prevent potential contamination by iNa. A–D, top panels: Currents recorded during control conditions (dark traces) and in the presence of 5 µM 293b (light traces). A–D, bottom panels: Mean current–voltage relations are presented with current density normalized by cell capacitance. A: iCa,L (n=7), currents were elicited by depolarizing clamp pulses from a holding potential –50 mV to various test potentials for 500 ms (bottom right), in the presence of 5 µM E-4031 to block iKr. B: iCa,T (n=7), during depolarizing clamp pulses from a holding potential of –80 mV to various test potentials (bottom right), in the presence of 5 µM E-4031 to block iKr. C: if (n=5) currents were triggered by hyperpolarizing pulses from a holding potential –40 mV to various potentials for 1 s (bottom right). D: ito (n=6), currents were elicited by depolarizing clamp pulses, after a 30 ms hyperpolarization pre-pulse to –80 mV, to various test potentials between –40 and +40 mV (bottom right), in the presence of 5 µM E-4031, 300 nM nisoldipine and 40 µM nickel to block iKr, iCa,L and iCa,T.

 
Depolarizing pulses from a holding potential of –50 mV elicit, under these conditions, iCa,L; an original recording in the absence and in the presence of 5 µM 293b is shown in Fig. 2A (top panels). After applying 5 µM 293b (for 5 min), iCa,L was unchanged, as witnessed by the current–voltage relations of iCa,L in the absence and in the presence of the drug (bottom panel). Mean current densities of peak iCa,L at 0 mV test potential in the absence and presence of 5 µM 293b were 11.3±2.1 and 11.1±2.0 pA/pF, respectively (n=7, P>0.05). This suggests that 5 µM 293b has no effect on iCa,L.

Fig. 2B shows calcium currents, recorded from a SAN cell in the absence and in the presence of 5 µM 293b, stimulated by depolarization from a holding potential of –80 mV to various test potentials. I–V relations obtained with this protocol, in the absence and presence of 5 µM 293b overlap. Mean current densities at –20 mV test potential are 14.6±2.0 pA/pF (control) and 14.0±1.8 pA/pF (n=7, P>0.05). Currents, elicited by this protocol, will contain contributions of both iCa,T and iCa,L. As the latter has been shown to be unaffected by the drug (Fig. 2A), these results indicate that 5 µM 293b has no significant effect on iCa,T either.

Fig. 2C shows an example of if, recorded in the absence and in the presence of 5 µM 293b. Mean current–voltage relations for the current in the absence and presence of the drug are indistinguishable. Mean peak if current densities, reached towards the end of a 1 s hyperpolarizing pulse to a test potential of –140 mV, were 11.3±2.2 pA/pF in the absence, and 10.6±1.9 pA/pF in the presence of 5 µM 293b (n=5, P>0.05). Thus, there was no significant change of if in the presence of 5 µM 293b.

The effect of 5 µM 293b on the transient outward current, ito, in SAN pacemaker cells was also studied. As shown in Fig. 2D, ito was elicited by 500 ms depolarizing pulses to various test potentials, from a holding potential of –80 mV. The following drugs were added to block iKr, iCa,L and iCa,T, respectively: 5 µM E-4031, 300 nM nisoldipine and 40 µM nickel. There is no significant difference between mean ito current densities in the absence and presence of 5 µM 293b (5 min) at any of the test potentials (see I–V curves). For example, the mean peak ito current densities at a test potential of +40 mV were 7.0±1.2 pA/pF (control) and 6.2±1.4 pA/pF (n=6, P>0.05). Thus 5 µM 293b did not affect ito in rabbit SAN cells.

In summary, 5 µM 293b – a concentration that affords sub-maximal block of iKs – does not have a significant effect on either iCaL, iCaT, if or ito in rabbit SAN cells. This is in keeping with earlier observations in ventricular myocytes, where concentrations of up to 50 µM had no significant effects on any current other than iKs [13].

3.3 293b-sensitive current during AP–clamp
The 293b-sensitive current during the rabbit SAN AP was studied using AP–clamp (Fig. 3). The pacemaker APs in panel 3A were recorded in current–clamp mode before switching to voltage–clamp, where the pre-recorded APs were used as the command potential for the current recordings (panels 3B and C). Several cycles (3–8) of the APs were required before the intrinsic rhythm of the cell synchronized with the cycle of the command APs. After a steady pattern of the control compensation current was obtained (Fig. 3B), 5 µM 293b was applied to sub-maximally block iKs. This resulted in an increase in the compensative inward current during the AP (Fig. 3C, note that compensation currents shown in 3B and C have the opposite polarity to membrane currents, for detail see Methods).

The 293b-induced change in compensation current was not due to a rundown of membrane current systems, since: (i) the inward shift in compensation current regularly occurred within tens of seconds after switching to the 293b containing solution (a time lag largely accounted for by the dead-space in the perfusion system) and it consistently reached a steady state within 2 min; (ii) similar changes in compensation current were never observed in the absence of the drug; (iii) during wash-out of 293b, the compensation current regularly returned towards control levels (an ‘ideal’ zero line was not normally reached within the standard 3 to 5 min follow-up observation period; probably caused, in part at least, by secondary responses to the intervention); and (iv) the same typical change in compensation current was observed in four cells after repeated exposure to the drug, performed up to 20 min after the end of the first intervention (note: only primary drug application data were analyzed in this study).

As the compensation current applied from the external current source has the opposite polarity compared to the ionic membrane current, the drug-sensitive current, iKs, was obtained by digitally subtracting the current recorded in the presence of the drug from the control current (Fig. 3D). This allowed us to establish that there is very little, if any, iKs during early diastolic depolarization (see inset of Fig. 3).

During the AP, the 293b-sensitive current activates near the peak of the action potential (Fig. 3, inset), slowly reaches a maximum during late repolarization, and deactivates towards the end of repolarization. In a total of 10 cells, a 293b-sensitive current was recorded in eight cells (cell capacitances between 27 and 62 pF, mean: 35±4.0 pF) with a peak current density range from 0.7 to 3.0 pA/pF (mean: 1.0±0.3 pA/pF). The remaining two cells (with cell capacitances of 15 and 23 pF) did not show a discernible 293b-sensitive current component.

3.4 Effect of 293b on spontaneous pacemaker activity
To address the role of iKs in pacemaker rate regulation, 293b was applied to spontaneously beating SAN cells in control conditions and during β-adrenergic stimulation (10 nM isoprenaline).

The effects of 5 µM 293b on control AP parameters are illustrated in Fig. 4A and summarised in Table 1 (left hand side, n=11). Sub-maximal block of iKs caused a small (5.3±1.6%) increase in LRD, but had no significant effect on overall CL and MDP, MSP, APD50, APD90 or DDR. It appears, therefore, that the role of iKs in setting pacemaker rate is negligible in rabbit SAN cells under control conditions.


Figure 4
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  		Fig. 4 Effect of 293b on spontaneous pacemaker activity in rabbit SAN cells under control conditions and during β-adrenergic stimulation by isoprenaline. A: Lack of effect of exposure to 5 µM 293b on the spontaneous activity of a SAN cell. Dark trace: intrinsic activity; light trace: in the presence of 293b. B: Effect of 5 µM 293b on the spontaneous activity of a β-adrenergically stimulated SAN cell (pre-treated with 10 nM isoprenaline). Dark trace: spontaneous activity during β-adrenergic stimulation; light trace: reduced beating rate after additional exposure to 293b.

 
Fig. 4B illustrates an example of the effect of the application of 5 µM 293b on the background of β-adrenergic stimulation. The cell was superfused by 10 nM isoprenaline for 2 min before 5 µM 293b was added. As expected, β-adrenergic stimulation accelerated spontaneous pacemaker activity of the cell (note different time scales in Fig. 4A and B). Under these conditions, addition of 5 µM 293b significantly decreases DDR, increases LRD, depolarizes MDP and increases CL, without significantly affecting APD50, APD90, THP, TTT and MSP (see, in particular, first pair of superimposed APs in Fig. 4B; for summary see Table 1, right). Thus, iKs influences pacemaker rate during β-adrenergic stimulation.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1 Selectivity of 293b
The characteristics of 293b -sensitive and -insensitive currents in rabbit SAN cells [16] in terms of V1/2 and k values, are similar to the E-4031-insensitive and E-4031-sensitive proportions of the delayed rectifier potassium current, measured previously in cardiac cells including rabbit SAN [4,9,13], and are understood to be consistent with those of iKs (293b-sensitive) and iKr (293b-insensitive), respectively (see also Refs. [12] and [13]).

In the present study we establish that the IC50 for 293b-block of iKs in rabbit SAN cells is 1.35 µM (Fig. 1). Using a concentration of 5 µM 293b, iKs is inhibited by 80%. Fig. 2 illustrates that this concentration of 293b has no significant effect on current densities and I–V relations of iCa,L, iCa,T, if and ito in rabbit SAN pacemaker cells.

This evidence suggests that 293b, at a concentration of up to 5 µM, is a selective blocker of iKs in rabbit SAN pacemaker cells.

4.2 293b-sensitive current during the action potential
The selectivity of 293b for iKs is an important pre-requisite for its use in the AP–clamp method. All changes in compensation currents could be clearly related to the timing of application of 293b. They were reversible and repeatable.

Under control conditions (Fig. 3B), the compensation current during most of the AP–clamp (and in particular for the segment where iKs is active, see Fig. 3, inset) is near zero, indicating good AP–clamp of the cells.

The drug-sensitive membrane current was obtained by digitally subtracting the current recorded in the presence of the drug (e.g. Fig. 3C) from the control current, (e.g. Fig. 3B). As shown in Fig. 3D (inset), iKs is not active during early diastolic depolarization. After the AP upstroke it increases in amplitude, reaches its peak during late repolarization, and inactivates near the end of repolarization of the AP. This is in keeping with the view that iKs contributes mainly to the late repolarization of the AP in SAN pacemaker cells.

In the present study, iKs current densities varied from cell to cell, ranging from no discernible current (two out of 10 cells) to a maximum of 3.0 pA/pF (mean current density of 1.0±0.3 pA/pF for the eight cells that showed iKs). While this population of cells is too small to draw separate cell-size related conclusions, our observations are in keeping with earlier reports that showed cell size-dependent expression of various ion currents in rabbit SAN (including iKs) [17–20].

Thus, we have established that iKs (in those cells that show discernible levels of the current) is active during the AP, with a peak during late repolarization, and that no iKs may be detected during early diastolic depolarization.

4.3 Contribution of iKs to pacemaker rate
Sub-maximal block of iKs by 5 µM 293b has no significant effect on intrinsic AP parameters of rabbit SAN pacemaker cells (see Table 1, left, and Fig. 4A). This is in accordance with the fact that iKs contributes little to total iK in rabbit SAN cells under control conditions [9]. However, this could be different during β-adrenergic stimulation, which has been reported to increase iK in many cardiac cell types, including rabbit SAN pacemaker cells [21–25], both directly and indirectly.

Direct effects include an increase in peak iK amplitude, a negative shift (by up to 10 mV) in the voltage dependence of iK activation, and faster deactivation kinetics [26]. In three AP–clamp experiments with additional application of 10 nM isoprenaline, we also observed an increase in the 293b-sensitive current to 119±26% (data not shown) reconfirming these earlier observations.

Indirect effects of β-adrenergic stimulation on iKs have been proposed to be associated with the higher beating rates, which may prevent complete deactivation of iKs, causing accumulation and increased current amplitudes [27]. Another indirect pathway relates to a β-adrenergic increase in intracellular Ca2+ concentration near the channel, which could raise total iKs [28,29].

To assess the contribution of iKs to spontaneous pacemaker activity during β-adrenergic stimulation, we repeated the application of 5 µM 293b after prior exposure of rabbit SAN cells to 10 nM isoprenaline (for detail see Fig. 4B and Table 1, right hand side). Under these conditions, sub-maximal block of iKs causes depolarization of MDP, reduction in DDR, increase in LRD and prolongation of CL. The expected increase in APD90 remained, due to parameter variability, marginally below statistically significant levels. LRD, however, which cancels out the error associated with the determination of AP-onset by subtracting APD50 from APD90, was significantly increased (by 28.7±10.2%). This is consistent with the timing of iKs during the SAN AP, as described above (see Fig. 3).

The observed effects, including the prolongation of CL, are compatible with the consequences of blocking a β-adrenergically enhanced iKs, delaying late repolarization and reducing MDP. The latter may cause slowing of spontaneous diastolic depolarization via secondary effects on other pacemaker currents. This includes if, whose contribution to pacemaking is enhanced during β-adrenergic stimulation (more negative MDP, increased availability of the current [30]). This explanation is in keeping with the action potential clamp data that show a maximum contribution of iKs to late repolarization, but negligible current during early depolarization (making a direct effect on DDR unlikely, see Fig. 3D, inset).

4.4 Context and potential relevance
The use of many antiarrhythmic drugs is, paradoxically, limited by their potential arrhythmogeneity. A major limitation of current class III antiarrhythmic drugs (iKr blockers) is their rate-dependent profile of AP prolongation, which results in maximal effects at slow rates and minimal effects during tachycardia [31]. This property limits efficacy in terminating rapid arrhythmias [32], while maximizing the risk of Torsade de Pointes arrhythmias, associated with bradycardia-dependent early afterdepolarization [33,34]. Reverse use-dependent effects on repolarization have been reported for the iKr blocker dofetilide in vitro [27,35,36] and in vivo [37]. Other iKr blockers, like E-4031, sotalol and UK-66914, also prolong repolarization preferentially at slow heart rates in vitro [38], and it has been suggested that iKs accumulation at increased frequencies decreases the relative importance of iKr, reducing the impact of iKr blockade and contributing to reverse use-dependent APD prolongation [27].

It has been proposed that iKs blockers might, in contrast to drugs that affect iKr, have a more favourable rate-dependent profile of APD prolongation [12,13]. Our findings would seem to support this notion.

There are, however, pronounced species-dependent differences in expression of iKr and iKs in SAN pacemaker cells, which have to be taken into account when addressing the potential value of pharmacological block of iKs and its effect on heart rate. Thus, in contrast to the rabbit, iK in pig SAN cells is largely derived from iKs. As a result, block of iKr by E-4031 (2–5 µM) hardly affects pig SAN cells, while the iKs blocker 293b inhibits pacemaker activity (IC50 for iKs block is 8.8 µM), causing arrest of spontaneous pacemaking at 30 µM [39].

It has been suggested that these species differences in iK-composition are related to the diverse intrinsic beating rates of different species, with ‘slower’ hearts expressing more iKs and faster ones – iKr (although it is not straightforward to judge whether the two observations would be causally linked, and if so, in which way). In contrast, first reports on human ventricular cells suggest that iKr is the principal component of iK in this ‘low heart rate’ species [40,41]. As yet, we are not aware of any data on human SAN cells in this context.

This issue of extrapolation from experiment to clinical relevance is further convoluted by the fact that little is known about inter-species differences in kinetic properties of the different sub-forms of iK. Also, there are reports on changed expression levels of various ion channels, including iK, during cardiac pathologies (reviewed in Ref. [42]). Further studies are clearly necessary to elucidate the role of iKr and iKs in pacemaker activity of SAN cells from different species, from mouse to human, and in different patho-physiological settings.

In conclusion, in rabbit SAN: there is dose-dependent inhibition of iKs by 293b (IC50: 1.35 µM) with no side effects on iCaL, iCaT, if and ito; iKs is activated during the normal pacemaker AP; its contribution to normal pacemaker rate is small, but becomes significant during β-adrenergic stimulation.

Time for primary review 28 days.


   Acknowledgements
 
We would like to thank Dr. Klaus Linz for help and advice on the AP–clamp technique and Dr. Uwe Gerlach for kindly providing the chromanol 293b. This work was supported by the British Heart Foundation and the Medical Research Council, London. PK is a Royal Society Research Fellow.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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