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Cardiovascular Research 1999 44(2):356-369; doi:10.1016/S0008-6363(99)00218-7
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Identification and properties of ATP-sensitive potassium channels in myocytes from rabbit Purkinje fibres

Peter E. Lighta,b,*, Jonathan M. Cordeiroa and Robert J. Frencha

aDepartment of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1
bDepartment of Pharmacology and Therapeutics, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1

* Corresponding author. Tel.: +1-403-220-4575; fax: +1-403-283-8731 plight{at}acs.ucalgary.ca

Received 16 December 1998; accepted 6 July 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Our goal was to identify the ATP-sensitive potassium (KATP) channels in cardiac Purkinje cells and to document the functional properties that might distinguish them from KATP channels in other parts of the heart. Methods: Single Purkinje cells and ventricular myocytes were isolated from rabbit heart. Standard patch-clamp techniques were used to record action potential waveforms, and whole-cell and single-channel currents. Results: The KATP channel opener levcromakalim (10 µM) caused marked shortening of the Purkinje cell action potential. Under whole-cell voltage-clamp, levcromakalim induced an outward current, which was blocked by glibenclamide (5 µM), in both Purkinje cells and ventricular myocytes. Metabolic poisoning of Purkinje cells with NaCN and 2-deoxyglucose caused a significant shortening of the action potential (control 376±51 ms; 6 min NaCN/2-deoxyglucose 153±21 ms). This effect was reversed with the application of glibenclamide. Inside-out membrane patches from Purkinje cells showed unitary current fluctuations which were inhibited by cytoplasmic ATP with an IC50 of 119 µM and a Hill coefficient of 2.1. This reflects ~five-fold lower sensitivity to ATP inhibition than for KATP channels from ventricular myocytes under the same conditions. The slope conductance of Purkinje cell KATP channels, with symmetric, 140 mM K+, was 60.1±2.0 pS (mean±SEM). Single-channel fluctuations showed mean open and closed times of 3.6±1.5 ms and 0.41±0.2 ms, respectively, at –60 mV and ~21°C. At positive potentials, KATP channels exhibited weak inward rectification that was dependent on the concentration of internal Mg2+. Computer simulations, based on the above results, predict significant shortening of the Purkinje cell action potential via activation of KATP channels in the range 1–5 mM cytoplasmic ATP. Conclusions: Purkinje cell KATP channels may represent a molecular isoform distinct from that present in ventricular myocytes. The presence of KATP channels in the Purkinje network suggests that they may have an important influence on cardiac rhythm and conduction during periods of ischemia.

KEYWORDS Arrhythmia; K-channels; Purkinje fiber


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
ATP-sensitive K+ (KATP) channels have been identified in a number of tissues and organs including the heart [1]. Under normoxic conditions, it is believed that these channels are predominantly closed, but that during ischemia or hypoxia, intracellular ATP levels decrease causing opening of KATP channels [2] (for reviews, see [3,4]). It has been suggested that activation of these channels will shorten the action potential duration (APD) [3], limit Ca2+ influx, and decrease contractility [5–7]; but see also discussion of physiological and clinical relevance for alternate viewpoints. These events may protect the myocardium from Ca2+ overload and excessive energy expenditure during conditions of ischemia [8].

During ischemia and upon reperfusion, cardiac arrhythmias are often observed [9]. The shortening of the APD by activation of KATP channels during ischemia, and the consequent reduction in refractory period, can increase the likelihood of initiation of arrhythmias. It has been shown that glibenclamide, a blocker of KATP channels, diminishes the ischemia-induced APD shortening [10]. Furthermore, prolongation of the APD with glibenclamide during ischemia produces an anti-arrhythmic effect [10,11].

Shortening of the Purkinje cell APD during ischemia may make the myocardium more susceptible to arrhythmias, particularly of the reentrant type. For example, recent three-dimensional modelling of Purkinje/muscle interactions demonstrate that the Purkinje system is critical in the initiation of reentrant arrhythmias [12]. Previous studies, using computer mapping of a heart subjected to ischemia and reperfusion, have also shown that both afterpotential and reentrant type arrhythmias occur [13] and have suggested that these may be initiated in the Purkinje conduction system [14].

KATP channels have been identified and investigated in many regions of the heart including atrium, ventricle, AV node [3,4] and SA node [15]. However, in single Purkinje cells, we are not aware of any reports of direct recordings of whole-cell or single-channel currents through ATP-sensitive K+ channels. Nonetheless, a variety of evidence suggests that Purkinje fibres do possess functional KATP channels, and that these can have an important influence on their electrical activity. For example; application of KATP channel activators [16–18] or hypoxia [19] has been shown to produce a shortening of the APD and a slight hyperpolarization of the membrane potential in Purkinje fibres. Given the potential importance of KATP channels in modulating Purkinje cell excitability and modifying the likelihood of potentially lethal arrhythmias [17,18], we set out to identify Purkinje cell KATP channels, and to describe some of the macroscopic and single-channel properties that may distinguish them from KATP channels in other cardiac tissues.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell isolation
Purkinje cells were prepared from rabbit ventricle using techniques described previously [20] with some modifications [21]. New Zealand white rabbits (Charles River, Quebec, Canada) were housed according to the guidelines published in the Canadian Council on Animal Care, Guide for the care and use of laboratory animals. Rabbits (male, 2.0–3.0 kg) were anaesthetized by injection of pentobarbital into the marginal ear vein and were euthanized by cervical dislocation. Hearts were then removed and perfused retrogradely through the aorta with nominally Ca2+-free Tyrode's solution of the following composition (mM): NaCl 126, KCl 5.4, MgCl2 5.0, NaH2PO4 1.0, glucose 22, HEPES 24, taurine 20, creatine 5, sodium pyruvate 5. This solution was equilibrated with 100% O2 and the pH was adjusted to 7.4 with NaOH. After 6–8 min of this nominally Ca2+-free perfusion, the heart was perfused for approximately 18 min with the solution described above supplemented with collagenase (273 U/ml, Worthington type II), protease (5.2 U/ml, Sigma type XIV), and 0.1 mM CaCl2. Following this enzyme digestion, the heart was perfused with enzyme-free Tyrode's solution containing 0.1 mM Ca2+ for about 5 min, to wash the enzyme from the tissue. The heart was then placed in a dissecting dish containing 0.1 mM Ca2+ Tyrode's solution. Purkinje fibres from both ventricles were dissected out and placed in a small dish containing fresh enzyme solution (described above). Dissociation of individual cells from the isolated fibres was aided by stirring of the enzyme solution with a teflon-coated stir bar. The temperature was maintained at 37°C. Periodically, aliquots (100–200 µl) of enzyme solution containing Purkinje cells in suspension were removed and added to about 5 ml of 0.1 mM Ca2+ Tyrode's solution. Fresh enzyme solution was added to the undigested Purkinje fibres to maintain a volume of approximately 2 ml. Digestion of the Purkinje fibres into populations of single myocytes required between 15–60 min under these conditions.

Ventricular cells were also isolated from the same hearts using the method described above. When the Purkinje strands had been dissected from both the right and left ventricles, the left ventricle was removed, minced and gently swirled in 0.1 mM Ca2+ Tyrode's solution. The cell suspension containing ventricular cells was then diluted with Tyrode's solution and stored in a beaker at room temperature until use. An aliquot of either Purkinje cells or ventricular cells was placed in a perfusion chamber mounted on the stage of an inverted microscope. Cells were left to settle for about 10 min to allow them to adhere to the bottom of the chamber, and were then superfused with oxygenated HEPES buffer.

2.2 Solutions
All solutions were made with double-distilled, de-ionized water. In recordings where transmembrane macroscopic currents and voltages were measured, cells were superfused with previously oxygenated HEPES buffer of the following composition (mM): NaCl 126, KCl 5.4, MgCl2 1, CaCl2 1.0, HEPES 24, and glucose 11. pH adjusted to 7.4 with 12.95 mM NaOH. The internal pipette solution was composed of (mM): potassium aspartate 90, KCl 30, K2ATP 5.0, HEPES 5.0, EGTA 10, MgCl2 1.0, and NaCl 15. pH was adjusted to 7.2 with KOH. In the series of action potential experiments with glibenclamide alone and with NaCN and 2-deoxyglucose, the pipette solution was as follows (in mM): KCl 113, NaCl 15, K2ATP 5, MgCl2 1, HEPES 10, KOH 8 (to give a final pH of 7.2).

Unless otherwise noted, the pipette solution used for most excised, inside-out, patch recordings contained the following (in mM): NaCl 140; KCl 5; HEPES 10; CaCl2 1; MgCl2 1; glucose 10 at pH 7.4. The standard bath solution contained (in mM): KCl 140; HEPES 10; EGTA 1; MgCl2 1.4; glucose 10. EGTA (1 mM) was included in the bath solution to prevent the Ca2+-induced run-down of KATP channels. The pH of the bath solution was adjusted to 7.4 with KOH. When currents were recorded in symmetrical K+, the bath solution was also used as the pipette solution. For experiments on magnesium dependence, the final magnesium concentration was adjusted by addition of 1 M MgCl2 stock solution.

2.3 Macroscopic current and action potential recording and acquisition
Both trans-membrane currents and voltages were recorded using fire-polished patch pipettes fabricated from borosilicate glass capillaries (O.D. 1.5 mm, I.D. 1.0 mm, A-M Systems Inc.). The pipettes resistance was 1–4 M{Omega} when filled with the internal solution and the series resistance was approximately 70% compensated in voltage clamp mode. The inclusion of aspartate caused a liquid junction potential of about –10 mV (measured in each new batch of pipette solution) which was compensated electronically at the start of each recording.

Transmembrane voltages and ionic currents were recorded using an Axopatch 1D amplifier (Axon Instruments, Foster City, USA). Action potentials were initiated by depolarizing 3 ms constant current pulses (0.5–2.0 nA) delivered through the pipette at a rate of 0.5 Hz.

Membrane potential and currents were monitored on a storage oscilloscope, and recorded and analyzed on a microcomputer using Cellsoft aquisition software (Dale Bergman, University of Calgary). All macroscopic current and action potential experiments were performed at 36°C.

2.4 Single-channel recordings and data acquisition
Standard tight-seal patch-clamp recording techniques [22] were used to record single-channel currents in the inside-out patch configuration. Pipettes were pulled from borosilicate glass (PG52151-4, World Precision Instruments Inc., Sarasota, FL, USA), their shanks near the tip were coated with a silicone resin (Sylgard 184, Corning, NY, USA) and the tips were fire polished. Normally pipettes had resistances of 2–7 M{Omega}, however, in several instances pipettes of 10–15 M{Omega} were used to obtain only a single channel in a patch. After the establishment of a seal (>10 G{Omega}), the pipette was rapidly pulled away from the cell, yielding an excised, inside-out patch. The inside face of the membrane patch was directly exposed to test solutions, via a multi-input perfusion pipette with a common outlet, at a flow rate of 100–150 µl/min. The time taken to change solutions was less than 2 s. All single-channel recordings were carried out at room temperature (20–22°C). For experiments using asymmetric K+ recording conditions, single channel currents were obtained at a holding potential of 0 mV.

Data were amplified (Axopatch 200, Axon Instruments Inc., Foster City, CA, USA), digitized (Neuro-corder DR-384, Neuro Data Instruments Corp., New York, NY, USA), and then stored on video tape. Raw data for ATP dose-response relations were replayed through a four-pole Bessel filter, low-pass filtered at 200 Hz (LPF-100, Warner Instruments Corp., Hamden, CT, USA) and sampled at 500 Hz using a computer interface (Axolab 1100, Axon Instruments Inc.) connected to an IBM PC-compatible computer for analysis.

2.5 Run-down of KATP channels
The activity of KATP channels in cardiac and other tissues "run-down" slowly with time after patches are excised into ATP-free solution. In order to slow the rate of run-down, patches were continuously exposed to 1 mM ATP upon excision, except for a brief exposure to 0 ATP at the beginning and end of experiments to estimate the number of channels in a patch, and the degree of run-down. Data from patches exhibiting >25% run-down were discarded.

2.6 Data analysis
KATP channel open probability was expressed as NPo, the product of N, the number of channels in the patch, and Po, the mean open probability. NPo was calculated by dividing the mean patch current (over a 10–30 s test period) by the mean unitary current amplitude (see below). For measurements of ATP sensitivity, NPo was usually expressed in normalized form for each patch, i.e. NPo(test [ATP])/NPo(zero ATP).

Single-channel conductances were measured under symmetrical conditions using the standard internal solution as pipette solution. Data were recorded at selected holding potentials, sampled at 2 kHz and filtered at 1 kHz using the acquisition procedure described above. Mean unitary current amplitudes were calculated from the difference between peaks in a multiple Gaussian fit to all points histograms constructed with data from data segments of 10–30 s in duration. Mean open and closed dwell times were generated from events lists (2818–10 150 events per distribution). For experiments on internal Mg2+ block of outward currents, data were recorded, at various holding potentials in symmetrical [K+], and the internal face of the membrane patch was exposed to either 0, 0.5, 1 or 2 mM Mg2+ in the absence of ATP. Single-channel current—voltage relations were determined as described above. All data were analyzed using pCLAMP v 5.5 and 6.0 software (Axon Instruments, Inc).

2.7 Computer simulations of action potentials
Action potentials in Purkinje cells were simulated using OXSOFT HEART version 4.4 (the model is updated from DiFranceso and Noble [23]). Calculations of the contribution of IKATP followed the work of Nichols and Lederer [24]. The KATP conductance was represented as an ohmic conductance, whose inhibition with increasing [ATP] is described by a saturating function with a Hill coefficient of 2 (note: the Hill coefficient from Purkinje KATP channels measured in this study is 2.1). Our own calculations used the slightly higher external [K+] of our experiments, and our measured IC50 for ATP inhibition (Purkinje, 0.119 mM). The model used, as a starting point, was the guinea pig ventricular action potential and repolarizing currents were adjusted to reproduce, as nearly as possible, the action potential waveforms seen for single Purkinje cells (Fig. 1). The major differences in repolarizing currents between ventricular and Purkinje cells were an increased Ito current and a decreased IK1 current in the Purkinje cell [21]. Accordingly, we increased the contribution of Ito to a value of 0.25 µS and decreased the IK1 conductance to one third of its value in ventricle. The contribution of the delayed rectifier (IKR) was reduced from 1 nA to 0.8 nA. These values were used in the calculations illustrated (Fig. 9), and were chosen to increase APD90 without drastically changing the resting potential. In the simulations, the contribution of IKATP to the total current was changed by varying the [ATP] in the range of 1–10 mM. This means that the simulated effects of IKATP on APD90 result from activation of only a very small fraction (Po~0.0001–0.01) of the available KATP channel conductance). In the simulations, reducing intracellular [ATP] mimics the result of activating IKATP with levcromakalim or NaCN/2-deoxyglucose in the Purkinje cell action potential experiments.


Figure 1
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  		Fig. 1 Representative examples of action potentials: A. in rabbit Purkinje cells, and B. in ventricular myocytes, recorded under control conditions and in the presence of 10 µM levcromakalim. The solid horizontal line represents 0 mV. Addition of 10 µM levcromakalim resulted in a shortening of APD in both cell types. The levcromakalim-induced shortening of action potential duration is reversed upon application of 5 µM glibenclamide. The cells were stimulated at a basic cycle length of 2 s and the bath temperature was 37°C.

 

Figure 9
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  		Fig. 9 A. Simulation of the Purkinje action potential and the effect of activating KATP channels by lowering intracellular ATP. The degree of KATP channel activation, as a fraction of the maximum, changes in a putative physiological range from ~0.0001 ([ATP]=10 mM) to ~0.01 ([ATP]=1 mM). The IC50 value for ATP-inhibition used was 0.119 mM, as measured from our single channel experiments. The displayed simulations were calculated as four individual action potentials (from the same starting conditions) with an instantaneous change in [ATP] from 10 to 5, 2.5 and 1 mM respectively. Thus, conditions for the simulated traces correspond to values of the ratio [ATP]/IC50 ranging from 8.4–84. Further increases in [ATP] yield predicted additional increases in action potential duration of no more than ~3%. B. Replotted ATP-dose response curve from Fig. 6B extrapolated to the millimolar range of [ATP]. C. Expanded section of the dose response curve shown in B (1–10 mM [ATP]), to illustrate the very low range of fractional KATP channel activity required to cause the changes in action potential durations simulated in A. See Methods and Results for more details.

 
2.8 Statistics
Statistical significance was evaluated by Student's paired t test. Differences with values of probability P<0.05 were considered to be significant. All values in the text are mean±S.E.M.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Cell morphology
Cells were identified on the basis of morphology and some of their electrical properties [21]. The shape and size of the rabbit Purkinje cells isolated using our technique differed significantly from ventricular cells from the same heart. Typical rabbit Purkinje cells were longer (approx 130–140 µm vs. 90–100 µm) and smaller in diameter than ventricular cells (about 10–15 µm vs. 15–20 µm). The resting input resistance of the Purkinje cell averaged 191±15 M{Omega} and the mean capacitance was 53±6 pF (n=15). In comparison, rabbit ventricular cells had an input resistance of 36±13 M{Omega} and a capacitance of 82±11 pF (n=15). From these values it was estimated that the macroscopic conductance at rest for Purkinje cells was 99 pS/pF and 339 pS/pF for ventricular cells.

3.2 Effects of ATP-sensitive K+ openers on action potentials
The action potential waveforms of both ventricular and Purkinje cells from rabbit myocytes were recorded. Fig. 1 shows representative action potentials obtained from a rabbit Purkinje cell (A) and from a rabbit ventricular cell (B) recorded under the same conditions. Both cells were stimulated at a basic cycle length of 2 s. When the two action potential waveforms were compared under steady-state control conditions, Purkinje cells exhibited a more prominent phase 1 repolarization, a more negative plateau, and the Purkinje cell APD was significantly longer (a mean of 402±33 ms, compared to 288±17 ms in ventricular cells, n=6 for both groups). Under control conditions, most Purkinje cells had a stable resting potential of –81.8±2.9 mV (n=11) in 5.4 mM K+. In normal [K+]o, Purkinje cells sometimes showed one or two early after-depolarizations (EADs) before fully repolarizing. The resting potential of these cells (–79.4±3.7 mV, n=3) was slightly more positive than cells not exhibiting EADs, although not significantly so.

Addition of 10 µM levcromakalim to the Purkinje cell resulted in a marked shortening of the APD. After 2 min (APD90 was reduced from 402±33 ms to 197±27 ms, n=6, Fig. 1A). Levromakalim had little effect on rapid phase 1 repolarization. Early after-depolarizations were never observed when Purkinje cells were exposed to levcromakalim. Exposure of ventricular cells to 10 µM levcromakalim after 2 min also resulted in a pronounced shortening of APD (APD90 was reduced from 288±17 ms to 141±14 ms, n=6, Fig. 1B). The percentage reduction in APD90 induced by levcromakalim was similar between Purkinje (51%) and ventricular cells (52%): these values were not significantly different from each other. The effects of levcromakalim on APD shortening were almost fully reversed by the ATP-sensitive K+ channel blocker glibenclamide (5 µM) 5 min after addition.

3.3 Effects of ATP-sensitive K+ openers on membrane currents
Fig. 2A,B (left side of figure) shows a family of membrane K+ currents recorded under control conditions from a representative Purkinje myocyte and ventricular myocyte, respectively. In these experiments, each myocyte was held at –80 mV and stepped for 1 s to membrane potentials between –120 and +60 mV in 10 mV increments. As expected, hyperpolarization activated an inwardly rectifying background K+ current (IK1) in both types of cells. The magnitude of IK1 was noticeably smaller in Purkinje cells (Fig. 2A) than ventricular myocytes (Fig. 2B), being approximately 1 nA and 3 nA at –120 mV, respectively (see Cordeiro et al. [21]). Voltage clamp steps in the depolarizing direction activated a large transient outward current (It) in the Purkinje cell. Application of 10 µM levcromakalim resulted in a substantial increase in the magnitude of outward current in both cell types after 2 min (right side of figure); there was also an increase in the magnitude of current in the inward direction.


Figure 2
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  		Fig. 2 Comparison of membrane currents from: A. a Purkinje cell, and B. a ventricular cell, under control conditions (left side) and in the presence of 10 µM levcromakalim (right side). In each experiment, the myocyte was held at –80 mV. Membrane currents were elicited by stepping the voltage from –80 mV to membrane potentials between –120 and +60 mV. Addition of levcromakalim resulted in a substantial increase in outward current in both cell types. Dashed line denotes zero current level.

 
The mean current—voltage relation from four Purkinje cells is illustrated in Fig. 3A. Under control conditions, the I–V curve shows a large inward component (IK1) at potentials negative to –80 mV; at potentials positive to –80 mV there is little current in the outward direction. Addition of 10 µM levcromakalim resulted in a substantial increase in the magnitude of current in the outward direction after 2 min application. Fig. 3B shows the mean current—voltage relation from 4 ventricular cells. In the absence of levcromakalim, the I–V relation curve shows a large inward component (IK1) at potentials negative to –80 mV, peaks at –60 mV and exhibits negative slope conductance at potentials between –60 and –20 mV. Addition of 10 µM levcromakalim for 2 min resulted in a marked increase in the magnitude of current in the outward direction; there was also an increase in inward current in the presence of 10 µM levcromakalim. The observed effects of levcromakalim were fully reversed by 5 min application of the KATP channel blocker glibenclamide (5 µM). In order to separate the background IK1 current from the levcromakalim-induced IKATP current, Fig. 3Aii and 3Bii show the differences between currents before and after addition of levcromakalim from the Purkinje and ventricular data sets respectively. In both groups, the subtracted currents reversed close to the expected equilibrium potential for potassium (–84.7 mV) calculated for the potassium concentrations and temperature used.


Figure 3
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  		Fig. 3 A comparison of current—voltage relations for A. a Purkinje cell, and B. a ventricular cell, recorded under control conditions (filled circles) and in the presence of drug (filled squares). Under control conditions (filled circles) Purkinje cells (A.i) exhibited an inward current (IK1) at potentials more negative than approximately –80 mV. Ventricular cells (B.i) had a notably larger IK1 than Purkinje cells. Addition of 10 µM levcromakalim resulted in a marked increase in the magnitude of outward current in both cell types (filled squares). This levcromakalim-induced increase in outward current is reversed upon application of 5 µM glibenclamide. In order to separate the background IK1 current from the levcromakalim-induced IKATP current, parts A.ii and B.ii show the difference between currents measured before and after addition of levcromakalim for the Purkinje and ventricular data sets respectively.

 
3.4 Effects of glibenclamide on action potential durations in normoxic conditions
In order to test the possibility that KATP channels are active in the resting Purkinje cell, we tested the effects of glibenclamide on action potential waveforms under normoxic conditions. In 6 Purkinje cells, glibenclamide alone did not alter the APD significantly. The APD under control conditions was 387±47 ms. After 10 min exposure to 5 µM glibenclamide, the APD was 393±42 ms. Fig. 4A shows the action potential waveform from a representative Purkinje cell, before and after application of glibenclamide.


Figure 4
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  		Fig. 4 Representative action potential waveforms from single Purkinje cells. A. Under normoxic conditions before and after the application of 5 µM glibenclamide. B. Before and after 4 and 6 min exposure to the metabolic inhibitors NaCN (2 mM) and 2-deoxyglucose (DG, 10 mM). The effects of 5 min application of 5 µM glibenclamide (glib), in the presence of NaCN and 2-deoxyglucose, on the action potential waveform are also shown. Note that the action potential is significantly shortened under conditions of metabolic stress (NaCN/DG) and this effect is almost fully reversible upon addition of the KATP channel blocker glibenclamide.

 
3.5 Activation of Purkinje KATP channels during metabolic compromise
We have demonstrated that the addition of KATP channel openers, such as levcromakalim, activate KATP channels in Purkinje cells under normoxic conditions leading to action potential shortening. However, we predict KATP channels are also capable of opening under conditions of metabolic compromise such as may occur during ischemia. In order to test this hypothesis, we exposed Purkinje cells to NaCN and 2-deoxyglucose, inhibitors of oxidative phosphorylation and glycolysis respectively. In 3 Purkinje cells, exposure to 2 mM NaCN and 10 mM 2-deoxyglucose caused APD to shorten from 376±51 to 153±21 ms after 6 min. Addition of glibenclamide (5 µM), in the continued presence of NaCN and 2-deoxyglucose, caused the APD to partially recover to a value of 321±44 ms, after 5 min application (Fig. 4B).

3.6 Single channel properties
In order to examine the single-channel properties of KATP channels from Purkinje cells, current recordings were made from inside-out membrane patches. When patches were excised into an ATP free solution, spontaneous channel activity was observed; subsequent application of internal ATP (1 mM) led to inhibition of the current (for example, see Fig. 5).


Figure 5
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  		Fig. 5 Current recording from an inside-out membrane patch containing at least 10 KATP channels. Removal of internal ATP (1 mM) causes reversible channel activation. Currents were recorded under asymmetric K+ conditions at a holding potential of 0 mV. Dashed line denotes zero current level. Data were sampled at 500 Hz and filtered at 200 Hz.

 
3.7 Single channel conductance
Using data from patches containing only one or two channels it was possible to construct an I–V plot under symmetrical K+ conditions. Purkinje cell KATP channels exhibited a slope conductance of 60.1±2.0 pS (n=6) at negative holding potentials. This value is slightly lower than the one observed for KATP channels from rabbit ventricular myocytes published previously under identical conditions [15] (see Fig. 6A), although there is no significant difference between the means for the two groups (P>0.05).


Figure 6
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  		Fig. 6 A. Current—voltage relationship for single KATP channels from Purkinje cells (n=6). Data for ventricle replotted for comparison from Han et al. [15]. B. ATP concentration-dependence curves for KATP channels from Purkinje cells (n=7) and ventricular myocytes (n=8) from rabbit heart. Channel activity was recorded from inside-out membrane patches containing multiple channels and open probability was expressed as NPo (see Methods section). Note the significantly greater IC50 in Purkinje cell KATP channels compared to ventricular myocytes. Data for ventricle replotted for comparison from Light et al. [25]. C. Dwell time analysis of single KATP channels from Purkinje cells. (i). Representative trace of a single KATP channel in symmetrical [K+] (140 mM) at a holding potential of –60 mV. Open (ii) and closed (iii) dwell time histograms constructed from a single channel trace 10 s in duration, recorded under the same conditions as in C(i). Solid curves denote fits to a single exponential. Data were sampled at 2 kHz and filtered at 1 kHz.

 
3.8 ATP-sensitivity
Increasing the ATP concentration on the internal face of inside-out patches inhibited KATP channel activity in a dose-dependent manner (see Fig. 6B). Fitting of the ATP dose-response data with Eq. (1) yielded an IC50 value for ATP inhibition of 119 µM and a Hill co-efficient of ATP binding of 2.1 (n=7), see Fig. 5. This value is approximately five-fold greater than that for KATP channels from rabbit ventricle (IC50=21 µM) previously published by Light et al. [25]) and the values are significantly different (P<0.001) from each other.


Formula (1)

NPo is the product of N (number of channels in the patch) and Po (open probability), Formula o(max) is the open probability in the absence of ATP and [ATP] is the test concentration of ATP. IC50 is the [ATP] at which half-maximal inhibition occurs, n is the Hill coefficient of cooperativity of ATP-binding.

3.9 Single channel kinetics
In several patches, it was possible to record current fluctuations from a single KATP channel. It was thus possible to measure the open and closed times of a single KATP channel (see Fig. 6C). Both the open and closed time histograms were fitted with a single exponential. The mean dwell times for the open and closed distributions were 3.6±1.5 and 0.41±0.2 ms respectively (n=4). These values are similar to those published previously from rat ventricle [26,27] and rabbit sino-atrial node [15].

3.10 Inward rectification: dependence on internal magnesium
Internal magnesium causes voltage-dependent block of KATP channels from a variety of tissues, resulting in weak inward rectification at holding potentials positive to the equilibrium potential for potassium [28,29]. (For reviews see [3,4].) We demonstrate that KATP channels from Purkinje cells also exhibit internal magnesium dependence of inward rectification using both steady-state and voltage-ramp protocols. Fig. 7 shows a single channel recording of a KATP channel at selected holding potentials in the presence and absence of internal magnesium (1.4 mM). At negative holding potentials, magnesium had little effect on the magnitude of the single-channel current. At positive holding potentials, there was a magnesium- and voltage-dependent reduction in current that was accentuated with increasing depolarization. In the absence of magnesium, and with symmetrical K+, the single-channel current—voltage relation was almost linear. The degree of outward current block by 1.4 mM magnesium was similar to that previously observed in KATP channels from guinea-pig ventricular myocytes [28]. Magnesium dependent block was observed in all single Purkinje cell KATP channels tested (n=7 patches).


Figure 7
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  		Fig. 7 Internal magnesium block of KATP channels from Purkinje cells. A. Inside-out patch recording of a single KATP channel at various holding potentials under symmetrical K+ (140 mM) in the presence and absence of 1.4 mM intracellular magnesium (recordings made with 0 mM internal ATP). B. Current—voltage relationship for the recordings shown in A. C. Voltage ramps on the same KATP channel as in A, in the presence and absence of intracellular magnesium. Voltage-ramps were 1 s in duration and were leak subtracted using ramp recordings made in the presence of 1 mM internal ATP to silence channel activity. Data were sampled at 1 kHz and filtered at 400 Hz.

 
In order to quantify the magnesium dependence of inward rectification, further experiments were performed on 3 patches containing single channels. The effects of a range of internal magnesium concentrations (0, 0.5, 1 and 2 mM) were evaluated at three positive holding potentials.

Fig. 8A shows the reduction in single channel current as internal magnesium is increased (holding potential=+60 mV). Fig. 8B shows how the magnesium effect is both concentration and voltage-dependent in nature. The voltage dependence of block is illustrated by plotting the fractional unitary current remaining at each internal magnesium concentration, (I(Mg)/I(control)), against voltage. The solid lines show fits to the data using a modified Boltzmann Equation:


Figure 8
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  		Fig. 8 Internal magnesium block of KATP channels from Purkinje cells is dependent on voltage and concentration. A. Single channel recordings from an excised inside-out patch containing a single Purkinje KATP channel at a holding potential of +60 mV (symmetrical K+) at various internal magnesium concentrations. B. Fractional current remaining, in the presence of different Mg concentrations, plotted against holding potential. The symbols denote data for the following magnesium concentrations tested: open circles=0.5 mM; filled triangles=1.0 mM; open squares=2.0 mM (n=3 patches). The dashed lines are individual fits to the data at each Mg2+ concentrations (see Eq. (2) and Results section).

 

Formula (2)

Here, I(Mg)/I(control) is the unblocked fraction of single channel current, I(Mg) and I(control) are the single channel amplitudes measured in the presence and absence of Mg, respectively, [Mg] is the internal magnesium concentration, Ko is the dissociation constant of magnesium at 0 mV, z is the valence of magnesium, {delta} is the apparent electrical distance from the cytoplasmic side to the magnesium binding/blocking site, E is the holding potential. F, R and T are the Faraday constant, the Gas constant and absolute temperature, respectively. A least squares fit to the global data set including all three Mg2+ concentrations yielded values for {delta} and Ko of 0.61 and 24.4 mM, respectively. These parameters gave an acceptable qualitative description of the magnesium block, however a more accurate representation of the data set was obtained by separately fitting the data for each Mg2+ concentration (dashed lines, Fig. 8B); this procedure yielded the values for {delta} and Ko given in Table 1. The inability of the simple single-ion blocking model to precisely fit the whole data set with a single set of parameters has precedent in other analyses of K channel block [30–32]. Specifically, in all of these studies, the apparent electrical distance, {delta}, increases with increasing blocker concentration. This probably reflects the competition of the blocker and potassium ions for the several ion binding sites now known to exist within a K channel [33]. In the case of the data of Horie et al. [28], there is evidence for ion—ion interactions in their observed increase in magnesium dissociation rate with increasing external potassium, but a single value of {delta} provided an adequate description of block by magnesium concentrations between 0.5 and 5 mM. This may reflect differences in the binding affinities of sites in KATP channels in the two preparations – guinea pig ventricular myocytes in the work of Horie et al. [28] and rabbit Purkinje cells in our own study. This minor difference provides a further hint of the possibility of small, but functionally important, molecular differences between KATP channels in Purkinje cells and ventricular myocytes.


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  		Table 1 Voltage-dependent block of single KATP channels by cytoplasmic Mg2+-parameters for individual Boltzmann fits

 
3.11 Action potential simulations
To explore further the effects of altering the activation of Purkinje KATP channels on action potential waveform configuration, we used a modified version of the guinea pig ventricular myocyte model developed by DiFrancesco and Noble [23], as implemented in OXSOFT HEART version 4.4. Specific details are provided in the Methods. Simulated action potentials for a range of ATP concentrations are shown in Fig. 9. Our results suggest the following:

The prominent phase 1 repolarization (and associated notch) and the long duration action potential in Purkinje cells are explained mainly by differences in the Ito and IK1 current magnitudes compared to ventricular cells [21] which were altered in the model according to experimental values for Ito and IK1 obtained directly from Purkinje cells [21]. The action potential duration is very sensitive to the intracellular ATP concentration, even in the physiological or mildly pathological millimolar range (Fig. 9). Decreasing [ATP], and consequent activation of KATP channels, results in significant action potential shortening. This suggests that the activated IKATP is contributing a significant proportion of the net transmembrane current during repolarization. The prominent effect of KATP channel activation on action potential duration occurs even though the actual fractional activation of IKATP is very small (~0.0005–0.01, corresponding to [ATP] in the range of 10 to 1 mM, Fig. 9B,C). Together with the fact that glibenclamide did not significantly change the APD in normoxic conditions, the simulations suggest that, in the Purkinje fibres during our action potential recordings, the ratio [ATP]/IC50≥100. This implies that the IC50 for ATP inhibition is slightly lower than the value we measured in excised patches due to the presence of one of the KATP channel's other cytoplasmic modulating factors. A decrease in IC50 to between 0.5x and 0.2x of our measured value would probably account for our observations. Energetically, this decrease represents a change of <1 kcal/mole in the free energy of ATP binding from the value implied by our measurements.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Our results show that isolated Purkinje cells from rabbit heart express ATP-sensitive K+ channels with pharmacological and biophysical properties similar to those in other cardiac cells. The ATP-sensitive K+ channels in Purkinje cells do, however, show a somewhat lower ATP sensitivity. This would allow the Purkinje cell KATP channels to play a stronger role in modulating action potential duration and cardiac rhythms during periods of hypoxia and ischemia.

4.1 Pharmacological results
We have demonstrated that glibenclamide has no effect on the Purkinje fibre action potential duration (APD) under normoxic conditions, but reverses action potential shortening induced by application of NaCN and 2-deoxyglucose. These results are similar to those reported previously [19]. Thus, our findings suggest that KATP channels are not active under normoxic conditions but may play an important role in modulating Purkinje fibre excitability under conditions of metabolic stress. In accordance with these findings, we show that glibenclamide (1) reverses the levcromakalim-induced APD shortening and (2) abolishes ATP-sensitive K+ channel activity induced by levcromakalim in whole-cell recordings. Our observations demonstrate that ATP-sensitive K+ channels are expressed in Purkinje tissue and this conclusion is confirmed by the single channel data presented in Figs. 4–8GoGoGoGo.

4.2 Single channel properties
We provide the first direct recordings from KATP channels in Purkinje tissue. Our experimental observations include: sensitivity of macroscopic currents and unitary current fluctuations to internal ATP, inhibition by micromolar glibenclamide, activation by the KATP channel opener levcromakalim, and inward rectification induced by internal magnesium. The magnitude of the single-channel conductance measured from Purkinje cells was 60.1±2.0 pS, which is similar to that measured in rabbit ventricular tissue (70 pS) under identical conditions. This value is also close to the range (70–90 pS) of unitary conductances previously observed in various heart preparations in the presence of symmetric K+ [3].

KATP channels from Purkinje cells exhibit single-channel burst kinetics similar to those reported previously for ventricular KATP channels [26,27]. These channels also display a characteristic block by internally applied magnesium. The degree of inward rectification depends on magnesium concentration. Our data are similar to previously published observations on magnesium block of guinea-pig ventricular KATP channels [28]. Some quantitative differences between characteristics of magnesium block in the two preparations are noted in the Results. In different ways, however, each of the studies provides evidence consistent with the notion that Mg2+ enters the transmembrane electric field and blocks a multi-ion pore, in which ion—ion interactions can occur.

The major difference between KATP channels from Purkinje cells and from ventricular tissue is the reduced sensitivity to ATP. Under identical recording conditions, Purkinje cells show a five-fold higher IC50 for ATP inhibition (119 µM) than rabbit right ventricular cells (IC50=21 µM, [25]). Other factors being equal, this implies that KATP channels in Purkinje cells operate at higher open probabilities and on a steeper section of the dose-response curve than those channels from ventricular myocytes. The experimentally determined dose-response relation underlies the dramatic modulation of action potential duration predicted in Fig. 9 as cytoplasmic [ATP] varies between 1 and 5 mM.

Previous studies have demonstrated that ATP-sensitive K+ channels can be modulated under various conditions. For example, ischemia/hypoxia have been shown to abbreviate action potential duration by activating ATP-sensitive K+ channels [3,19]. KATP channel activity is clearly controlled by intracellular ATP, but it is now generally thought that the ATP/ADP ratio is the major determinant of channel activity [3,38]. In addition, KATP channels can be modulated by lactate [34], adenosine [35,36], pH [37] and protein kinase C [25,39]. Further detailed study of other factors affecting Purkinje KATP channel activity would be interesting, and may suggest strategies for treatment of some forms of arrhythmia.

One possible explanation for the difference in ATP sensitivity may be that Purkinje cells express a different channel isoform. At the molecular level, there is now evidence that there are different KATP channel isoforms with differing ATP sensitivities [40]. Recent cloning has revealed that KATP channels are composed of at least two subunits, a sulfonylurea receptor, SUR, and an inwardly rectifying potassium channel, Kir6.2 [41]. It is known that SURs exist in at least three isoforms (SUR1, 2A, and 2B) and that there is preferential expression of one or more of these isoforms in different tissues [40]. Thus, it is possible that the differences in ATP-sensitivity among KATP channels from Purkinje cells, ventricle and SA node [25,15], are due to differential expression of the subunit isoforms that constitute the KATP channel. However, this possibility remains to be tested and other intrinsic factors modulating the properties of KATP channels from various regions of the heart cannot be discounted.

4.3 Physiological and clinical relevance
Under different circumstances, individual drugs targeted towards KATP channels may be either pro- or anti-arrhythmic, as well as having significant metabolic effects. For example, glibenclamide has been observed to exacerbate reperfusion arrhythmias due to after-depolarizations, but to abolish re-entrant arrhythmias (for a review, see [42]). Similarly, KATP channel activators may be either pro- or anti-arrhythmic. In the case of the Purkinje network, potassium channel openers such as levcromakalim may be anti-arrhythmic because the reduction of action potential duration would tend to reduce the likelihood of EAD-induced arrhythmias. Membrane hyperpolarization induced by KATP openers generally reduces or abolishes abnormal automaticity (spontaneous impulse generation arising from unusually depolarized diastolic potentials [42]). However, without actually knowing the membrane potential and other cellular variables during ischemia, it is difficult to predict the precise end effect of a specific pharmacological treatment.

Cardioprotection due to reduction of metabolic activity has been attributed to KATP channels. Various authors have suggested that, in working ventricular myocytes, activation of these channels shortens the action potential duration (APD) [24] thereby limiting Ca2+ influx and thus decreasing contractility [2,5–7]. During ischemia, these events may protect the myocardium from Ca2+ overload and excessive energy expenditure [8], but some recent studies have questioned this hypothesis (e.g. [43–45]). In the case of Purkinje cells, we suggest that the direct electrical effects of modulating KATP channel activity are likely to be more important to overall cardiac excitability and mechanical function than are the metabolic consequences.

It is known that hypoxic conditions significantly shorten the Purkinje action potential duration [19] and results from our study indicate that: (1) as KATP channels in Purkinje cells have a relatively high IC50 for ATP inhibition they may operate on a steeper segment of the ATP-dose response curve than ventricular KATP channels (see Figs. 5B and 9BGo,C); (2) because Purkinje cells have a higher background resistance and exhibit action potentials with a more prolonged plateau phase than ventricular myocytes [21], the introduction of a repolarizing current such as IKATP is expected to lead to a more pronounced action potential shortening; (3) taking the two latter considerations together, even changes of [ATP] in the millimolar range would significantly shorten the action potential (see simulations of the Purkinje action potential in Fig. 9A). Thus, even relatively mild periods of ischemia may induce a significant KATP channel-mediated response. This combination of factors may provide one reason why arrhythmias tend to be initiated in the Purkinje conduction network, or at its junctions with the myocardium, during ischemia or reperfusion [12]. In addition, regional activation of Purkinje KATP channels may contribute to the dispersion of refractoriness. Another consideration is that although the levels of glycolytic enzyme in Purkinje fibres and myocardial cells are comparable, the levels of enzymes involved in oxidative phosphorylation are lower in Purkinje fibres [46]. This suggests that during ischemia, Purkinje fibres may be forced to support much of their energy requirement (ATP production) anaerobically through glycolysis. This would raise intracellular H+ and lactate levels, both of which are known to further activate KATP channels [34,37]. In addition, the fact that Purkinje fibres are less reliant on oxidative phosphorylation and possess larger glycogen stores suggests that this cell type may be less susceptible to damage occurring during ischemic episodes [46].

4.4 Conclusions
We have identified KATP channels in Purkinje cells of rabbit heart and shown that they exhibit all of the general biophysical and pharmacological properties of their counterparts in other regions of the heart. However, the KATP channels of Purkinje cells require about a five-fold higher ATP concentration for 50% inhibition than do those of ventricular myocytes. This fact, combined with the higher input resistance and sustained plateau-phase of the action potential of Purkinje cells, suggests that Purkinje KATP channels are likely to be important in the modulation of cardiac rhythm and conduction in the working heart. Activation of Purkinje KATP channels may contribute to Purkinje tissue-derived arrhythmic activity that is frequently observed during ischemia and early reperfusion.

Time for primary review 26 days.


   Acknowledgements
 
This study was supported by operating grants from the Medical Research Council of Canada (MRC) and the Heart and Stroke Foundation of Canada and by an MRC Group Grant for research on the Biophysics of Ion Channels and Transporters (P.I., Dr. W.R. Giles). We thank Dr. Giles for discussions, support and encouragement during the course of the work. J.M. Cordeiro is a recipient of a Heart and Stroke Foundation of Canada Fellowship and an Alberta Heritage Foundation for Medical Research (AHFMR) Fellowship. R.J. French is an MRC Distinguished Scientist and an AHFMR Medical Scientist.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
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