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Cardiovascular Research 2003 57(2):497-504; doi:10.1016/S0008-6363(02)00668-5
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Copyright © 2003, European Society of Cardiology

Modulation of the hyperpolarization-activated current (If) by calcium and calmodulin in the guinea-pig sino-atrial node

Lauren Rigg, Paul A.D Mattick, Bronagh M Heath and Derek A Terrar*

Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK

* Corresponding author. Tel.: +44-1865-271-613; fax: +44-1865-271-853. derek.terrar{at}pharm.ox.ac.uk

Received 17 June 2002; accepted 10 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
The aim of this study was to investigate possible regulation of the hyperpolarization-activated current (If) by cytosolic calcium in guinea-pig sino-atrial (SA) node cells. Isolated SA node cells were superfused with physiological saline solution (36 °C) and the perforated patch voltage-clamp technique used to record If activated by hyperpolarizing voltage steps. A 10-min loading of SA node cells with the calcium chelator BAPTA (using 10 µM BAPTA-AM) significantly reduced the amplitude of If at all potentials studied (69±8% at –80 mV, n=6). BAPTA loading also shifted the voltage of half-activation (Vh) of the conductance from –83±2 mV in control to –93±2 mV in BAPTA (n=6) without significantly altering the slope of activation. The calmodulin antagonists W-7 (10 µM), calmidazolium (25 µM) and ophiobolin A (20 µM) caused similar reductions in If amplitude (73±4, 86±9 and 59±6% at –80 mV, n=6, 5 and 4, respectively) and shifts in Vh (11±3, 14±3 and 8±2 mV). In cells pre-treated with W-7, exposure to BAPTA caused no further reduction in current amplitude (n=6). If current amplitude was unaffected by the calmodulin dependent kinase (CaMKII) inhibitor KN-93 (1 µM) although this CaMKII inhibition did reduce L-type calcium by 48±19% at 0 mV (n=3). These results are consistent with a role for calcium and calmodulin in the regulation of If, via a mechanism that is independent of CaMKII. Alterations in intracellular calcium during the cardiac cycle may be involved in fine tuning the voltage-dependent properties of If and may thus determine its relative contribution to pacemaking in the SA node.

KEYWORDS Calcium (cellular); Ion channels; Membrane currents; Sinus node


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The hyperpolarisation-activated current (If) is one of several ionic currents thought to contribute to pacemaking in the sino-atrial (SA) node [1,2]. However, there has been much debate regarding the relative contribution of If to the generation of spontaneous activity, largely as a consequence of conflicting evidence concerning the threshold of activation and voltage-dependence of this current [3–5]. Several possibilities to explain the different degrees of activation of If have been proposed and include: differences in the level of intracellular calcium [6], size and shape of SA node cells from which If was recorded [7,8] and concentration of cyclic nucleotides in whole-cell patch pipette filling solutions.

A role for intracellular calcium in the regulation of If in pacemaker cells of the heart was originally proposed by Hagiwara and Irisawa [6]. Using the whole-cell patch clamp technique, they demonstrated that If was sensitive to changes in the level of calcium in the pipette internal solution. With an internal solution of pCa 10, If was decreased to ~20% of the current recorded at the beginning of whole-cell recording, whereas the amplitude of If was increased when the pipette contained a pCa 6 solution. The increase in calcium also shifted the voltage-activation of If to more positive potentials, whereas a decrease in pipette calcium caused a negative shift in the current–voltage relationship. Since the increase in If by internal calcium was unaffected by a non-selective protein kinase inhibitor and the calmodulin antagonist, calmidazolium, it was proposed that calcium had a direct effect on the gating properties of If. Subsequent experiments performed by Zaza et al. [9] were contrary to this conclusion. They used the inside-out macropatch technique and demonstrated that calcium did not have a direct effect on the If current. To date, there have been no further reports investigating the role of calcium in the modulation of If in the SA node.

The hyperpolarization-activated current is not only expressed in pacemaker cells of the heart; If (otherwise known as Ih) is widely distributed in the nervous system and plays a substantial role in determining rhythmic spontaneous behaviour in certain neurones. For example, in thalamocortical cells, Ih not only contributes to the discharge of single neurones, it also underlies sleep-related oscillatory activity [10].

It has been reported in thalamocortical cells that elevation of intracellular calcium upregulates Ih [11]. This is thought to be mediated by an increase in the levels of cAMP as a consequence of enhanced, calcium-stimulated adenylate cyclase activity [12]. However, there is still conflicting evidence regarding a role of calcium in Ih modulation in other areas of the brain; for instance, while chelation of intracellular calcium decreased Ih in neocortical neurones [13], primary afferent neurones did not seem responsive to levels of calcium in the pipette solution [14]. It is conceivable that differences in the regulation of If in various tissues may be the result of differing patterns of expression of either adenylate cyclase subtypes (e.g. calcium and non-calcium regulated) or different subtypes of hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels underlying whole cell If currents.

The present experiments were designed to test whether calcium plays a role in the regulation of the hyperpolarization-activated current in the guinea-pig SA node and to establish, at least in part, the mechanisms by which calcium exerts its actions.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell isolation
SA node cells were isolated using a combination of previously described techniques [15,16]. Briefly, male guinea-pigs (weighing 400–450 g) were killed by cervical dislocation following stunning in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (H.M.S.O.). The heart was rapidly removed and perfused by the Langendorff technique with a solution containing zero calcium for 3 min (composition (mmol/l): NaCl 137, KCl 5, NaHCO3 12, glucose 5, Na pyruvate 1, NaH2PO4 0.4, MgCl2 1, NaOH 1, EGTA 0.1, pH 7.4, 95% O2/5% CO2, 36 °C). This was subsequently replaced with a solution of the same composition, lacking EGTA (referred to as solution A) but containing 25 mg collagenase (Type 2, activity 308 U/mg, Worthington Biochemicals). Following collagenase perfusion for 12xthe weight of the heart in grams (maximum exposure, 30 min), the atria were removed and further dissected to reveal the SA node region in solution A (room temperature, 22–23 °C). The SA node region was identified as being bordered by the crista terminalis, superior and inferior vena cava and inter-atrial septum, cut into small strips (2x5 mm) perpendicular to the crista terminalis and dispersed in 2 ml of a solution high in potassium (composition (mmol/l): KCl 70, K2ATP 5, MgSO4 5, K+ glutamate 5, taurine, 20, trisphosphocreatine 5, EGTA 0.04, succinic acid 5, KH2PO4 20, glucose 10, HEPES 5, pH 7.2 with KOH) and stored at 4 °C for at least 1 h before use. SA node cells used in the present study were dissociated from the entire guinea-pig SA node and therefore represent a mixed population of cells with corresponding heterogeneity in shape; most cells were characteristically spindle shaped but ‘spider’ shaped cells were also used in these experiments [8,17]. Irrespective of their shape, all healthy SA node cells showed faint striations, well defined membranes and displayed regular spontaneous activity when superfused with physiological saline solution (solution B) (composition (mmol/l): NaCl 118.5, KCl 4.2, NaHCO3 14.5, KH2PO4 1.18, MgSO4·7H2O 1.18, glucose 11.1, CaCl2 2.5, pH 7.4, 95% O2/5% CO2, 36 °C).

2.2 Electrophysiology
Membrane currents were measured using the perforated patch clamp technique (Axopatch 200 B amplifier, Axon Instruments, USA). Patch pipettes (3–5 M{Omega}) were filled with a solution containing (mmol/l): KCl 150, MgCl2 5, K2ATP 1, HEPES 3, pH 7.2 with KOH. Amphotericin B (Sigma-Aldrich) was used at 240 µg/ml. Perforation took between 5 and 10 min after seal formation. A representative sample of the series resistance was taken from 20 cells and was found to be 37±5 M{Omega}. The series resistance compensation was corrected to above 40% and predicted to at least the same value. The experimental temperature was 36±1 °C. If was activated by step hyperpolarizations from a holding potential of –40 mV (–10-mV increments, 2–3-s duration, interval between pulses 10 s) and measured as the current between the instantaneous current recorded at the start of the pulse and the steady-state current at the end of the pulse. Control recordings were not taken until the series resistance was stable and data were rejected if the series resistance changed significantly during experimentation. Current data were normalised to the maximum current activated under control conditions and transformed into conductance values using the equation below, after the reversal potential of If was found to be –19.2±1.4 mV (n=10), using methods previously described [18]:

Formula
where gf is the conductance, If the current, E the membrane potential and Ef the reversal potential for current through channels conducting If. Activation curves for each cell were constructed by plotting normalised conductance as a function of membrane voltage. Data points were fitted by a Boltzmann function where Vh represents the voltage of half activation and k is the slope factor.

2.3 Drugs
1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA-AM) and KN-93 were obtained from Calbiochem-Novabiochem and were prepared in dimethyl sulphoxide (DMSO) as a 10-mM stock. W-7 and W-5 were obtained from Sigma and were prepared in distilled water as a 10-mM stock. Ophiobolin A and calmidazolium were obtained from sigma and prepared in DMSO as 10-, 20- and 25-mM stock solutions, respectively.

2.4 Statistics
Data are expressed as mean±S.E.M. and n represents the number of cells used. Student's paired t-test was used to determine drug effects and observations taken to be significantly different if P<0.05. Current amplitudes at –80 mV before and after drug are stated in many cases in this paper, since a repetitive voltage step to this voltage was use to monitor the progress of drug action during its onset; however, it should be noted that this amplitude will be a altered by both a shift in the Vh and any alteration in current density.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Modulation of intracellular calcium influences If
The membrane permeable acetoxymethyl (AM) ester of the calcium chelator, BAPTA [19], was used to investigate the role of calcium in the modulation of If. A reduction in If amplitude could be detected within a couple of minutes of BAPTA perfusion and recordings were taken after 10 min; a trace from a typical cell after 10-min exposure to BAPTA is shown in Fig. 1A. The left trace shows If under control conditions, and the right following a 10-min exposure to 10 µM BAPTA-AM. BAPTA loading significantly reduced current amplitude at all potentials studied; for example at –80 mV, If was reduced by 69±8% from –101±16 to –28±8 pA (n=6, P<0.05). Fig. 1B shows the effect of BAPTA on the voltage-dependent activation of If. BAPTA loading shifted the voltage of half-activation (Vh) from –83±2 mV in control to –93±2 mV in BAPTA (n=6, P<0.05) without significantly altering the slope of activation (control, 8±1 mV; drug, 8±1 mV, n=6, P0.05).


Figure 1
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  		Fig. 1 (A) Current traces in control and following exposure to 10 µM BAPTA-AM for 10 min. Cell capacitance 42 pF. (B) Conductance–voltage relationship in control (filled squares) and BAPTA loaded cells (open squares), n=6. Current normalised to max control current.

 
3.2 Effect of calmodulin antagonists on If
To determine whether calmodulin plays a role in the regulation of If the effects of the calmodulin antagonist, W-7 ([N-(6-aminohexyl)-5-chloro-1-napthelenesulphonamide, HCl]), were investigated. Fig. 2A shows current traces from a typical cell in control (left), following a 5-min (trace 2) and 10-min exposure (trace 3) to 10 µM W-7. Calmodulin inhibition significantly reduced the amplitude of the current at all potentials studied; at –80 mV, If was reduced by 73±4% from –143±39 to –42±14 pA (n=6, P<0.05). The effects of W-7 stabilised within 5 min and there was no additional decrease in current at 10 min (n=6). The effect of W-7 was almost completely reversible on washout (trace 4). Fig. 2B shows the effect of W-7 on the voltage-dependent activation of If. W-7 consistently shifted the voltage of half activation to more negative potentials without having any significant effect on the slope of activation (Vh control –79±3 mV and drug –90±3 mV, n=6, P<0.05; slope control, 11±1 mV, drug 14±2 mV, n=6, P0.05). The less potent calmodulin antagonist W-5 ([N-(6-aminohexyl)-1-napthelenesulphonamide, HCl]) did not shift the voltage activation of If (Vh control –100±2 mV and drug –105±3 mV n=3, P0.05).


Figure 2
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  		Fig. 2 (A) Current traces in control (left), 5-min and 10-min W-7 (centre traces) and following a 5-min wash period (far right). Cell capacitance 46 pF. (B) Conductance–voltage relationship in control (filled squares) and following 5 min in W-7 (open squares) (n=6). Current normalised to max control current.

 
In addition to the napthelene sulphonamides, the effects of a structurally different calmodulin antagonists were investigated. Calmidazolium (25 µM) and ophiobolin A (20 µM), other well-known calmodulin antagonists, caused 14±2 mV (n=4, P<0.05) and 8±2 mV (n=5, P<0.05) negative shifts in the Vh of If, without altering the slope of activation (P0.05). Calmidazolium (25 µM) and ophiobolin A (20 µM) also reduced If amplitude measured at –80 mV (86±9%, n=5 and 59±6%, n=4).

In a separate set of control experiments, 0.2% DMSO (double the peak DMSO concentration used in this study) caused no significant change in If amplitude or voltage dependence over a 15-min period (n=3, P0.05).

3.3 Lack of effect of BAPTA in the presence of W-7
Fig. 3A shows typical current traces recorded on step hyperpolarizations to –80 mV from a holding potential of –40 mV from an SA node cell in control, following a 10-min exposure to 10 µM W-7 and in the presence of W-7 and 10 µM BAPTA-AM. There was no significant change in If current amplitude in the presence of BAPTA loading following exposure to W-7; at –80 mV mean current amplitude in W-7 was –41±15 pA and in the presence of W-7 and BAPTA was –47±23 pA (n=6, P0.05). There was also no change in either Vh or slope of activation of If (Fig. 3B) in the presence of W-7 and BAPTA, compared to those data in the presence of only W-7 (Vh control –91±3 mV and drug –89±2 mV, n=6, P0.05).


Figure 3
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  		Fig. 3 (A) Raw current traces recored from a single SA node cell, after steps to –80 mV. Cell capacitance 31 pF. (B) Conductance–voltage relationship in the presence of combination of W-7 and 10 µM BAPTA-AM (10-min exposure) (n=6).

 
3.4 Lack of effect of CAMKII inhibition on If
To test whether the regulation of If by calmodulin is mediated through a specific kinase, the effects of a calmodulin kinase II (CaMKII) inhibitor, KN-93, were investigated [20]. Fig. 4A illustrates current traces recorded in the absence (left) and presence (right) of 1 µM KN-93 (10 min). Fig. 4B shows the effect of KN-93 on the voltage-dependent activation of If. There was no significant decrease in the amplitude of If or an alteration of the voltage-dependent properties of activation following exposure to the kinase inhibitor (n=6, P0.05). In three of these six cells, If was recorded simultaneously with the L-type calcium current (ICaL). Although there was no decrease in If amplitude in these cells, peak calcium current at 0 mV was reduced by 48+19% (P<0.05, n=3). This indicates KN-93 inhibition of CaMKII under these experimental conditions, leading to an effect on ICaL similar to that reported in rabbit SA node cells [21], being attributed to CaMKII regulation of ICaL.


Figure 4
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  		Fig. 4 (A) Current traces in control (left) and following exposure to 1 µM KN-93 (right). Cell capacitance 34 pF. (B) Conductance–voltage relationship in control (filled squares), and in KN-93 (open squares), n=6. Current was normalised to max control current. Inset: Peak calcium current (holding potential –40 mV, step to 0 mV) in the absence (black line) and presence (grey line) of 1 µM KN-93.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The results presented here are consistent with the hypothesis that cellular calcium does play a role in the regulation of If. It seems likely that calmodulin may be involved in this process since three structurally unrelated antagonists decreased If and shifted its voltage-dependent activation to more negative potentials. However, If was not affected by inhibition of CaMKII. It is expected that alterations in intracellular calcium during the cardiac cycle are involved in fine tuning the voltage-dependent properties of If and may thus determine its relative contribution to pacemaking.

As mentioned in the Introduction, conflicting data exist regarding the role of calcium in the regulation of If, both in pacemaker cells of the heart [6,9] and in the brain [11,14]. However, in the experiments presented here, chelation of intracellular calcium with BAPTA significantly reduced the amplitude of If and altered the voltage dependence of activation. These findings are consistent with the observations of Hagiwara and Irisawa [6] and Schwindt et al. [13] and support the hypothesis that intracellular calcium does modulate If in the guinea-pig SA node. It is possible too, that the observed effects of BAPTA on If may have been underestimated in these experiments since conditioning prepulses (simulating the normal action potential) were not given prior to recording If current–voltage relationships. One may expect that during repetitive firing of nodal action potentials, intracellular calcium may rise to levels in excess of that present during the voltage-clamp procedure.

So, how does calcium modulate If? One way in which calcium could exert its effect on the If channel could be via a calmodulin-dependent pathway. Although calmodulin has no intrinsic enzymatic activity, it regulates a wide variety of basic cellular processes [22]. In the present experiments, the effects of three, structurally distinct, calmodulin antagonists were tested; each decreased current amplitude at a given test potential, there being a greater effect of these antagonists at mid-activation voltages rather than voltages close to maximal activation. The reduction in current amplitude seems likely to be due to the observed negative shift in the voltage-dependence of If in the presence of calmodulin inhibition (there were negative Vh shifts of 11±1, 15±3 and 8±3 mV in the presence of W-7, calmidazolium and ophiobolin A, respectively). There was no significant change in current amplitude or voltage dependence when SA node cells were exposed to BAPTA loading in the continued presence of W-7. This is consistent with the hypothesis that the effects of calcium on If are mediated largely through a W-7 sensitive mechanism thought to involve calmodulin and not through a direct action on the channel. Lack of a direct effect of calcium on If is also supported by previous data from inside-out macropatch experiments showing that alterations in levels of free calcium at the cytosolic side of the membrane did not affect If [9].

The conclusion that calcium may be working via a calmodulin dependent pathway appears to differ from the work of Hagiwara and Irisawa, who did not detect an effect of calmodulin-dependent pathways on If [6]. They showed that 1 µM calmidazolium did not alter the ability of high internal calcium to increase If. In addition, the effects of high calcium on If were not altered in the presence of protein kinase inhibitors. Several factors might be taken into account when comparing previous experiments with those reported here. Firstly, the concentration of calmidazolium (1 µM) used in the Hagiwara and Irisawa experiments may not have been large enough to cause significant suppression of calmodulin-dependent pathways. In cardiac myocytes, calmidazolium has been used in various concentrations up to 100 µM [23,24]. In our experiments, 25 µM calmidazolium caused a significant reduction in If amplitude and a shift in If voltage dependence, as was the case with W-7 but not the less potent, W-5. Secondly, it is not clear whether calcium-calmodulin is acting directly on If channels or whether there is a modulation of a downstream signalling cascade such as adenylate cyclase, as in thalamocortical cells (see later discussion). If this is the case, it is possible that in the experiments of Hagiwara and Irisawa employing the whole-cell patch clamp technique, dialysis by the patch pipette may have interfered with the cytoplasmic pathways underlying the calcium-dependence of If. The present experiments were performed using the perforated patch clamp technique where dialysis is not expected to occur.

It is conceivable that all three, structurally unrelated, antagonists used in the present study may have caused the observed decrease in If current by non-specific means. For example W-7 may be acting by channel block. If this were the case, depression of maximal If would be expected, and this seemed not to be the case (Fig. 2). Another non-specific effect might be inhibition of If by the solvent, but DMSO alone had no effect on the amplitude and voltage-dependence of If. Therefore, it seems likely that a calmodulin-dependent pathway is involved in the regulation of If in pacemaker cells.

Calcium-calmodulin is a rather promiscuous complex and is responsible for the activation of various cellular proteins. Recent studies have suggested that calcium-calmodulin dependent protein kinase (CaMKII) can modulate various ion channels in the heart: the transient outward K+ current [25] and the L-type calcium current [21,26]. It seemed likely that this may also be the case for If. However, the present study demonstrates that, at concentrations found to inhibit ICaL in the rabbit SA node, KN-93 (selective antagonist of CaMKII) had no effect on either the amplitude or voltage dependence of If (Fig. 4).

The observed negative shift in If activation is similar to that reported for the effect of acetylcholine (Ach) on If in isolated SA node cells; acting via muscarinic receptors, Ach mediates its actions largely through a decrease in the levels of cAMP [27]. It is conceivable that changes in the level of cyclic nucleotides could account for the observed modulation of If in the experiments reported here. Indeed, calcium-mediated upregulation of If in thalamocortical neurones is thought to be mediated via stimulation of a calcium sensitive adenylate cyclase (AC) and a subsequent increase in levels of cAMP [12].

There are numerous AC isoforms, which are heterogeneous in terms of regulation and cellular expression. Certain AC isoforms are calcium stimulated, this being mediated by calmodulin [28]; this would allow alterations of intracellular calcium to underlie production of cAMP. The presence of these specific AC isoforms in the SA node, which are not found in other cardiac tissue, would not be surprising considering the inhomogeneity of the SA node compared to other cardiac tissues. If this were the case then inhibition of calmodulin would decrease activation of calcium-stimulated AC, lower levels of cAMP and thus shift If activation to more negative potentials. These interesting possibilities remain for future investigation.

In conclusion, the data presented here are consistent with a role for calcium in the regulation of If. It is likely that changes in intracellular calcium during the cardiac cycle fine tune If and allow it to make a significant contribution to pacemaking. It should be borne in mind in future experiments that both the levels of calcium buffering (e.g. during whole-cell patch clamp experiments) and the administration frequency of standard voltage-clamp protocols will affect cytosolic calcium and thus the amplitude of If. Indeed, Zaza et al. [29] demonstrated in his experiments that coupled the action potential clamp with the perforated patch, that the amplitude of If was substantially larger than had been previously recognised using standard voltage-clamp protocols.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by The Wellcome Trust.


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

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