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Departments of 1 Medicine3 Anaesthesiology, University of Montreal4 Department of Pharmacology and Therapeutics, McGill University2 Research Center, Montreal Heart Institute, Montreal, Quebec, Canada
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Abstract |
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–84 mV with 5.4 mM[K+]o and changed by 55.7 ± 2.4 mV per decade [K+]o change. IKH was exquisitely Ba2+ sensitive, with a 50% inhibitory concentration (IC50) of 2.0 ± 0.3 µM (versus 76.0 ± 17.9 µM for instantaneous inward-rectifier current, P < 0.01), and showed similar Cs+ sensitivity to instantaneous current. IKH was potently blocked by tertiapin-Q, a selective Kir3-subunit channel blocker (IC50 10.0 ± 2.1 nM), was unaffected by atropine and was significantly increased by isoproterenol (isoprenaline), carbachol and the non-hydrolysable guanosine triphosphate analogue GTP
S. IKH activation by carbachol required GTP in the pipette and was prevented by pertussis toxin pretreatment. Tertiapin-Q delayed repolarization in atropine-exposed multicellular atrial preparations studied with standard microelectrodes (action potential duration pre- versus post-tertiapin-Q: 190.4 ± 4.3 versus 234.2 ± 9.9 ms, PV; 202.6 ± 2.6 versus 242.7 ± 6.2 ms, LA; 2 Hz, P < 0.05 each). Seven-day atrial tachypacing significantly increased IKH (e.g. at –120 mV in PV: from –2.8 ± 0.3 to –4.5 ± 0.5 pA pF–1, P < 0.01). We conclude that IKH is a time-dependent, hyperpolarization-activated K+ current that likely involves Kir3 subunits and appears to play a significant role in atrial physiology.
(Received 13 January 2004;
accepted after revision 12 March 2004;
first published online 12 March 2004)
Corresponding author S. Nattel: Research Center, Montreal Heart Institute, 5000 Belanger Street E, Montreal, Quebec, Canada H1T 1C8. Email: nattel{at}icm.umontreal.ca
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Introduction |
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Methods |
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Adult mongrel dogs of either sex (20–35 kg) were anaesthetized with pentobarbital (30 mg kg–1I.V.) and artificially ventilated with room air. Hearts and adjacent lung tissue were quickly excised through a left lateral thoracotomy and immersed in oxygenated Tyrode solution (composition below) at room temperature. Removal of the heart and lungs produced circulatory arrest, resulting in effective and humane killing. A left atrial (LA) preparation with the PVs intact was perfused via the left circumflex coronary artery, and subjected to either standard fine-tipped microelectrode recording of action potentials (APs) or cardiomyocyte isolation with collagenase-containing solutions, as previously described (Ehrlich et al. 2003). Six dogs were subjected to atrial tachycardia-induced remodelling induced by 1 week of atrial pacing at 400 beats min–1 after ablation of the AV node, as previously described (Li et al. 1999). All animal care and handling procedures followed the guidelines of the Canadian Council on Animal Care.
To isolate LA and PV cardiomyocytes, the proximal circumflex artery was cannulated and the distal ends of PV myocardial sleeves (approximately 1–1.5 cm from the PV–LA junction) were marked with silk thread prior to subsequent enzyme perfusion with collagenase (100 U ml–1, Worthington, type II), in order to facilitate localization of PV sleeves after enzymatic digestion. After a period of 45 min, epicardial tissue was removed and the underlying muscular sleeve of PVs was found to be well digested, with the smooth muscle layer still intact and unaffected by the isolation procedure. With this method, PVs were well-perfused and single cardiomyocytes could be isolated from all veins. Cardiomyocytes obtained from PVs were morphologically similar to LA cardiomyocytes isolated from the LA free wall in the same dogs. All comparisons were based on PV and LA cardiomyocytes isolated from each dog on each experimental day. After isolation, cells were stored at 4°C and studied on the same day. For standard microelectrode experiments, intact tissue preparations including the LA and adjacent PVs were mounted in a chamber and perfused via the circumflex artery with oxygenated Krebs solution at 36 ± 0.5°C (Kneller et al. 2002).
Electrophysiology
Currents were recorded with the whole-cell patch-clamp technique at 36 ± 0.5°C, as previously described (Yue et al. 1996). All junction potentials were zeroed prior to formation of gigaohm seals. The compensated series resistance and capacitive time constant (
) averaged 3.9 ± 0.1 M
and 257 ± 81 µs, respectively, and voltage errors across the series resistance did not exceed 5 mV. Capacitance was assessed using 5 mV, 10 ms hyperpolarizing steps from a holding potential (HP) of –60 mV. Junction potentials averaged 11.8 ± 0.9 mV and were not routinely corrected. Cell capacitance averaged 81 ± 4 pF for PV and 69 ± 8 pF for LA cardiomyocytes (n= 83, 29 cells, respectively, P= n.s.). Atrial tachycardia did not affect cell capacitance (84 ± 10 versus 91 ± 10 pF, n= 9 and 12 for PV and LA cells, respectively, P= n.s.). Original recordings are shown in terms of current amplitude, but mean data are presented as current density (pA pF–1) to control for variability in cell size.
Currents were recorded with hyperpolarizing and depolarizing pulses (generally 4 s duration) from a HP of –40 mV to selected test potentials (TPs). Recordings were repeated 3 times, and mean values obtained. For the determination of reversal potentials, tail currents were recorded after 1.6 s pulses to –120 mV followed by 3.2 s depolarizations to TPs between –110 and +20. All voltage protocols were delivered at 0.1 Hz.
Fine-tipped microelectrodes (resistance 15–20 M when filled with 3 M KCl) coupled to a high input-impedance amplifier were used to record APs as previously described (Kneller et al. 2002).
Solutions
Tyrode solution contained (mM): NaCl 136, KCl 5.4, MgCl2 1, CaCl2 1, NaH2PO4 0.33, Hepes 5 and dextrose 10 (pH 7.35 with NaOH). The cell-storage solution contained (mM): KCl 20, KH2PO4 10, dextrose 10, mannitol 40, L-glutamic acid 70, ß-OH-butyric acid 10, taurine 20, EGTA 10 and 0.1% bovine serum albumin (pH 7.3, KOH). Nifedipine (5 µM) was used to suppress L-type Ca2+ current (ICa) in all experiments. 4-Aminopyridine (4-AP, 2 mM) was added to suppress transient outward current (Ito). Atropine was added as indicated to the extracellular solution to suppress muscarinic receptor-activated currents. Na+ current (INa) contamination was avoided by using a HP of –40 mV for recording of hyperpolarization-induced currents and by substitution of equimolar Tris-HCl for external NaCl for tail-current recordings. When different external K+ concentrations were applied, the osmolarity was kept constant by proportionate reduction of NaCl content in the solution. The standard internal solution contained (mM): potassium aspartate 110, KCl 20, MgCl2 1, MgATP 5, GTP (lithium salt) 0.1, Hepes 10, sodium phosphocreatine 5 and EGTA 5.0 (pH 7.3 with KOH). In experiments with K+-free internal solution, potassium aspartate was replaced by equimolar caesium aspartate and KCl by CsCl and pH was set to 7.3 with CsOH. For standard-microelectrode experiments, a solution containing (mM): NaCl 120, KCl 4, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaCl2 1.25 and dextrose 5 (95% O2–5% CO2, pH 7.4) was used to perfuse the tissue.
Stock solutions of BaCl2 (1 M) and CsCl (1 M) were produced initially and used throughout the experiments. Stock solution of isoproterenol was prepared under protection from light on the day of experiments and freshly prepared ascorbic acid (100 µM) was added in order to prevent isoproterenol oxidization. Carbachol (1 µM) was dissolved in Tyrode solution, tertiapin-Q in 0.1% acetic acid. GTPS (0.1 mM) was used in place of GTP in internal solutions for some experiments. For experiments involving pertussis toxin (PTX, stock solution dissolved in distilled H2O) cells were incubated at 37°C in 1.5 mg l–1 PTX for at least 9 h prior to experiments. Parallel controls were performed with cells from the same isolates incubated in the same fashion and the same solution, but without PTX. Vehicle alone did not affect the current. Unless otherwise specified, drugs were obtained from Sigma.
Western blot, immunofluorescence studies and confocal imaging
After isolation of single cardiomyocytes, cells were suspended in lysis buffer (5 mM Tris pH 7.4, 2 mM EDTA, 5 mg ml–1 trypsin inhibitor, 0.1 mg ml–1 benzamidine, 0.43 mg ml–1 leupeptin). After homogenization (2 x 10 s bursts with a Polytron homogenizer) and centrifugation (20 min, 16000 r.p.m), pellets were resuspended in a buffer (75 mM Tris, 12.5 mM EDTA, 2 mM MgCl2). Proteins were fractionated on 7.5% SDS-PAGE gels, transferred to polyvinyl difluoride (PVDF) membranes and blotted with anti-Kir 3.1 (1: 1000), anti-M2 receptor (1: 500, both from Alomone), anti-G
i-3 (1: 500, Santa Cruz) and anti-Kir 3.4 antibody (1 µg ml–1, kind gift of Dr Krapivinsky). Bands were visualized with enhanced chemiluminescence. All immunoblot band intensity measurements were normalized to the GAPDH band intensity of the loaded sample (anti-GAPDH 1: 5000, RDI).
LA and PV cardiomyocytes were seeded on glass coverslips (prepared with 15 µg ml–1 laminin) for 1 h, fixed with 2% paraformaldehyde for 20 min, washed 3 times (5 min) with phosphate-buffered saline (PBS), then blocked with 2% normal donkey serum (Jackson Laboratories) and permeabilized with 0.2% Triton X-100 for 1 h in an incubation chamber. Cells were incubated with primary antibodies (anti-Kir 3.1 1: 200, anti-Kir 3.2 1: 400, both from Alomone), anti-Kir 3.4 1.3 µg ml–1) overnight at 4°C, followed by three washes with PBS (5 min) and incubation with anti-rabbit secondary antibody (conjugated with tetra-methyl-rhodamine-isothiocyanate (TRITC)) for 1 h at room temperature, Molecular Probes). Cells were examined on an inverted laser-scanning microscope (LCM 510, Zeiss, Germany). TRITC was excited at 543 nm with a He–Ne laser and emitted fluorescence signals at 566 nm (red).
Specificity of primary antibodies for Kir 3.1, 3.2 and 3.4 was validated by immunofluorescent studies of transfected and non-transfected mammalian cells (Chinese hamster ovary cells; American Type Culture Collection, Manassas, Virginia). Transfected cells showed clear staining, whereas no staining of non-transfected cells was observed with any of the primary antibodies (not shown).
Data analysis
Clampfit 6.0 (Axon) and Graph Pad Prism 3.0 were used for data analysis and non-linear curve fitting. Bands from immunoblots were analysed using QuantityOne software and immunofluorescence data were analysed using LSM Image Browser. Data are presented as means ±S.E.M. and statistical comparisons were performed with Student's t test. P < 0.05 was considered to indicate statistical significance.
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Results |
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Upon voltage steps from –40 mV, 25% of LA and PV cardiomyocytes showed instantaneous inward currents with small inactivating components and strong inward rectification typical of IK1 (Fig. 1A). In the remaining 75% of cells, we observed time-dependent inward currents that activated over several seconds upon hyperpolarization, with outward tail currents upon repolarization (Fig. 1B). We quantified the instantaneous current component present immediately upon resolution of the capacitive current, as well as the slow time-dependent component, which we identified with IKH. When the term IKH is used without additional qualification in this paper, it refers to the slowly time-dependent component. The slowly activating time-dependent current reversed at –72 mV (–84 mV with correction for the junction potential, compared to the calculated Nernst potential of –84.9 mV) and showed strong inward rectification (Fig. 1C). IKH was greater in PV cardiomyocytes (e.g. average at –120 mV: –2.8 ± 0.3 in PV versus–1.9 ± 0.2 pA pF–1 in LA, n= 27 and 26, respectively, P < 0.01), for both inward and outward (inset of Fig. 1C) components. Current activation kinetics were well-fitted by mono-exponential functions, with time constants decreasing at more negative potentials and no significant kinetic differences between LA and PV cardiomyocytes (Fig. 1D).
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Tail currents recorded upon steps to –40 mV after hyperpolarization to negative potentials were contaminated by activating Na+ current (INa, Fig. 2A). Replacement of extracellular NaCl with equimolar Tris-HCl eliminated the inward INa component without altering the outward current tail (Fig. 2A). For example, IKH tail amplitude (determined by back-extrapolation to the onset of the pulse to –40 mV) following a hyperpolarization to –120 mV was –185 ± 53 pA before and –182 ± 62 pA after Tris-HCl substitution (n= 5 cells studied under both conditions, P= n.s.). Tail-current density at –40 mV was a function of prepulse potential (Fig. 2B), suggesting voltage-dependent activation with a half-activation voltage (V50) of –93 ± 4 mV and a slope factor of –15.2 ± 2.2 (Boltzmann-distribution fit). Replacement of extracellular Na+ with Tris failed to significantly alter hyperpolarization-induced inward current, as illustrated in Fig. 2C and D. In five cells studied in this fashion, IKH at –120 mV averaged –3.1 ± 0.9 and –3.0 ± 0.9 pA pF–1 (P= n.s.) in the presence and absence of extracellular Na+, respectively. As an additional approach to analysing activation voltage dependence, we evaluated IKH current–voltage data obtained as shown in Fig. 1C according to the relationship:
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The reversal potential of the time-dependent component (Fig. 1C: –72.7 ± 1.6 mV, n= 6; –84 mV after correction for junction potential) was indicative of high K+ selectivity. With increasing [K+]ext., tail currents reversed at increasingly positive potentials (Fig. 2E). Tail current reversal potentials were quantified by linear regression of tail currents against voltage, based on data elicited with the protocol shown in the inset in Fig. 2E. A 10-fold change in external K+ led to a 55.7 ± 2.4 mV decade–1 shift in Erev (n= 4, Fig. 2E) compared to 61.5 mV decade–1 predicted by the Nernst equation for a K+-specific current at 37°C. Figure 2F and G show IKH recorded in one PV cell before and after exposure to nominally K+-free extracellular solution. In this and four other cells studied in the same fashion, elimination of extracellular K+ strongly suppressed IKH. When IKH was recorded with K+-free pipette solution (K+ replaced by Cs+), inward current remained but outward tail currents were absent (n= 3, Fig. 2H).
Figure 3 illustrates the response of IKH to extracellular Ba2+ and Cs+. Ba2+ inhibited IKH appreciably at a concentration of 1 µM in most cells and full inhibition was generally seen at 10 µM (Fig. 3A). Mean concentration–response data for inhibition of the instantaneous component and time-dependent IKH are shown in Fig. 3B. Ba2+ inhibited time-dependent IKH more potently than the instantaneous current (IC50, 76.0 ± 17.9 µM for instantaneous current versus 2.0 ± 0.3 µM for IKH at –120 mV, n= 8 cells each, P < 0.01). Figure 3C shows block of IKH as a function of time during pulses to –120 mV in four cells. The time-dependent current was calculated at each time point as the difference between the current immediately following hyperpolarization and the current level at the time point indicated. Fractional inhibition was calculated for each time point as the time-dependent current under control conditions minus the time-dependent current in the presence of Ba2+, divided by the time-dependent current under control conditions. Block showed minimal time dependence, suggesting that the difference in IC50 between instantaneous and time-dependent current is more likely to be due to intrinsic differences in sensitivity of different currents to block by Ba2+ than to a time-dependent blocking mechanism. Figure 3D shows the response of instantaneous current and IKH to Cs+. Both were highly and equally sensitive: at –120 mV, IC50 values averaged 139.0 ± 34.2 versus 184.0 ± 18.2 µM for instantaneous and time-dependent current, respectively (n= 6 cells each, P= n.s.).
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Isoproterenol was applied extracellularly at concentrations of 10, 100 and 1000 nM. Figure 4A shows the effect of 1 µM isoproterenol on IKH in a PV cell. A clear and reversible increase was seen, with steady-state effects achieved rapidly (within 2 min). Mean current density at –100 mV increased from –0.9 ± 0.1 pA pF–1 (control) to –1.2 ± 0.1 pA pF–1 in the presence of 10 nM isoproterenol, –1.4 ± 0.2 pA pF–1 with 100 nM isoproterenol and –1.5 ± 0.1 pA pF–1 with 1000 nM isoproterenol (n= 14 cells, P < 0.05 versus control for each concentration). Washout returned mean current amplitude to –0.9 ± 0.2 pA pF–1. Mean percentage changes from baseline at each isoproterenol concentration and upon washout are shown for PV cells in Fig. 4B. Isoproterenol also had concentration-dependent effects on holding current, which increased from 44 ± 11 pA pF–1 under control conditions to 65 ± 14 pA pF–1 (59 ± 14% increase) at 10 nM isoproterenol, 70 ± 13 pA pF–1 (89 ± 33% increase) at 100 nM isoproterenol and 87 ± 14 pA pF–1 (144 ± 37% increase) at 1000 nM (P < 0.05 versus control for all). Holding current changes were also reversible after washout.
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In view of the response to carbachol, we considered the possibility that channels composed of Kir3 subunits carry IKH. Tertiapin-Q is a 22-amino acid peptide synthesized from honeybee venom that blocks Kir3-based currents at nanomolar concentrations without affecting Kir2 currents (Jin & Lu, 1999), and has been found to block acetylcholine-dependent current in the heart in a highly selective fashion (Drici et al. 2000). Figure 5 shows a family of currents recorded in a PV cardiomyocyte under control conditions (A) and after exposure to 200 nM tertiapin-Q (B). The inhibitory effect was completely reversible upon washout of the drug (data not shown). Tertiapin-Q completely and reversibly abolished time-dependent IKH. Figure 5C shows mean current–voltage relations for the time-dependent component of the current in seven PV cardiomyocytes before and after tertiapin-Q. Whereas the time-dependent current was eliminated after 1 µM tertiapin-Q application, mean instantaneous current decreased by 25%, a change that was not statistically significant. These data indicate that the time-dependent component is entirely tertiapin-Q sensitive, whereas a statistically non-significant minority of the instantaneous current is blocked by tertiapin-Q, supporting the notion that the instantaneous component is largely carried by a conductance (probably IK1) that is distinct from IKH. Mean concentration–response data for the effect of tertiapin-Q on time-dependent IKH at –120 mV in four PV cardiomyocytes are shown in Fig. 5D, and provide an IC50 of 10 ± 2 nM, close to the reported IC50 for Kir 3.1/3.4 currents of 8 nM (Jin & Lu, 1999).
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Role of IKH in AP repolarization
Given the high selectivity of tertiapin-Q for Kir3 channels and its potent inhibition of IKH, we evaluated the effect of tertiapin-Q on repolarization of LA and PV APs in multicellular canine atrial preparations in the presence of 200 nM atropine to prevent any contribution of endogenously released acetylcholine. Figure 5E and F show representative APs before and after 100 nM tertiapin-Q in LA and PV. Mean AP duration characteristics are provided in the table at the bottom of Fig. 5, and indicate that tertiapin-Q significantly prolonged AP duration in both regions.
Potential role of G proteins in IKH regulation
In the light of a possible contribution of Kir3 channels to IKH current, we studied the effects of a variety of interventions targeting G proteins. In each case, experiments were performed in one cell under control conditions followed by another cell from the same batch studied in the presence of an intervention, in order to exclude data contamination by inter-day and inter-isolate differences in IKH amplitude and response. When GTP was not included in the pipette solution, basal IKH was not altered. However, in the absence of pipette GTP the addition of carbachol failed to further activate IKH (Fig. 6A and B). For example, at –120 mV current density in cells patched with GTP-free pipettes averaged –8.3 ± 0.7 versus–8.3 ± 1.1 pA pF–1(P= n.s., n= 4) before and after 1 µM carbachol, respectively. In the same batches of cells studied on the same days with GTP-containing pipettes, IKH increased from –6.2 ± 1.0 to –10.0 ± 1.2 pA pF–1(P < 0.05, n= 4) upon exposure to carbachol.
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S for GTP in the pipette. As illustrated in Fig. 6C and D, the inclusion of GTPS significantly increased IKH in the absence of cholinergic stimulation compared to current recorded with pipettes containing regular GTP (e.g. at –120 mV from –3.8 ± 0.6 to –8.2 ± 0.8 pA pF–1, P < 0.05, n= 6 cells each). In addition, GTPS fully prevented any further IKH response to carbachol, suggesting that IKH was already maximally activated in the presence of GTPS. Preincubation with 1.5 mg l–1 PTX also attenuated the effect of carbachol (Fig. 6E and F). For example, at –120 mV carbachol increased IKH density from –6.2 ± 1.8 to –11.5 ± 1.8 pA pF–1 in cells incubated without PTX at 37°C for >9 h (P < 0.05 for carbachol effect, n= 6). In cells preincubated with PTX at the same temperature and time, IKH density averaged –6.4 ± 0.8 pA pF–1 before carbachol and –5.5 ± 1.2 pA pF–1 in the presence of carbachol at –120 mV (P= n.s., n= 6). The response of IKH tail current to carbachol was significantly reduced in PTX-treated cells, but not abolished. For example, following hyperpolarization to –120 mV tail currents averaged 2.1 ± 0.7 and 8.4 ± 1.6 pA pF–1(P < 0.01) before and after carbachol under PTX-free conditions and 1.8 ± 0.6 and 3.4 ± 0.9 pA pF–1 (P < 0.01) before and after carbachol in PTX-preincubated cells (n= 6 for each). Overall, carbachol increased IKH tail currents following a pulse to –120 mV by 538 ± 170% in cells incubated without PTX versus 127 ± 37% (P < 0.05versus change in absence of PTX) in PTX-treated cells.
Potential role of IKH in atrial tachycardia-induced remodelling
Figure 7A shows mean ±S.E.M.IKH density–voltage relations in control dogs and dogs subjected to 7 day atrial tachycardia (AT) for LA and PV cardiomyocytes. IKH was increased modestly in LA cardiomyocytes. Larger increases in IKH were seen in PV cells.
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Possible molecular basis for IKH
The expression of Kir3.1, 3.2 and 3.4 channel subunits was evaluated in isolated LA and PV cardiomyocytes with Western blot and semiquantitative immunohistochemical methods. Immunohistochemical studies confirmed the presence of Kir3.1 and 3.4 on isolated LA and PV cardiomyocytes, with clear membrane staining (Fig. 8A and B). Kir3.2 staining was fainter and no outer membrane distribution was observed (Fig. 8C). Quantitative analysis of immunofluorescence indicated no significant differences between LA and PV cardiomyocytes in Kir3 subunit immunofluorescence intensity (right panels). Kir3 subunit expression as measured by Western blotting of isolated cardiomyocyte membrane preparations was not significantly different in LA versus PV (Fig. 9A and B). Atrial tachypacing reduced Kir3.4 expression, but did not significantly affect Kir3.1. M2 muscarinic receptor and inhibitory G protein (Gi) expression was down-regulated by atrial tachycardia-induced remodelling (Figs 9C and D), consistent with the decreased response of the instantaneous component to carbachol shown in Fig. 7C.
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Discussion |
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We characterized in detail a time-dependent inwardly rectifying K+-current, IKH, in canine atrial cardiomyocytes. IKH sensitivity to Ba2+ is instantaneous rather than time dependent, favouring conductance by Kir3 channels over Kir2 (Yamada et al. 1998). IKH sensitivity to tertiapin-Q resembles that seen with Kir3.1/3.4 channels (Jin & Lu, 1999), pointing to possible constitutive, agonist-independent Kir3.1/3.4 activity. IKH is subject to modulation by important endogenous neurotransmission systems (adrenergic and cholinergic), and by a recognized atrial arrhythmogenic intervention (atrial tachycardia-induced remodelling).
Possible molecular basis
IKH is an inwardly rectifying, highly K+-selective conductance sensitive to Ba2+, properties compatible with several inward-rectifying Kir subunits. Tertiapin-Q is a highly selective inward-rectifier K+ channel blocker that inhibits Kir1 channels with an IC50 of 2 nM and Kir3.1/3.4 with an IC50 of 8 nM, but has minimal effects on Kir2.1 channels at micromolar concentrations (Jin & Lu, 1999). Acetylcholine-dependent K+ current (IKACh) in native cells, based on Kir3.1 and 3.4 subunit heteromers, is suppressed by tertiapin-Q with IC50 values ranging from 8 to 30 nM (Drici et al. 2000; Kitamura et al. 2000), concentrations with no effect on IKr, IKs, Ito, IK1 or IKATP (Drici et al. 2000; Kitamura et al. 2000). The action of tertiapin-Q on IKACh is independent of muscarinic receptor activation state (Yamada, 2002). IKH-like kinetics have been noted for IKACh in human atrial myocytes (Heidbuchel et al. 1987) and Kir3.1/3.4 channels activated by Gß subunits (Reuveny et al. 1994). Kir1.1 subunits carry tertiapin-sensitive currents (Jin & Lu, 1999) and are detectable in PV cardiomyocytes (Michelakis et al. 2001); however, Kir1-based currents lack IKH kinetics (Schuck et al. 1994) and are more sensitive to tertiapin-Q than Kir3 current (Jin & Lu, 1999), IKACh (Drici et al. 2000; Kitamura et al. 2000) or IKH. All of these observations suggest that IKH is carried by constitutive Kir3 subunit activity.
PV expression of Kir3.1 and Kir3.4 proteins was not different from LA (Figs 8 and 9), suggesting that larger PV IKH may be due to regulatory factors, rather than differences in ion channel expression. Signalling events besides direct Gß modulation of Kir3 channels may be important for determining basal and agonist-stimulated current (Yamada et al. 1998). Kir3-channel activity is increased by protein kinase A (PKA) and inhibited by phosphatases (Mullner et al. 2000). Phosphatidylinositol phosphates increase current through Kir3 channels and are regulated by Gq-mediated signalling (Kobrinsky et al. 2000). Channel activity is regulated by intracellular sodium and chloride (Mirshahi et al. 2003) and by extracellular and intracellular pH (Mao et al. 2003). Stretch also inhibits Kir3 channels (Zhang et al. 2003). Ca2+–calmodulin facilitates GTPase activity of regulators of G protein signalling (RGS) proteins by suppressing inhibitory effects of phosphatidylinositol-3,4, 5-trisphosphate (PIP3) on RGS4 activity (Ishii et al. 2002). G protein-regulated K+ currents showed time dependence like IKH, suggesting that similar complex lipid–protein interactions may regulate IKH kinetics and function. Such regulation may be important in maintaining basal IKH activity, as neither PTX nor absence of GTP affected IKH in the absence of carbachol. Alterations in these signalling pathways may occur during atrial tachypacing and may lead to increased basal IKH. Further experiments are needed to define the exact mechanisms of IKH regulation.
Relationship to previous studies
Chen et al. (2001) observed time-dependent inward currents upon hyperpolarization of PV cardiomyocytes, and believed them to be If. These currents may correspond to IKH; however, since they were not characterized in the Chen study, it is impossible to be sure. Dobrev et al. (2001) noted IKACh down-regulation in atrial myocytes from patients with AF. Kir3.4 mRNA levels were reduced, but protein expression was not assayed. We observed decreased M2 receptor and Gi protein expression with atrial tachycardia-induced remodelling, which could account for IKACh down-regulation. We also saw slight decreases in Kir3.4, but not Kir3.1, protein expression with atrial tachycardia-induced remodelling.
Many properties of IKH suggest that it is carried by constitutively active IKACh channels. Evidence for constitutive IKACh channel activity in cardiomyocytes has been presented by Heidbuchel et al. (1992a, b). The lack of change in baseline IKH with GTP-free pipettes indicates that the constitutively active, time-dependent current does not require intracellular GTP under basal conditions. However, an important role for G protein regulation is indicated by the maximally increased current upon inclusion of GTPS in the pipette. The attenuation of the carbachol response by PTX preincubation and its abolition in the presence of GTP-free or GTPS-containing pipette solutions indicates that the signal transduction pathway coupling muscarinic receptor stimulation to IKH requires PTX-sensitive G proteins.
Potential significance
Tertiapin-Q prolonged AP duration recorded with standard microelectrode techniques from multicellular LA and PV preparations in the presence of atropine to exclude contributions from endogenous acetylcholine, pointing to a potentially significant role for IKH in LA and PV cardiomyocyte repolarization. Since atrial tachycardia-induced remodelling increases IKH, it may contribute to AF-promoting action potential abbreviation caused by persistent atrial tachyarrhythmias (Yue et al. 1997; van der Velden et al. 2000).
The autonomic nervous system (parasympathetic and sympathetic) is known to contribute to atrial arrhythmogenesis. ß-Adrenergic stimulation hyperpolarizes atrial myocytes (Boyden et al. 1983); however, isoproterenol typically inhibits IK1 (Koumi et al. 1995; Zhang et al. 2002). The increase in IKH caused by ß-adrenergic stimulation shown here is a potential contributor to AF promotion and atrial myocyte hyperpolarization resulting from adrenergic stimulation. IKH is also a candidate to participate in cholinergic AF promotion. Atrial tachycardia-induced remodelling is a significant factor in clinical AF (Nattel, 2002). Ionic current changes that may contribute to remodelling-induced APD-abbreviation include decreased ICa and increased IK1 (Yue et al. 1997; Bosch et al. 1999; Dobrev et al. 2001). The present findings add IKH up-regulation as a potential contributor to atrial-tachycardia induced AP abbreviation. The larger IKH in PV versus LA cells after tachycardia-induced remodelling (Fig. 7) suggests that IKH may contribute to the role of PVs in AF maintenance (Wu et al. 2001).
Our study suggests that ß-adrenoceptor activation can stimulate Kir3-based channels in cardiomyocytes. Kir3 channels are opened by the Gß heterodimer in response to Gi activation by M2 muscarinic receptors (Lim et al. 1995). It was believed that ß-adrenoceptors were incapable of modulating these currents in native tissues (Trautwein et al. 1982). Some investigators found an increase in IKACh with isoproterenol application (Kim, 1990; Sorota et al. 1999; Mullner et al. 2000). Others attributed the effect of isoproterenol on IKACh to isoproterenol-activated IK,ATP (Wang & Lipsius, 1995). The present study suggests that ß-adrenoceptor stimulation of Kir3-based current occurs in LA and PV cells, and that the organization of signalling may therefore be cell-type dependent.
Potential limitations
We defined IKH based on the slowly activating time-dependent current component. The complete abolition of this time-dependent component by tertiapin-Q justifies its consideration as a distinct entity. In some experiments, we contrasted the time-dependent IKH component with instantaneous current. The instantaneous component is carried largely by IK1; however, there was some evidence for a potential contribution from IKH. Since tertiapin-Q produced statistically non-significant and relatively minor effects on instantaneous current, it is reasonable to contrast the latter component with the slowly activating time-dependent current; nevertheless, it must be recognized that the distinction between the components is imperfect.
While we showed IKH to have properties of a Kir3-based current modulated by PTX-sensitive G proteins, many mechanistic aspects remain to be elucidated. Such issues as the basis for the large constitutive current, the fundamental mechanism of its time dependence, the detailed G protein-coupled regulation, the molecular determinants of up-regulation by atrial tachycardia and the differential expression of IKH in PV and LA cardiomyocytes are important and unresolved, and need further investigation.
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, PV; •, LA cardiomyocytes.
) and after (
) substitution of extracellular sodium with Tris-HCl. Removal of Na+ eliminated the fast inward Na+ transient but did not affect the tail current. B, tail currents recorded in Na+o-free solution, normalized to values at test potential of –140 mV. Half-activation voltage obtained with Boltzmann fits to normalized tail currents was –93.1 ± 4.6 mV, slope factor –15.2 ± 2.2 (n= 5 cells). C and D, removal of external sodium did not affect time-dependent inward IKH. A family of recordings obtained in the same cell with test pulses to –120, –100, –90 and –80 mV before (C) and after (D) substitution of NaCl by equimolar Tris-HCl is shown (similar results were obtained in 5 PV cardiomyocytes). E, determination of tail current reversal (voltage protocol in inset) at various external K+ concentrations. The reversal potential became more positive as [K+]o increased. Linear regression (
P < 0.05 versus LA.
