Published online before print August 2, 2001, doi: 10.1161/hh1601.094979
© 2001 American Heart Association, Inc.
Cellular Biology |
A TREK-1–Like Potassium Channel in Atrial Cells Inhibited by ß-Adrenergic Stimulation and Activated by Volatile Anesthetics
From the Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, Valbonne, France.
Correspondence to Prof Michel Lazdunski, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. E-mail ipmc{at}ipmc.cnrs.fr
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Abstract |
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Many members of the two-pore-domain potassium (K+) channel family have been detected in the mammalian heart but the endogenous correlates of these channels still have to be identified. We investigated whether IKAA, a background K+ current activated by negative pressure (stretch) and by arachidonic acid (AA) and sensitive to intracellular acidification, could be the native correlate of TREK-1 in adult rat atrial cells. Using the inside-out configuration of the patch-clamp technique, we found that IKAA, like TREK-1, was outwardly rectifying in physiological K+ conditions, with a conductance of 41 pS at +50 mV. Like TREK-1, IKAA was reversibly activated by clinical concentrations of volatile anesthetics (in mmol/L, chloroform 0.18, halothane 0.11, and isoflurane 0.69). In cell-attached experiments, IKAA was inhibited by chlorophenylthio-cAMP (500 µmol/L) and also by stimulation of ß-adrenergic receptors with isoproterenol (1 µmol/L). In addition, TREK-1 mRNAs were detected in all cardiac tissues, and the TREK-1 protein was immunolocalized in isolated atrial myocytes. Such a background potassium channel might contribute to the positive inotropic effects produced by ß-adrenergic stimulation of the heart. It might also be involved in the regulation of the atrial natriuretic peptide secretion.
Key Words: potassium channels • volatile anesthetics • ß-adrenergic receptor • heart cells
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Introduction |
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Cardiac potassium (K+) channels have extensively been studied for years. In the working atrial and ventricular myocytes, they belong to the following two major families: (1) the voltage-dependent channels (Ito, IKr, IKs, etc), which are involved in the different repolarization phases of the cardiac action potential (AP), and (2) the background K+ channels, which stabilize the resting membrane potential at a hyperpolarized level and regulate AP duration in physiological conditions (IK1), during sympathetic stimulation of the heart (IKACh), or during pathological events such as ischemia (IKATP, etc) (for a review, see Carmeliet1).
During the last decade, a large number of K+ channel 
subunits were cloned and further characterized in expression systems such as Xenopus oocytes and mammalian cell lines (COS, HEK, etc). Some of these cloned channels were proposed as molecular substrates for the endogenous cardiac K+ currents on the basis of their biophysical and pharmacological properties. For instance, Kv4.2 is suggested to be a molecular correlate of Ito, whereas HERG and KvLQT1/Isk correspond to IKr and IKs, respectively.2–6 IRK1 (Kir2.1) generates a current similar to that of IK1, whereas GIRK1 (Kir3.1) forms a complex with GIRK4 (Kir3.4) to reproduce IKACh.7,8 Kir6.2 coassembles with the sulfonylurea receptor SUR2A to reconstitute the cardiac KATP channel.9,10 However, not all cloned K+ channel subunits shown to be expressed in the heart have been associated with an endogenous channel. This is particularly the case for the expanding family of mammalian two-pore-domain potassium (K2P) channels.11 Of the 11 functional members that have now been cloned, the following seven were detected in the mammalian heart, at low or high levels, by Northern and reverse transcriptase–polymerase chain reaction (RT-PCR) experiments: TWIK-1 (human), TWIK-2 (human and rat), TREK-1 (mouse), TASK-1 (human, mouse, and rat), TASK-3 (rat), THIK-1 (rat), and TALK-2 (human).12–23
TREK-1 is one of the most interesting members of the K2P channel family. It is activated by changes in membrane tension (stretch), by arachidonic acid (AA) and other polyunsaturated fatty acids, and by intracellular acidification.15,24,25 It looks similar to a background potassium channel that was previously recorded in adult rat atrial myocytes.26 This channel, also sensitive to intracellular acidosis and activated by stretch, produces an outwardly rectifying current, named IKAA, on application of AA. Despite the lack of specific effectors for TREK-1, this K2P channel presents an interesting property; it is activated by volatile anesthetics,27 whereas all cardiac potassium channels studied until now are either inhibited or not sensitive to these agents (Ito,28 IK,29–31 IK1,32 and IKATP,33,34 with the exception of IKACh, which is activated by halothane.35 The purpose of this work is to establish the presence of the TREK-1 protein in the heart using specific antibodies to demonstrate that TREK-1 is the channel responsible for IKAA and that IKAA, like TREK-1,24,27 is activated by clinical concentrations of volatile anesthetics and is inhibited by increases of cAMP that activate protein kinase A (PKA) and that can be triggered by ß-adrenergic stimulation.
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Materials and Methods |
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Cell Isolation
Male Wistar rats (200 to 300 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (90 mgxkg-1). Heart dissociation was performed following a previously described protocol.33 Oxygenated bicarbonate-buffered solution, mixed with 0.06% collagenase (type II, Worthington) and 0.03% hyaluronidase (type IV-S, Sigma) for 20 to 45 minutes and containing the following, was used (in mmol/L): NaCl 125, KCl 5, MgSO4 2, KH2PO4 2, NaHCO3 25, and glucose 10. Isolated myocytes were stored at 4°C in modified KB medium containing the following (in mmol/L): potassium glutamate 90, KH2PO4 30, MgCl2 1, pyruvic acid 5, taurine 20, glucose 20, and HEPES 10 (pH adjusted to 7.2 with KOH).
Electrophysiological Experiments
Whole-cell, inside-out, and cell-attached configurations of the patch-clamp technique were performed on atrial cells at room temperature (20°C to 22°C) with a RK300 patch-clamp amplifier (Bio-Logic). Pipettes (3 to 5 M
) were Sylgard-coated when used for single-channel experiments. The standard intracellular solution contained the following (in mmol/L): KCl 150, MgCl2 2, EGTA 5, and HEPES 10 (pH adjusted to 7.2 with KOH). For cell-attached experiments, the same solution without EGTA was used as bathing solution. The basic extracellular solution contained the following (in mmol/L): NaCl 145, KCl 5, MgSO4 2, and HEPES 10 (pH adjusted to 7.2 with NaOH). AA, lysophosphatidylcholine (LPC), chlorophenylthio-cAMP (CPT-cAMP), glibenclamide, and tetraethylammonium (TEA) were all purchased from Sigma-Aldrich Chimie. The solutions containing volatile anesthetics were prepared as previously described.27 Negative pressure was applied to the pipette via a calibrated syringe. Whole-cell recordings were digitized and analyzed with pClamp software. Single-channel recordings, filtered at 3 kHz, were digitized using a DAT recorder (Bio-Logic) and further analyzed with Biopatch software (Bio-Logic). Maximum open probabilities of a single channel (POmax, measured over 5 seconds every 20 or 30 seconds, during a burst of openings) and amplitude histograms were determined using filtered signals at 1 kHz. Results, expressed as mean±SEM, were considered as significant when P<0.05 with the Student t test.
Reverse Transcriptase–Polymerase Chain Reaction
Adult rat hearts were dissected, and total RNAs from four different tissues (atrium, septum, left ventricle, and right ventricle) were extracted by the guanidinium isothiocyanate method. Five micrograms of RNA was reverse transcribed in a final volume of 40 µL. In a first step, these cDNAs were used as template for PCR by using primers deduced from the mouse TREK-1 sequence, as follows: sense primer, 5'-TCAAGCACATAGAAGGCTGG-3', and reverse primer, 5'-TCAGGTGGTTCACAGACAGG-3'. The amplified DNA corresponding to rat TREK-1 DNA was subcloned and then sequenced. The deduced proteic sequence represents 100% identity with mouse TREK-1 over 145 amino acids. In a second step, the following two rat-specific primers were deduced from this sequence and used for PCR amplification: sense primer, 5'-GCCCTGGACGCCATCTAC-3', and reverse primer, 5'-GTTACCCGCCAGCTCTGCA-3'. One microliter of each sample was used as template. PCR conditions were 32 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C. The amplified fragments were transferred onto nylon membrane and probed with a 32P-labeled internal primer specific for TREK-1, 5'-GGATTTGGCGATTATGTGGCA-3'. For control, actin was amplified and analyzed according to the same protocol.
Membrane Preparations and Western Blot
Rat ventricular and atrial microsomes were prepared from adult rats.36,37 Tissues were minced and homogenized, and nuclei and debris were pelleted by centrifugation at 1000g. The supernatant was then centrifuged (8000g for 20 minutes), and again the supernatant was recovered and then treated with 0.5 mol/L KCl (4°C for 30 minutes). Microsomes were recovered after centrifugation (60 000g for 30 minutes). Homogenates of COS-7–transfected cells were prepared as previously described.38 Aliquots of solubilized ventricular and atrial microsomes (25 µg) and COS-7–transfected cell homogenates (5 µg) were fractionated on 10% SDS-PAGE gels and subjected to Western blotting. Immunoblot analysis was performed as described previously, using rabbit polyclonal antibodies directed against TREK-1.38
Immunohistochemistry
Immunohistochemical studies were performed on freshly isolated adult rat atrial myocytes. Cells were plated on two-chamber Falcon culture slides and fixed with 4% paraformaldehyde in PBS. Cells were permeabilized in 0.3% Triton X-100 for 15 minutes and then incubated in blocking buffer (5% normal goat serum and 0.05% Triton X-100 in PBS) (3 hours at room temperature). Cells were hereafter incubated with TREK-1 antibodies (1:3000 to 1:5000)38 at 4°C overnight. Development was performed using either the biotinylated horseradish peroxidase–diaminobenzidine (DAB) method (Vectastain Elite ABC kit, Vector Laboratories) or the Alexa Fluor 488 goat anti-rabbit IgG antibody (Molecular Probes).
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
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Voltage-Clamp Recording of a Potassium Current Activated by Chloroform
To our knowledge, among all K+ channels that have been detected by molecular biology techniques in the heart, TREK-1 is the only one to be activated by chloroform. We used this property to investigate the possible presence of a TREK-1–like potassium current in adult rat atrial cells. Voltage-clamp experiments were performed using a protocol with two depolarizing steps (Figure 1A). The current recorded at -40 mV was rapidly increased by chloroform (0.18 mmol/L), whereas both peak and steady-state outward currents at +40 mV were inhibited. This initial inhibition at +40 mV is probably due to an effect on voltage-dependent potassium channels, as already reported for halothane in other preparations.28,29,39 When chloroform application was maintained, a noninactivating outward current finally developed at +40 mV. This current, not sensitive to 10 µmol/L glibenclamide, was mainly carried by potassium ions as indicated by the reversal potential (-80 mV in this example) of the current-voltage curve obtained under chloroform (Figure 1B). Chloroform effects were completely reversible and reproduced in five other cells. Isoflurane (0.69 mmol/L) also produced a strong increase of a similar noninactivating potassium current (n=3, not shown).
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) and at end of voltage steps to -40 mV (
) and +40 mV (
) during chloroform application (CHCl3, 0.18 mmol/L, horizontal bar). Labeled arrows indicate recording time at which traces shown in the top panel were obtained. B, Current-voltage curves of Iss (measured at the end of voltage steps) in control conditions (
), using 1-second voltage steps from -120 to +40 mV (20-mV increments) from a holding potential of -80 mV, every 6 seconds. Inset, Current-voltage curve of chloroform-induced Iss.
Identification of IKAA
In inside-out patches, we first used negative pressure (stretch) and AA stimulation to confirm the presence of IKAA.26 In physiological K+ conditions, stretch (P
12 mm Hg) instantaneously activated a channel (Figure 2A) characterized by a flickering behavior and bursting openings, with a mean open time of 1.63±0.18 ms at +40 mV (n=4). This channel was not voltage-sensitive from -10 to +40 mV. In the particular recording shown in Figure 1, the maximum open probability (POmax) averaged over six different voltages was 0.36±0.01. Channel activity was not affected by 10 µmol/L glibenclamide or by 10 mmol/L TEA, a blocker for KATP and voltage-sensitive K+ channels, respectively, and it was not calcium-dependent, as we used calcium-free extracellular solutions in addition to the presence of a calcium chelator (EGTA, 5 mmol/L) in intracellular solutions. AA (10 µmol/L) applied to the cytosolic surface of the patch was able to activate the channel with a low activity remaining after washout of the fatty acid (Figure 2B). The corresponding current-voltage curves were outwardly rectifying both in physiological and in symmetrical K+ conditions, with chord conductances at +50 mV of 41±1 pS (n=4) and 118±4 pS (n=3), respectively (Figure 2C).
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Volatile Anesthetics Activate IKAA
The 41-pS conductance ofIKAA in physiological K+ conditions is close to the 48-pS conductance previously measured for TREK-1 expressed in COS cells.24 To further demonstrate that TREK-1 generates IKAA, we first examined the effect of three volatile anesthetics that are known to activate TREK-127 (chloroform, halothane, and isoflurane) on the native cardiac channel in the inside-out configuration. Chloroform (0.18 mmol/L), applied to the cytosolic side of the patch, activated a channel (Figure 3A) with a POmax (calculated from amplitude histograms in Figure 3B) of 0.33±0.04 (at 0 mV, n=3) (Figure 3C). Chloroform-induced openings occurred in bursts (mean open time of 1.6 ms in this example) and were highly flickering as shown in Figure 3D (inset). This chloroform-activated current did not display voltage sensitivity from -10 to +40 mV, as indicated by the POmax of 0.36±0.01 averaged over this voltage range. The current-voltage curve established under chloroform (Figure 3D) was perfectly superimposable on the one obtained under AA and stretch. Channel activity was significantly reduced after chloroform washout (Figures 3A and 3C), but it was then always possible to reactivate the channel with stretch. As illustrated in Figure 4A, halothane (0.11 mmol/L) was also able to induce bursting openings but only when the channel had been preactivated (POmax=0.13±0.02 versus POmax=0.03± 0.01 in control conditions, at 0 mV, n=3, P<0.05). Isoflurane (0.69 mmol/L) activated the channel in the same way as chloroform (Figure 4B); POmax=0.26±0.04 versus POmax=0.01± 0.01 in control conditions (at 0 mV, n=3, P<0.05). The current-voltage curves of isoflurane- and chloroform-induced currents were perfectly superimposable and also superimposable to the current-voltage curves obtained after activation by AA and stretch. Washout of the isoflurane-induced effects was rapid (Figure 4C), and the channel could then be reactivated by stretch.
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IKAA Is Regulated by the PKA-Dependent Pathway
TREK-1 is potently inhibited by intracellular increases of cAMP via a PKA activation that leads to a phosphorylation of a well-identified serine residue situated in the cytoplasmic C-terminal region.24 We investigated whether IKAA could also be regulated by cAMP. Spontaneous channel activity was never recorded in cell-attached experiments. Therefore, the analysis of cAMP effects was first carried out after application of AA. When IKAA was activated by AA (10 µmol/L) (Figure 5), additional application of 500 µmol/L CPT-cAMP, a permeant analog of cAMP, fully inhibited channel activity within 1.5 to 2 minutes (n=3). We then examined whether CPT-cAMP could regulate IKAA when it had been induced by chloroform. Extracellular application of 0.18 mmol/L chloroform activated the same flickering current (POmax=0.14±0.01 at 0 mV, n=4) as AA, an effect that was again inhibited (Figure 6A) after a 1.5- to 2-minute application of CPT-cAMP (Figure 6B). We also recorded the CPT-cAMP inhibition of IKAA after activation of this current by stretch (P12 mm Hg, n=2, not shown).
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Because in cardiac cells cAMP is produced by adenylate cyclase from cytosolic ATP, as a result of ß-adrenergic stimulation, we tested whether the inhibition of IKAA could be produced by stimulation with isoproterenol, a specific agonist of ß-adrenergic receptors. In cell-attached experiments, extracellular application of isoproterenol (1 µmol/L) strongly inhibited the AA-induced current within 14.2±0.7 seconds (n=4) (Figures 7A and 7B).
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TREK-1 Is Expressed in Cardiac Preparations
RT-PCR techniques indicate that the mRNA for TREK-1 was present in atria and ventricles as well as in the septum of adult rat heart (Figure 8A). To determine whether TREK-1 proteins could also be detected, rat ventricular and atrial microsomes were separated on SDS polyacrylamide gels and immunoblotted with affinity-purified polyclonal anti–TREK-1 antibodies. Figure 8B shows that the anti–TREK-1 antibody recognizes a single band at 45 kDa, which is the same size as that obtained in membranes from TREK-1–transfected COS cells that express this channel at a high level in their plasma membrane.24 The presence of TREK-1 in the plasma membrane was confirmed in a second set of experiments in which isolated atrial myocytes, the same preparation used in parallel for electrophysiology, were fixed and immunostained with anti–TREK-1 antibodies. Figure 8C illustrates typical experiments, showing the staining of atrial cells (a and c). No labeling was seen in atrial cells subjected to the same staining protocols, except that the primary antibody was omitted (b).
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Discussion |
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This work provides biophysical and pharmacological evidence indicating that IKAA, the atrial background potassium current activated by membrane tension (stretch) and by AA,26 is probably generated by the TREK-1 channel or a very closely related member of the K2P channel family, as follows. (1) IKAA in symmetrical K+ conditions has a single-channel conductance of 118 pS, close to the 101 pS measured for TREK-1 in COS cells.24 (2) Both the native channel that generates IKAA and the TREK-1 channel are activated by stretch and by AA.15,24,26 Moreover, in physiological K+ conditions, the resulting current is outwardly rectifying in both cases (Figure 1C and Patel et al24), and conductances are also very similar, 41 pS for IKAA and 48 pS for TREK-1 (in COS cells24). (3) Neither IKAA nor the current generated by TREK-1 in a heterologous system is sensitive to voltage in physiological K+ conditions. Both the native channel and TREK-1 display flickering openings occurring in bursts as long as the stimulus is maintained. (4) Both the native currents corresponding to IKAA and to TREK-1 are sensitive to intracellular acidification,25,26 and in both cases decreasing pHi leads to an increase in stretch sensitivity. (5) TREK-1 is potently activated by LPC40; similarly, we have demonstrated in cell-attached experiments an activation of IKAA with extracellular application of 10 µmol/L LPC (n=3, not shown). (6) Neither IKAA nor TREK-115 is inhibited by glibenclamide or TEA or is Ca2+-sensitive.
None of the other cloned K2P channels, demonstrated to be present in the heart and analyzed for their biophysical and pharmacological properties,12,14,16,20–23 display characteristics as close to IKAA as do those of TREK-1.24 The only difference is the stretch sensitivity (P1/2=-12 mm Hg for IKAA26 and P1/2=-23 mm Hg for ITREK-1). The most probable interpretation of this difference between the native and the cloned channel is that there are additional subunits that associate with the TREK-1 protein to modify its sensitivity to pressure just as ß-type subunits change the voltage sensitivity (and/or kinetics) of voltage-sensitive K+ channels and the Ca2+ sensitivity of KCa2+ channels.
Another key element indicating the TREK-1 nature of IKAA is provided by its unique sensitivity to volatile anesthetics. The activity of several of the K2P channels that have been shown to be present in the heart is modified by volatile anesthetics but in a different way. TASK-1 is not sensitive to chloroform but is activated by halothane and isoflurane.27 TWIK-2 is inhibited by chloroform and by halothane.14 THIK-1 is not sensitive to chloroform and is inhibited by halothane.22 TALK-2 is inhibited by chloroform and by halothane but is slightly activated by isoflurane.23 Among the K2P channels present in the heart, only TREK-1 activity is increased by chloroform, halothane, and isoflurane.27 In inside-out experiments, we have found that IKAA was also reversibly activated by clinical concentrations of (in mmol/L) chloroform 0.18, halothane 0.11, and isoflurane 0.69. This indicates, as also previously observed for TREK-1,27 that these anesthetics do not exert their effect via second-messenger molecules but rather directly on the channel macromolecule.
In addition to its sensitivity to volatile anesthetics, TREK-1 is inhibited by cAMP via PKA phosphorylation of Ser333 in the C-terminal part of the channel structure.24 The native cardiac channel generating IKAA can also be regulated by cAMP. In cell-attached experiments, CPT-cAMP, the permeant analog of cAMP, produced a complete inhibition of IKAA when the current had been activated by either AA (Figure 4), chloroform (Figure 5), or stretch. Interestingly, the same inhibitory effect was observed after stimulation of the atrial-cell ß-adrenergic receptors with the specific agonist isoproterenol, a treatment that is known to increase the intracellular cAMP content.
All of these results taken together provide very strong evidence that IKAA is the endogenous correlate of TREK-1 in adult rat atrial myocytes. In addition, RT-PCR, Western blot, and immunohistochemistry experiments independently demonstrate the presence of TREK-1 in adult rat heart. The mechanosensitive IKAA channel is also present in ventricular cells with a single-channel conductance of 106±12 pS at +60 mV and 72±10 pS at -60 mV and the same pressure and pH sensitivity as the atrial channel.26 The mechanism of regulation of this ventricular IKAA channel via AA production was recently analyzed.41 In the adult human heart, the expression of TREK-1 is much less important than in rodent heart but it could be significantly upregulated during development or in aging and/or heart diseases.
In the working atrial and ventricular myocytes, the main background K+ current is generally considered to be carried by the inwardly rectifying current IK1. Under physiological conditions, it stabilizes the resting membrane potential near the K+ equilibrium and participates in the final repolarization phase of the AP. ITREK-1, as an outwardly rectifying current, will balance any membrane depolarization and will contribute to the regulation of the AP duration. Because of its stretch sensitivity, we suggest that this current could be a negative feedback after stretch activation of nonselective cationic channels.42 TREK-1 would then have a beat-to-beat regulation of atrial function. It is particularly interesting to note that both IK143 and ITREK-1 (the present study) are inhibited by the specific ß-adrenergic agonist isoproterenol via a phosphorylation process involving cAMP-dependent kinase. Then, the positive inotropic effect exerted by ß-adrenergic agonists on the heart will not only be produced by activation of voltage-dependent Ca2+ channels but will also be the result of prolonged Ca2+ entry into the cell by inhibition of IK1 and ITREK-1 activities.
Because ITREK-1 is present in atrial cell and is activated by physiological levels of stretch, it might be involved in the regulation of atrial natriuretic peptide (ANP) secretion. Stretch is known to be the main stimulus eliciting this secretion (for a review, see Ruskoaho44) which plays an important role in the control of blood pressure. The initial depolarization process associated with the intracellular Ca2+ increase that is necessary for ANP secretion is expected to be triggered by stretch activation of nonselective stretch-activated cationic channels.42 Half-maximal activation of these cationic channels occurs for a pressure of 1.5 mm Hg, whereas it occurs 12 mm Hg for the K+ channel IKAA.26 This suggests that a significant activation of ITREK-1 by stretch can only occur secondary to the activation of these cationic channels. The function of TREK-1 could then be to act as a negative feedback for ANP secretion, and a ß-adrenergic stimulation inhibiting TREK-1 could then be expected to enhance ANP release from atrial myocytes, an effect that has indeed been observed.44
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Acknowledgments |
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This work was supported by the Centre National de la Recherche Scientifique, the Ministère de la Recherche et de la Technologie, the Fondation pour la Recherche Médicale, and the Association Française contre les Myopathies. We are grateful to R. Mengual for his assistance with gas chromatography and to V. Lopez for excellent secretarial assistance.
Received January 8, 2001; accepted June 21, 2001.
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