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Circulation Research. 1999;85:707-715

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(Circulation Research. 1999;85:707-715.)
© 1999 American Heart Association, Inc.

Cellular Biology

A Novel KATP Current in Cultured Neonatal Rat Atrial Appendage Cardiomyocytes

Anne Baron, Laurianne van Bever, Dominique Monnier, Angela Roatti, Alex J. Baertschi

From the Department of Physiology, Centre Médical Universitaire, Geneva, Switzerland.

Correspondence to Alex J. Baertschi, Department of Physiology, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland. E-mail Alex.Baertschi{at}medecine.unige.ch


*    Abstract
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 *Abstract
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Abstract—The functional and pharmacological properties of ATP-sensitive K+ (KATP) channels were studied in primary cultured neonatal rat atrial appendage cardiomyocytes. Activation of a whole-cell inward rectifying K+ current depended on the pipette ATP concentration and correlated with a membrane hyperpolarization close to the K+ equilibrium potential. The KATP current could be activated either spontaneously or by a hypotonic stretch of the membrane induced by lowering the osmolality of the bathing solution from 290 to 260 mOsm/kg H2O or by the K+ channel openers diazoxide and cromakalim with EC50 {approx}1 and 10 nmol/L, respectively. The activated atrial KATP current was highly sensitive to glyburide, with an IC50 of 1.22±0.15 nmol/L. Recorded in inside-out patches, the neonatal atrial KATP channel displayed a conductance of 58.0±2.2 pS and opened in bursts of 133.8±20.4 ms duration, with an open time duration of 1.40±0.10 ms and a close time duration of 0.66±0.04 ms for negative potentials. The channel had a half-maximal open probability at 0.1 mmol/L ATP, was activated by 100 µmol/L diazoxide, and was inhibited by glyburide, with an IC50 in the nanomolar range. Thus, pending further tests at low concentrations of KATP channel openers, the single-channel data confirm the results obtained with whole-cell recordings. The neonatal atrial appendage KATP channel thus shows a unique functional and pharmacological profile resembling the pancreatic ß-cell channel for its high affinity for glyburide and diazoxide and for its conductance, but also resembling the ventricular channel subtype for its high affinity for cromakalim, its burst duration, and its sensitivity to ATP. Reverse transcriptase–polymerase chain reaction experiments showed the expression of Kir6.1, Kir6.2, SUR1A, SUR1B, SUR2A, and SUR2B subunits, a finding supporting the hypothesis that the neonatal atrial KATP channel corresponds to a novel heteromultimeric association of KATP channel subunits.


Key Words: KATP channel • sulfonylurea receptor • cardiac atrium • atrial natriuretic peptide • patch clamp


*    Introduction
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 up arrowAbstract
 *Introduction
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Atrial cardiomyocytes release atrial natriuretic peptide (ANP), a hormone that plays a major physiological role as an antihypertensive and cardioprotective agent.1 2 Both stretch and hypoxia are potent stimuli for ANP secretion.2 3 In isolated rat heart, the stretch- and hypoxia-induced ANP secretion are inhibited by K+ channel openers such as diazoxide or pinacidil.4 Regulatory effects of sulfonylureas have also been described that either potentiate or inhibit stretch-induced ANP secretion, depending on the experimental conditions and the concentrations used (References 4 and 54 5 and J.H. Jiao, P. Baumann, A. Baron, A. Roatti, R.A. Pence, A.J. Baertschi, unpublished data, 1995–1999). Taken together, these findings suggest that ATP-sensitive K+ (KATP) channels might be important regulators of stimulated-ANP secretion.

Because KATP channels are inhibited by physiological concentrations of cytosolic ATP, they are thought to couple membrane excitability to the metabolic state of the cell. These widely distributed channels are involved in various physiological functions, including secretory processes such as the glucose-stimulated insulin secretion by pancreatic ß-cells6 7 and the release of neurotransmitters, growth hormone, and renin.6 8 9 10

The KATP channel is formed by the association of the following 2 types of protein subunits: (1) inward rectifying K+ channel subunits (Kir), constituting the pore of the channel and containing the major ATP binding site, and (2) sulfonylurea receptor (SUR) regulatory subunits.11 12 13 14 Until now and regardless of animal species, 2 Kir subunits, Kir6.1 and Kir6.2,12 15 and 5 SUR subunits, SUR1A, SUR1B (GenBank accession No. AF039595), SUR2A, SUR2B, and SUR2C,14 16 17 18 19 have been cloned. The association SUR1A/Kir6.2 has been shown to form KATP channels with a 1-for-1 stoichiometry, the functional channel being an octamer.20 21 The association Kir6.2/SUR1A is found in the pancreatic ß-cell KATP channel involved in insulin secretion, whereas SUR2A and SUR2B subunits have been proposed as components of the ventricular and the vascular smooth muscle KATP channels, respectively.

Concerning the heart, discrepancies persist regarding the pharmacological properties of ventricular KATP channels (see Discussion), and very few data have been published on atrial KATP channels. Zünkler et al22 described an opening effect of cromakalim and a low affinity for tolbutamide on human atrial myocytes, whereas Hamada et al23 and Song et al24 reported a high-affinity inhibitory effect of glyburide on guinea pig atrial myocytes. Interestingly, van Wagoner25 showed that rat atrial KATP channels were stretch-activated, a phenomenon that could play a major physiological role in these stretch-sensitive secretory cells.

We report here novel functional and pharmacological properties of the atrial appendage KATP channel. These were investigated in primary cultured neonatal rat atrial appendage myocytes by means of patch-clamp recordings of cellular and unitary KATP currents. The expression of Kir and SUR isoforms was determined by performing reverse transcriptase–polymerase chain reaction (RT-PCR) on atrial appendage cardiomyocyte mRNA. A preliminary account of the work has been published in abstract form.26


*    Materials and Methods
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 *Materials and Methods
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Atrial Myocyte Cultures
Atrial appendage myocytes from 2- to 3-day-old rats were cultured as described previously27 for 2 to 4 days in the presence of 1 µmol/L dexamethasone and 1 µmol/L triiodothyronine in a 5% CO2 incubator. Cultured atrial myocytes were shown to synthesize ANP by immunostaining (not shown).

Patch-Clamp Recording of KATP Current
KATP currents were recorded on culture days 2 to 4 at room temperature from initially beating atrial myocytes using the whole-cell and the inside-out configurations of the patch-clamp technique28 with hardware and software from Axon Instruments. Borosilicate glass pipettes had a resistance of 2 to 4 M{Omega} for whole-cell recordings and 5 to 10 M{Omega} for inside-out recordings. For whole-cell recordings, the standard pipette solution contained (in mmol/L) KCl 121, CaCl2 1.5, EGTA 10, MgCl2 1.3, ATP 0 to 5, glucose 10, KOH 34, and HEPES 10 (pH 7.45 with KOH), and the bathing solution contained (in mmol/L) KCl 5, CaCl2 1, MgCl2 1, NaCl 118, glucose 10, and HEPES 10 (pH 7.5 with NaOH). The osmolality of the hypotonic solution (stimulus) was 260 mOsm/kg H2O, and sucrose was added to yield a solution of 290 mOsm/kg H2O. Currents were filtered at 2 kHz and sampled at a frequency of 0.8 kHz. For inside-out recordings, the pipette solution contained (in mmol/L) KCl 140, CaCl2 1, MgCl2 1, and HEPES 5 (pH 7.3 with KOH), whereas the bathing solution contained (in mmol/L) KCl 5, KOH 15.5, NaCl 135, MgCl2 1, EGTA 5, glucose 10, and HEPES 5 (pH 7.3 with KOH). Currents were filtered at 1 kHz and sampled at 6 kHz. Burst kinetics analysis and open and close time duration histograms were only performed where a single channel was present on the membrane patch.

Chemicals and Drugs
EGTA, ATP, glyburide, diazoxide, and cromakalim were all purchased from Sigma.

RT-PCR Analysis for KATP Channel Subunits
Total RNA from 4-day-old cultured neonatal atrial appendage myocytes was extracted by Trizol reagent (GIBCO-BRL) and subjected to RQ1 DNase (Promega) digestion. cDNAs were synthesized with oligo(dT) primers and Superscript II RT (200 units, GIBCO-BRL) at 42°C. PCR was carried out in a Biometra personal cycler in a final volume of 50 µL containing 1 µL of the RT reaction, 1.5 units of Taq polymerase (GIBCO-BRL), 1.5 mmol/L MgCl2, 250 µmol/L of each dNTP, and 20 pmol of each primer. The PCR conditions were the following: initial denaturation at 94°C for 3 minutes followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension step at 72°C for 10 minutes. The nature of the PCR products (see also legend to Figure 7Down) was confirmed by double-strand sequencing on both strands.



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  		Figure 7. RT-PCR for rat Kir and SUR mRNA (same gel, same culture for all lanes). Expected fragment sizes in top panel were the following: ß-actin, 497 bp (lane 2); ANP, 535 bp (lane 4); Kir6.1 (primers based on GenBank accession No. D42145), 236 bp (lane 6); and Kir6.2 (D96039), 443 bp (lane 8). Expected fragment sizes in the lower panel were the following: SUR1A (L40624), 402 bp, and SUR1B (AF039595), 288 bp (with SUR1A-1B primers) (lane 10); SUR2A (D83598), 249 bp (with SUR2A-specific primers) (lane 12); SUR2A, 349 bp, and SUR2B (AF087838), 173 bp (with SUR2A-2B primers) (lane14); and SUR2A (D83598), 358 bp, and SUR2C (AF003531), 252 bp (with SUR2A-2C primers) (lane 16). The primers used to detect the SUR2C rat mRNA were based on the mouse mRNA SUR2C, following the SUR2 nomenclature suggested by Ashcroft and Gribble14 ; their sequences are 100% homologous to the rat mRNA SUR2A sequence (D83598). The primers used are listed in the online Materials and Methods (see http://www.circresaha .org). PCR reactions were performed for each primer pair with (+) and without (-) RT. Negative and positive controls of the PCR were performed, including a PCR without DNA template (water control, Ctrl-, lane 17) and PCRs on expression vectors containing mouse cDNA SUR2C, 252 bp (lane 18); and SUR2A, 358 bp (lane 19) with the SUR2A-2C primers.

An expanded Materials and Methods section is available online at http://www.circresaha.org.


*    Results
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 *Results
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KATP Whole-Cell Current on Neonatal Atrial Appendage Myocytes
Whole-cell KATP currents were recorded on initially beating atrial appendage cardiomyocytes during a 10-second voltage ramp (from –70 or –80 mV to +90 mV), with a holding potential of –40 mV, to prevent the activation of voltage-dependent channels. The EGTA-buffered low Ca2+ concentration in the patch pipette (10 nmol/L estimated free Ca2+) prevented the activation of Ca2+-dependent currents. The membrane potential at 0 pA current was measured after each current recording.

Whole-cell capacitance was measured to estimate the changes in atrial myocyte membrane surface in culture. Assuming a specific capacitance of 1 µF/cm2, the membrane surface increased from a mean value of 1180 µm2 on culture day 2 to 1600 µm2 on day 3 and 2000 µm2 on day 4. In the presence of 2 mmol/L internal ATP, KATP current densities were 105±12 pA/pF (n=21), 77±8 pA/pF (n=16), and 78±9 pA/pF (n=19) on days 2, 3, and 4, respectively.

In the absence of ATP in the pipette, a weakly inward rectifying outward current developed 1 to 2 minutes after breaking the patch membrane (Figure 1ADown and 1BDown, •), correlating with a cell hyperpolarization (Figure 1BDown, {circ}) reaching a mean maximal value of –64.6±3.2 mV (n=18; for resting potentials, see online Materials and Methods, http://www.circresaha.org). The slow activation is probably due to the long time constant for the diffusion of cellular ATP into the pipette. This activation was usually transient and the current amplitude progressively decayed with time. Figure 1CDown shows the saturable relation between the current amplitude and the subsequent membrane hyperpolarization, with {approx}20% of the maximal current inducing a maximal hyperpolarization. Increasing the external K+ concentration from 5 to 30 mmol/L (substitution of Na+ by K+) shifted the current reversal potential from –80 to –40 mV, thus following the predicted Nernst equilibrium potential for K+ (Figure 1DDown). The probability of K+ current activation depended on the pipette ATP concentration: 65.4% (18/28), 57% (69/121), 40% (26/65), and 13.1% (8/61) of the recorded cells in the presence of 0, 1, 2, and 5 mmol/L internal ATP, respectively. According to the {chi}2 test, the probability of KATP current activation is significantly (P<0.001) tilted toward low percentages at high internal ATP. The mean maximal subtracted current amplitude, measured at +50 mV, and the mean maximal potential, measured at 0 pA, were not significantly different for ATP concentrations ranging from 0 to 2 mmol/L, 1065±107 pA and –64.6±3.2 mV (n=18) in the absence of ATP, but significantly decreased in the presence of 5 mmol/L internal ATP to reach 32.8±1.1% (current) and 52.8±2.5% (potential), respectively, of the control values measured in the absence of ATP (P<0.001, n=8). These results show that the atrial appendage KATP channel is influenced by ATP. However, the KATP current activation in the presence of 5 mmol/L ATP may reflect some cellular heterogeneity of KATP channel properties, a minority of myocytes being less sensitive to ATP. Alternatively, it may reflect nonspecific effects of the whole-cell configuration, such as dilution of cytosolic regulators or membrane stretch by the pipette. The accurate ATP sensitivity of the atrial KATP channel, determined on unitary currents, is shown in Figure 6DDown.



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  		Figure 1. Atrial appendage KATP current. A, Spontaneous activation of an inward rectifying outward current after establishment of the whole-cell configuration at 0 mmol/L internal ATP. Durations of whole-cell recording were 30 seconds (a), 2 minutes (b), 3 minutes (c), and 6 minutes (d). Voltage-ramp protocol is shown at the top. B, Same experiment as shown in panel A. Current amplitude at +50 mV (•) and potential at 0 pA ({circ}) have been plotted as a function of time of whole-cell recording. a, b, c, and d refer to the original current traces shown in panel A. C, Same experiment as in panels A and B. The current amplitude at +50 mV was plotted as a function of subsequent membrane potential at 0 pA. D, K+ selectivity. The bathing K+ concentration was changed from 5 to 30 mmol/L after the current was fully activated at 0 mmol/L internal ATP. Currents, after subtraction of leak (a) control current, were expressed as percentage of maximal current and plotted as I-V curves. The current reversal potential followed the shift of the calculated Nernst equilibrium potential for K+.



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  		Figure 6. ATP and glyburide sensitivity of the atrial appendage KATP channel. A, Effect of ATP on the KATP channel activity. The channel Po is plotted as a function of recording time. Each bar represents the Po calculated over a 15-second period. Before starting the experiment, the channel activity was refreshed by a 5 mmol/L ATP containing bath solution. The membrane potential was -80 mV. Original current traces a, b, c, and d are shown. B, Mean±SEM Po as a function of ATP concentration. n is indicated in italics. C, Effect of glyburide on the KATP channel activated by diazoxide. The channel Po is plotted as a function of recording time. Each bar represents the Po calculated over a 15-second period. Diazoxide was applied in the presence of 0.1 mmol/L ATP, and the membrane potential was -90 mV. Original current traces a, b, c, and d are shown. D, Mean±SEM Po as a function of glyburide concentration. n is indicated in italics.

Activation of Neonatal Atrial Appendage KATP Current by a Hypotonic Stretch
When no current activation occurred after 5 to 6 minutes of whole-cell recording with ATP in the pipette, KATP currents could be activated by a hypotonic stretch of the membrane, with the osmolality of the bathing solution being reduced from 290 to 260 mOsm/kg H2O (Figure 2ADown and 2BDown). Figure 2CDown shows the saturable relation between the current amplitude and the subsequent membrane hyperpolarization. The mean maximal subtracted current, measured at +50 mV, and the mean maximal membrane potential, measured at 0 pA, were 1159±155 pA and –64.3±2.5 mV (n=17) with 1 mmol/L ATP. In 5 of the 17 cells, glyburide (10 µmol/L) was applied and fully inhibited the stretch-activated current (example in Figure 2BDown). Figure 2DDown shows the mean control current amplitude, in the presence of 1 mmol/L internal ATP, and its inhibition after application of glyburide (0.1 to 1 µmol/L).



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  		Figure 2. Activation of the atrial appendage KATP current by hypotonic stretch. A, Original current traces of the experiment shown in panel B. B, In the presence of 1 mmol/L internal ATP, activation of the KATP current by perfusion of hypotonic solution (260 mOsm/kg H2O). Note lack of activation during the preceding 6-minute interval. Current amplitude at +50 mV (•) and maximal potential at 0 pA ({circ}) were plotted as function of time of whole-cell recording. a, b, and c refer to the original current traces shown in panel A. The current is inhibited by 1 µmol/L glyburide (Glyb). C, Same experiment as in panels A and B. Leak-subtracted current amplitude at +50 mV was plotted as a function of membrane potential at 0 pA. D, Hypotonic stretch-induced current is totally inhibited by 0.1 to 1 µmol/L glyburide. The maximal leak-subtracted current at +50 mV was expressed as mean±SEM. n is indicated in italics.

Inhibition of Neonatal Atrial Appendage KATP Current by Glyburide
Figure 3ADown and 3BDown shows the effect of 1 and 10 nmol/L glyburide on the atrial KATP current spontaneously activated in the presence of 1 mmol/L internal ATP. This glyburide-sensitive current showed a saturable relation with membrane potential measured at 0 pA (Figure 3CDown), similar to that seen for spontaneously activated or hypotonic stretch-activated KATP currents (Figures 1CUp and 2CUp). Figure 3DDown illustrates the percentage of remaining current as a function of glyburide concentration and shows a high sensitivity of the atrial appendage KATP current to glyburide, with IC50=1.22±0.15 nmol/L. The affinity for glyburide was similar whether the KATP current was activated spontaneously (•) or by a hypotonic stretch ({circ}). In the presence of 1 nmol/L glyburide, the KATP current was 49.9±10.2% (n=8) and 47.9±3.8% (n=5) of the control current, when activated spontaneously or by a hypotonic stretch, respectively, in the presence of 1 mmol/L internal ATP.



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  		Figure 3. Inhibition of atrial appendage KATP current by glyburide. A, Original current recordings showing inhibition by glyburide of KATP current spontaneously activated in the presence of 1 mmol/L internal ATP. a, control maximal current; b, glyburide 1 nmol/L; c, glyburide 10 nmol/L. B, Same experiment as in panel A. Current amplitude at +50 mV (•) and potential at 0 pA ({circ}) were plotted as function of time of whole-cell recording. a, b, and c refer to the original current traces shown in panel A. C, Same experiment as in panels A and B. The leak-subtracted current amplitude at +50 mV was plotted as a function of membrane potential at 0 pA. D, Inhibition of KATP current as a function of glyburide concentration. Current amplitude was expressed as a percentage of the maximal leak-subtracted control current amplitude at +50 mV and plotted as mean±SEM (n=3 to 13). KATP currents were activated spontaneously (•), by hypotonic stretch ({circ}), or by K+ channel openers ({square}) in the presence of 1 mmol/L internal ATP. Corresponding DMSO concentrations were without any inhibitory effect on KATP current.

Activation of Neonatal Atrial Appendage KATP Current by K+ Channel Openers
The KATP current could be activated in the presence of 1 to 5 mmol/L internal ATP by diazoxide (Figure 4ADown and 4BDown, b current) and cromakalim (not shown), 2 K+ channel openers. Maximal cromakalim and diazoxide-activated currents were of similar amplitude, and both were fully inhibited by glyburide (Figure 4ADown and 4BDown, c current), with an affinity in the nanomolar range (Figure 3DUp, {square}). Figure 4CDown and 4DDown shows the mean current amplitude activated by various concentrations of cromakalim (Figure 4CDown) and diazoxide (Figure 4DDown) in the presence of 2 mmol/L internal ATP. In the presence of either cromakalim or diazoxide, the mean maximal activated current, measured at +50 mV, was 1010±101 pA and the mean maximal membrane potential, measured at 0 pA, was –64.6±1.6 mV (n=39). Although the exact EC50 was not determined, both cromakalim and diazoxide activated the KATP current with approximate EC50 values of 10 and 1 nmol/L, respectively, corresponding to a mean current amplitude of 444±149 pA (n=6) and 401±84 pA (n=7), with P<0.05 compared with the mean maximal current amplitude. DMSO was required to dissolve these drugs, but its final concentration in the perfusion medium was never >1 mmol/L. At 1 µmol/L or 1 mmol/L, DMSO by itself had no effect on KATP current activation. In fact, 100 µmol/L diazoxide activated a mean maximal KATP current of 875±166 pA (n=9) after 2 to 3 minutes of bath application, whereas the mean subtracted current amplitude was 4±14 pA (n=4) after 2 to 4 minutes of application of the corresponding DMSO concentration (1 mmol/L).



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  		Figure 4. Activation of atrial appendage KATP current by K+ channel openers. A, Original current recordings of a glyburide-sensitive diazoxide-activated current in the presence of 1 mmol/L internal ATP. a, Control; b, diazoxide (100 µmol/L); c, diazoxide (100 µmol/L)+glyburide (10 µmol/L). B, Same experiment as in panel A. Current amplitude at +50 mV (•) and potential at 0 pA ({circ}) were plotted as a function of time of whole-cell recording. a, b, and c refer to the original current traces shown in panel A. C, Mean maximal leak-subtracted current amplitude at +50 mV as function of concentration of cromakalim (2 mmol/L internal ATP; n=6 to 12). *P<0.05 compared with current activated by 10 µmol/L cromakalim. D, Mean maximal leak-subtracted current amplitude at +50 mV as function of concentration of diazoxide (2 mmol/L internal ATP; n=5 to 10). *P<0.05 compared with current activated by 100 µmol/L diazoxide.

Unitary Neonatal Atrial Appendage KATP Current
Although usually closed in the cell-attached configuration, KATP channels were activated on excision of the membrane patch (inside-out configuration) in the absence of ATP in the bath solution. The channel activity ran down in 2 to 3 minutes and could be refreshed on washout of an ATP-containing solution. Figure 5ADown shows the I-V curve and the original current recordings in the absence of ATP, with 140 mmol/L K+ in the pipette solution and a Nernst K+ equilibrium of +48 mV. The KATP channel opened in long-lasting bursts of 133.8±20.4 ms (n=15 patches), with a unitary conductance of 58±2 pS (n=11) for negative potentials. A similar conductance was measured on the rare openings recorded on cell-attached patches, 58±4 pS (n=6). Within bursts, the channel showed a flickering activity, with rapid openings and closings (Figure 5BDown). The mean opening duration and the mean closing duration within bursts were estimated to be 1.40±0.10 ms and 0.66±0.04 ms (n=15) respectively, between -120 and -70 mV and in the absence of ATP.



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  		Figure 5. Inside-out recording of atrial appendage KATP channel. A, Unitary current amplitude plotted vs patch membrane potential. In this experiment, the channel conductance was 58 pS between 0 and -100 mV. Original currents are shown, recorded at -90 mV in the presence of 140 mmol/L K+ in the pipette solution (Nernst K+ equilibrium +48 mV) and in the absence of ATP in the bathing solution. The bottom trace is a detail from the middle trace (between open arrowheads). B, Open time (top) and close time (bottom) histograms of burst openings from the same experiment as in panel A. The mean open time and the mean close time were 1.73 and 0.54 ms, respectively. Note that the 1-kHz filtering, which is currently used in this type of experiment,19 22 25 can lead to an overestimation of the channel opening and closing time constants.

The sensitivity to ATP of the channel has been examined after the channel activity was refreshed by an ATP-containing medium. After refreshment, the channel showed a high open probability (Po), {approx}0.7, which could be abolished by 1 mmol/L ATP, whereas 0.1 mmol/L only exerted a partial inhibition (Figure 6AUp). The effects of ATP concentration ranging from 0 to 5 mmol/L on the atrial KATP channel Po are represented on Figure 6BUp. The half-maximal inhibition is obtained in the presence of 0.1 mmol/L cytosolic ATP, the KATP channel Po being reduced from 0.49±0.11 (n=6) to 0.23±0.09 (n=6).

The atrial appendage KATP channel could be activated first by diazoxide, reaching a mean maximal Po of 0.31±0.1 (n =8), and then inhibited by nanomolar concentrations of glyburide (Figure 6CUp and 6DUp). The channel Po is reduced to 31.11±0.05% (n=3) and 16.17±5.58% (n=4) of the control value, in the presence of 1 and 10 nmol/L glyburide, respectively, thus confirming the affinity for glyburide measured on whole-cell current. The inhibition by glyburide could be reversed (not shown), but usually required a 4- to 10-minute washout, especially for high concentrations.

KATP Channel Subunits Expressed by Neonatal Atrial Appendage Cardiomyocytes
RT-PCR was performed on RNA extracts from primary cultured atrial cardiomyocytes (Figure 7Up). To reveal the expression of the splice variants SUR1A and SUR1B, SUR2A and SUR2B, and SUR2A and SUR2C, specific primers were chosen on each side of the splicing site, thus amplifying 2 fragments of different size when both isoforms were expressed. Atrial cardiomyocytes, characterized by the expression of ANP (lane 4), expressed both Kir6.1 (lane 6) and Kir6.2 (lane 8), and SUR1A and SUR1B (lane 10). SUR2A is expressed, as shown by the 249-bp fragment amplified by the SUR2A-specific primers (lane 12), as well as the SUR2B (173-bp) isoform amplified with the SUR2A-2B primers (lane 14). The short splice variant SUR2C is not expressed by rat atrial cardiomyocytes, the SUR2A-2C primers only amplifying a 358-bp SUR2A fragment (lane 16). Positive controls were performed on expression vectors containing either the SUR2C (lane 18) or the SUR2A cDNA (lane 19), thus showing that the SUR2A-2C primers were effective. Negative controls without RT were obtained for all primer pairs (see lanes 1, 3, 5, 7, 9, 11, 13, and 15), indicating that the PCR products were not due to contamination with genomic DNA. This expression pattern was obtained from 3 different cell cultures, and similar results were found with whole atrial extracts (not shown).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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The Neonatal Atrial Appendage KATP Channel as a New Subtype
We have shown that neonatal atrial appendage cardiomyocytes display a cellular KATP current that is activated by hypotonic stretch of the membrane and shows a high affinity for the sulfonylurea glyburide (IC50=1.2 nmol/L) as well as for the K+ channel openers cromakalim and diazoxide, with a higher affinity for diazoxide. The channel displays a flickering pattern activity within long-lasting bursts (134-ms mean duration), a unitary conductance of 58 pS in the presence of a high K+ level on the extracellular side of the membrane, and an IC50 for ATP {approx}0.1 mmol/L. Confirming the results obtained on whole-cell current, single activated KATP channels were inhibited by glyburide in the nanomolar range. Neonatal atrial appendage cardiomyocytes displayed a wide expression pattern of KATP channel subunits, including all Kir6.x and SUR isoforms except for the SUR2C subtype. Together, these results lead us to propose the neonatal atrial KATP channel as a new pharmacological and functional subtype.

Pharmacological Properties of Neonatal Atrial KATP Current
As an attempt to determine which SUR subtype is involved in atrial KATP channel function, we compared its pharmacological properties with data obtained by other groups on ventricular cardiomyocytes, pancreatic ß-cells, and reconstituted KATP currents (TableDown). The pancreatic KATP current, and its corresponding functional subunit association SUR1A/Kir 6.2, yields a high affinity for glyburide (nmol/L range) and for diazoxide but a low affinity for cromakalim, the latter being sometimes even described as ineffective.6 7 11 12 18 29 30 31 32 The SUR2A subunit, which confers high affinity for cromakalim, total insensitivity to diazoxide, and low affinity for glyburide (µmol/L range), has been described as the ventricular subtype.13 18 33 However, the affinity of ventricular KATP channels for glyburide remains controversial, with IC50 values varying from the nmol/L to the µmol/L range, depending on the experimental conditions and the metabolic state of the cell.6 7 11 34 35 36 37 38 39 40 41


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  		Table 1. Pharmacological Properties of KATP Channels

The pharmacological properties of the neonatal atrial appendage KATP current presented here appear to differ from both the pancreatic and ventricular subtypes, the sensitivity to diazoxide constituting a distinctive feature compared with ventricular KATP channels.6 7 11 37 The high affinity for diazoxide and cromakalim should be interpreted by taking into account the experimental conditions. Indeed, the activity of these 2 channel openers depends on cytosolic ATP, involving a decrease in the channel sensitivity to ATP42 and possibly a phosphorylation.43 44 Thus, the affinities for openers we measured in the presence of 2 mmol/L internal ATP on whole-cell current are expected to be higher than the affinities on intact cells, in which the cytosolic ATP level is thought to be higher.

The most recently cloned SUR2 subtype, SUR2B, considered to be the smooth muscle subtype, confers sensitivity to diazoxide in reconstituted KATP channels.16 19 42 Further pharmacological characterization of the SUR2B subtype is needed to speculate whether it could be involved in the neonatal atrial appendage KATP channel formation.

Single Atrial Appendage KATP Channel Properties: Burst Kinetics and Conductance
Neonatal atrial KATP channels display characteristic features shared by all KATP channels: weak inward rectification, flickering activity within bursts, fast rundown after excision of the membrane patch, and refreshment on washout of cytosolic ATP. However, burst kinetics, unitary conductance, and ATP sensitivity have been shown to depend on the subtypes constituting the channel.11 18 45 The pancreatic KATP channel has been described as opening in bursts that are shorter than those of the ventricular channel: {approx}20 to 40 ms burst duration for the pancreatic channel versus 212 ms for the ventricular one. In contrast, the intraburst kinetics were similar for both types, with a mean open time of 1 to 4 ms and a mean closed time of 0.2 to 0.6 ms.6 33 34 45 46 47 The atrial KATP channel has a mean open time of 1.4 ms and a mean closed time of 0.7 ms within bursts of 133.8±20.4 ms (n=15) duration. This latter value, although closer to what has been reported for the ventricular KATP channel, is significantly lower (P<0.05, 2-tailed t test) than the ventricular burst duration reported by Alekseev et al,45 212.5±23.6 (n=7). The sensitivity of the neonatal atrial appendage KATP channel to ATP, with an IC50 {approx}100 µmol/L, is closer to the 20 to 100 µmol/L value reported for the ventricular channel6 45 48 than to the high-affinity 10 to 30 µmol/L value reported for the pancreatic channel.6 18 However, in terms of channel conductance, the atrial appendage KATP channel differs from the 70- to 90-pS ventricular channel by its lower conductance of 58 pS, a value similar to what has been reported for the pancreatic channel. Recently, Babenko et al49 showed virtually identical functional and pharmacological properties for the human ventricular KATP channel and the SUR2A/Kir6.2 reexpressed channel, a finding that further supports the notion that the atrial appendage KATP channel does differ from the ventricular KATP subtype.

The neonatal atrial appendage KATP channel appears to differ not only from the adult ventricular KATP channel, but also from the neonatal ventricular one. The latter shares the following properties with the adult ventricular channel: a unitary conductance of 75 pS, an IC50 for ATP of 4.8 µmol/L, openings in bursts with a flickering pattern, and similar open and close time duration. This channel also shows a very low sensitivity to diazoxide, which could constitute a difference compared with adult channel properties. On neonatal ventricular myocytes, 0.5 mmol/L diazoxide induces a small, whole-cell KATP current of 5 pA/pF in 50% of the cells, representing 17% of the maximal current induced by pinacidil.50 This effect of diazoxide is 1 million times lower than for neonatal atrial appendage KATP channels. However, the high sensitivity of the neonatal atrial appendage KATP channel to diazoxide that was observed in whole-cell recordings remains to be verified by single-channel analysis, given that the macroscopic current may contain channels not presented in the unitary recordings.

There seems to be no difference between atrial appendage and ventricular cardiomyocytes with respect to density of the KATP channels on the plasma membrane. Assuming a mean maximal cellular KATP current of 1000 pA at +50 mV, a unitary inward conductance of the atrial appendage KATP channel of 11 pS in the presence of 5 mmol/L extracellular K+,46 and a half-reduced outward conductance of 5.5 pS (inward rectification), the density of atrial KATP channels can be estimated to be 1500 channels per cell, a value similar to the 2000 to 3000 channels per cell reported for ventricular myocytes.48

KATP Channel Subunits Expressed by Neonatal Atrial Appendage Cardiomyocytes
On the basis of the mixed pharmacological and functional properties of atrial appendage KATP channels, we performed RT-PCR on RNA extracts from primary cultured neonatal atrial appendage cardiomyocytes to determine which KATP subunits were expressed. Our results show a wide expression pattern, with the 2 Kir6.x isoforms, Kir6.1 and Kir6.2, and 4 SUR isoforms, SUR1A, SUR1B, SUR2A and SUR2B. SUR2C was not expressed in rat neonatal atrial myocytes, thus confirming the results of Chutkow et al,17 who reported that the SUR2C isoform was not expressed in rat tissues. The expression of Kir6.2 and Kir6.1 was already reported in rat heart.12 15 SUR1A expression was reported in rat heart,12 to a lower degree than SUR2A,17 18 whereas SUR2A and SUR2B have also been shown to be both expressed in atrium of mouse heart.19 The tissue distribution pattern of SUR1B, recently cloned from a rat pancreatic cell line (GenBank accession No. AF039595), is not yet described, and its expression by rat atrium could constitute a characteristic feature. The inclusion of dexamethasone and T3 in the culture medium is not responsible for this expression pattern, because in their absence the same subunits were expressed, and the same sensitivity to glyburide and diazoxide was observed in the electrophysiological recordings (not shown).

The results do not allow a conclusion on the neonatal atrial KATP channel composition, but they support the view that atrial KATP channels are a heteromultimeric association of several SUR subtypes showing mixed pharmacological and functional properties with regard to other known KATP channel types. For example, a mixture of SUR1A and SUR2B could explain several properties of the neonatal atrial KATP channel, as follows: conductance, diazoxide, and glyburide sensitivity of the SUR1A type, and bursts kinetics, ATP, and cromakalim sensitivity of the SUR2A type. However, the contribution of SUR1B and the possibility of a new uncloned SUR subtype must also be taken into account.

Possible Physiological Functions of the Atrial Appendage KATP Channels
The physiological role of cardiac KATP channels in ischemic preconditioning and hypoxia-triggered events has been well documented,6 7 11 35 40 but atrial KATP channels also appear to be involved in cardiac secretion. Opening of atrial KATP channels, either pharmacologically (diazoxide or pinacidil) or metabolically (2-D-deoxyglucose), abolishes the stretch-stimulated ANP secretion in isolated heart and cultured atrial appendage cardiomyocytes (Reference 44 and J.H. Jiao et al, unpublished data, 1995–1999). It is tempting to draw analogies with KATP channel–triggered secretion of insulin by pancreatic ß-cells. However, KATP channels, normally open in resting pancreatic ß-cells, are closed in cardiac myocytes,7 44 in which they could only modulate stimulated ANP release. The links between membrane stretch, KATP channel activation, and ANP release are still unknown. Our results confirm van Wagoner's25 finding that stretch opens atrial KATP channels. Other potent stimulators of ANP release, such as endothelin and hypoxia,2 3 4 27 are known to open cardiac KATP channels.6 51 52 This suggests that activation of KATP channels could be a common mechanism for feedback inhibition of stimulated ANP release.


*    Acknowledgments
 
This study was supported by the Swiss National Science Foundation (Grant 31-49798.96) and the following foundations: Société Académique de Genève, Horten, de Reuter, Sandoz, and the Roche Research Foundation. We thank William A. Chutkow for providing the expression vectors containing SUR2A and SUR2C cDNA, Uta Schmidt for helpful discussion, and Dr Rui de Sousa for numerous suggestions on the manuscript.

Received December 14, 1998; accepted August 11, 1999.


*    References
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