Published online before print October 30, 2008, doi: 10.1161/CIRCRESAHA.108.178186
© 2008 American Heart Association, Inc.
Cellular Biology |
Differential Structure of Atrial and Ventricular KATP
Atrial KATP Channels Require SUR1
From the Department of Cell Biology and Physiology (T.P.F., H.T.K., R.M., G.C., C.G.N.), Washington University School of Medicine, St Louis, Mo; Department of Molecular Physiology and Biophysics (M.A.M.), Vanderbilt University School of Medicine, Nashville, Tenn; Department of Medicine and Pathology (D.J.L.), Albert Einstein College of Medicine, New York; and Department of Pediatrics (W.A.C.), New York University School of Medicine.
Correspondence to Colin G. Nichols, PhD, Department of Cell Biology and Physiology, Box 8228, Washington University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110. E-mail cnichols{at}wustl.edu
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Abstract |
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The isoform-specific structure of the ATP-sensitive potassium (KATP) channel endows it with differential fundamental properties, including physiological activation and pharmacology. Numerous studies have convincingly demonstrated that the pore-forming Kir6.2 (KCNJ11) and regulatory SUR2A (ABCC9) subunits are essential elements of the sarcolemmal KATP channel in cardiac ventricular myocytes. Using a novel antibody directed against the COOH terminus of SUR1 (ABCC8), we show that this KATP subunit is also expressed in mouse myocardium and is the dominant SUR isoform in the atrium. This suggests differential sarcolemmal KATP composition in atria and ventricles, and, to test this, KATP currents were measured in isolated atrial and ventricular myocytes from wild-type and SUR1–/– animals. KATP conductance is essentially abolished in SUR1–/– atrial myocytes but is normal in SUR1–/– ventricular myocytes. Furthermore, pharmacological properties of wild-type atrial KATP match closely the properties of heterologously expressed SUR1/Kir6.2 channels, whereas ventricular KATP properties match those of heterologously expressed SUR2A/Kir6.2 channels. Collectively, the data demonstrate a previously unappreciated KATP channel heterogeneity: SUR1 is an essential component of atrial, but not ventricular, KATP channels. Differential molecular make-up of the 2 channels underlies differential pharmacology, with important implications when considering sulfonylurea therapy or dissecting the role of cardiac KATP pharmacologically, as well as for understanding of the role of diazoxide in preconditioning.
Key Words: diazoxide • sarcolemmal • mitochondrial • ABCC8 • ABCC9
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Introduction |
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ATP-sensitive potassium (KATP) channels are expressed in a diverse set of excitable tissues and provide a direct molecular link between metabolism and function. By responding to changes in the ratio of [ADP] to [ATP] in the cell, KATP channels modulate cell membrane excitability, controlling Ca2+ entry and Ca2+-dependent cell functions. All KATP channels share the same structural blueprint1: an inward rectifier potassium channel (Kir6.x) and a sulfonylurea receptor (SURx) coassemble in 4:4 stoichiometry to form the KATP channel complex. Kir6.x encodes the binding site for inhibitory ATP and forms the conducting pore of the channel. SURx confers sulfonylurea sensitivity to the channel and determines efficacy of potassium channel openers (KCOs) such as diazoxide and pinacidil, and its nucleotide binding folds are essential for nucleotide diphosphate-dependent stimulation.2–4 Although the same overall architecture is maintained for all KATP channels, specific components depend on tissue type: pancreatic β-cell KATP channels are formed by coassembly of SUR1 and Kir6.2,5–9 whereas SUR2A coassembles with Kir6.2 to generate ventricular sarcolemmal channels.10–14 Subunit composition determines, in part, the dynamic range of channel activity. For example, channel complexes containing SUR1 are more sensitive to stimulation by ADP than those containing SUR2A,15 which, in part, may underlie the observation that pancreatic β-cell KATP channels (SUR1+Kir6.2) respond to lowered blood glucose, whereas ventricular KATP channels (SUR2A+Kir6.2) respond only to pathological stimuli or extreme stress, like ischemia or hypoxia.
However, SUR1 transcripts and protein have been reported in the heart,16,17 and several observations suggest a functional role in sarcolemmal KATP. For example, antisense oligonucleotides to SUR1 suppress KATP channel activity in cultured neonatal myocytes,18 and atrial myocytes are more sensitive to the SUR1-specific KCO diazoxide19,20 than ventricular myocytes. SUR1–/– hearts show improved functional recovery after ischemia,21 whereas extensive arrhythmias and sudden death are observed in mice overexpressing SUR1, but not SUR2A, in the heart.22 In this study, we have examined the regional expression of SUR1 and the functional properties of KATP channels, in wild-type (WT) and SUR1–/– hearts. The striking results demonstrate that sarcolemmal KATP is markedly different in structure in the atria and ventricles, SUR1 being an essential component of the atrial channel but having no obvious role in the ventricle. These findings have significant implications for the pharmacology and pathophysiology of atrial versus ventricular KATP channels.
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Materials and Methods |
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The generation of SUR1–/– and SUR1 transgenic (TG) mice have been described elsewhere.23,24 All procedures complied with the standards for the care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996), and protocols were approved by the Animal Studies Committee at Washington University School of Medicine.
Quantitative RT-PCR
Expression of SUR1 and SUR2A mRNA was examined using real-time RT-PCR.23 Total RNA was isolated from cardiac atrial or ventricular tissue using TRIzol according to the protocols of the manufacturer (Invitrogen). Isolated RNA was then treated with DNAseI to digest residual genomic DNA and further purified using a silica-based column protocol (Rneasy, Qiagen). [RNA] was determined spectrophotometrically (Nanodrop Technologies Inc). Reverse transcription and PCR were carried out in a single tube in an ABI Prism 7000 sequence detection system (Applied Biosystems Inc). A total of 75 ng of template RNA was used in all reactions. Following the reverse-transcription reaction (45 minutes, 48°C), 40 cycles of PCR were carried out. Double-stranded DNA was fluorescently labeled with SYBR green (Applied Biosystems Inc). Gene-specific primers for SUR1, SUR2A, and β-actin were designed using the PrimerExpress software (ABI) and purchased from Integrated DNA Technologies. Reactions with each primer pair and template were performed in duplicate. Following baseline correction, a fluorescence threshold was established, and the cycle when this threshold was crossed (Ct) was determined for each reaction. To control for variability in RNA quantity, the normalized value, 
Ct, for each sample was calculated using the formula Ct=Ct(SUR)–Ct(actin). Relative mRNA expression is reported as 2–Ctx1000.
Generation of Anti-SUR1 Antibody
Antibodies to SUR1 were raised and purified using standard protocols. Briefly, a peptide (CKDSVFASFVRADK) corresponding to the final 13 amino acids of the mouse SUR1 subunit and containing an amino-terminal cysteine for downstream purification reactions was synthesized, and antibodies to the COOH-terminal peptide were raised in rabbits (Strategic Biosolutions) and affinity-purified against the peptide (Sulfolink, Pierce) according to manufacturer protocols.
Protein Isolation and Analysis
Recombinant channel protein expressed in COSm6 cells was analyzed as described.15 Hearts were excised and washed in ice cold PBS, and atrial appendages/ventricular tissue (or ventricular myocytes, isolated as below) were separated and placed in cardiac homogenization buffer (300 mmol/L sucrose, 10 mmol/L Tris, pH 7.4) supplemented with protease inhibitors. Tissue was disrupted using a Polytron homogenizer. Crude membrane extract was obtained by ultracentrifugation at 65 000 rpm for 60 minutes, and pellet was resuspended in COS cell lysis solution (150 mmol/L NaCl, 20 mmol/L HEPES, 5 mmol/L EDTA, 1% Nonidet P-40, pH 7.4) supplemented with protease inhibitors. Protein concentration was determined by the bicinchoninic method (Pierce). For Western analysis, proteins (typically 50 µg) were separated by 7.5% polyacrylamide gel electrophoresis and transferred to poly(vinylidene fluoride) membrane. Antibodies were used at 1:1000 dilution, and signal was visualized with either the SuperSignal West Femto or Supersignal West Pico ECL substrates (Pierce).
For immunohistochemistry, freshly isolated cardiomyocytes were attached to laminin-coated glass cover slips and fixed with 10% formalin for 15 minutes, followed by 100% methanol overnight at 4°C. Following fixation, cells were permeabilized with PBS containing 0.1% Triton X-100. Following washing (3x for 5 minutes), cells were incubated with anti-SUR1 antibody (1:300) and goat serum (1:1000) at room temperature. After washing (3x for 5 minutes) with PBS containing goat serum (1:1000), samples were incubated with 2° antibody (goat anti-rabbit) labeled with Alexa-488 for 45 minutes at room temperature. Following washing and mounting, cells were visualized on a Zeiss LSM 510 laser scanning confocal microscope.
Electrophysiology
Myocytes (isolated as described previously25) were placed in a recording chamber containing Tyrode solution (mmol/L: NaCl, 137; KCl, 5.4; NaH2PO4, 0.16; glucose, 10; MgCl2, 0.5; HEPES, 5.0; NaHCO3, 3.0; pH 7.3 to 7.4) with additions as described. Intracellular (pipette) solution contained (mmol/L: KCl, 20; K-aspartate, 130; MgCl2, 1; HEPES, 10; EGTA, 10; pH 7.3 to 7.4). Patch clamp electrodes (1 to 3 M
, when filled with electrode solution) were fabricated from soda lime glass microhematocrit tubes (Kimble 73813). PClamp 8.2 software and DigiData 1322 converter (Axon Instruments) were used to generate command pulses and collect data. Cell capacitance and series resistance were estimated using a 5 to 10 mV hyperpolarizing square pulse from a holding potential of –70 mV following establishment of the whole-cell recording configuration. Current was evoked with a slow voltage ramp protocol from –120 to 40 mV over 4 seconds (Vhold=–70 mV during interpulse periods), filtered at 5 kHz. Total conductance was calculated from the slope in a 10-mV window around the reversal potential. Series resistance was electronically compensated by 80% (to less than 2 M) to minimize measurement errors. However, given the size of the KATP current, reported conductance may be an underestimation of the true magnitude of the conductance, particularly in WT cells.
Inside-out patch-clamp experiments were made at room temperature, using a microfluidic capillary chip-based platform (Dynaflow, Cellectricon Inc). DF-16 ProII chips were used, allowing synchronized control of switching between 16 experimental solutions. Laminar flow at each solution outlet of the microfluidic chip prevents mixing, and a computer-controlled stepper motor is used to move the chip relative to the patch pipette, allowing for relatively rapid solution exchange around the membrane patch. The pipette (extracellular) and bath (cytoplasmic) solution (Kint) used in excised patch experiments had the following composition: 140 mmol/L KCl, 1 mmol/L EGTA, 10 mmol/L HEPES, pH 7.3. Diazoxide and Pinacidil were stored as 100 mmol/L stocks in DMSO. ATP and ADP (potassium salts) were stored as 100 mmol/L stocks in Kint solution, with the pH of each stock solution adjusted to 7.3. All stock solutions were stored at –20°C and diluted into working concentrations on each experimental day. Membrane currents were recorded at –50 mV. Data were filtered at 1 kHz, digitized at 5 kHz, and stored directly on computer hard drive using Clampex version 9 software (Axon Inc). Mean data are presented throughout the text as stimulated Irel (current amplitude, normalized to the maximum KATP current per patch).
Data Analysis
Data were analyzed using ClampFit and Microsoft Excel software. Results are presented as means±SEM. Statistical tests and probability values (ANOVA, Tukey post hoc test, where appropriate) are denoted in figure legends.
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Regional SUR Transcription in the Mouse Heart
Although its is widely accepted that Kir6.2 and SUR2A subunits generate the cardiac sarcolemmal KATP current, various studies indicate that SUR1 subunits may also be expressed in cardiac myocytes.16,26,27 We examined SUR isoform expression, and functional properties of KATP channels, in atria and ventricles of WT and SUR1 knockout (SUR1–/–) mice. Quantitative RT-PCR (primers targeted to exon 2) indicates that SUR1 transcription is at similar levels in atria and ventricle (Figure 1A), even though deletion of exon 1 (SUR1–/–)23,24 leads to abolition of protein expression (see below). As a positive control, SUR1-overexpressing animals23 reveal
100x levels of SUR1 transcript, mirrored by similarly increased protein levels (see below). Two important features are revealed for SUR2A (Figure 1B). Firstly, SUR2A transcript levels are considerably (10x) higher in the ventricle than atrium in WT animals, as also recently reported by Marionneau et al.28 Secondly, SUR2A transcription and atrial/ventricular ratio are unaffected either by SUR1 knockout or by SUR1 overexpression.
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SUR1 Protein Expression and Distribution in the Mouse Heart
Several studies have reported SUR1 protein in the heart,16,17 but none has used the critical SUR knockout tissue control. There are concerns regarding the specificity of anti-KATP subunit antibodies,29 and our experience with commercial anti-SUR1 antibodies suggested that nonspecificity was a potentially significant problem. We therefore raised a novel antibody against SUR1 and extensively verified specificity. Western blot analysis on proteins isolated from COSm6 cells expressing FLAG epitope-tagged SUR1 subunits shows that anti-FLAG m2 and anti-SUR1 antibodies detect the same core and higher-order glycosylated proteins, at the predicted molecular weight for SUR1 (Figure 2A). In addition, the antibody specifically recognizes SUR1, and no cross-reactivity is observed in samples from cells transfected with SUR2A or SUR2B (Figure 2B).
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Having confirmed specificity, proteins isolated from WT and SUR1–/– hearts were probed for SUR1. Significant core glycosylated (150 kDa30) SUR1 is present in WT, but not SUR1–/–, hearts (Figure 2C). In addition, using high-sensitivity detection, higher-order glycosylated proteins, as well as a specific shorter protein 100 kDa, are detected in only WT hearts, and these same bands are all enhanced in hearts overexpressing SUR1 (SUR1 TG) (Figure 2C).23 None of these bands is present in knockout hearts, with the exception of a very faint band at 150 kDa using high-sensitivity detection and a single major nonspecific band at 60 kDa, present at similar levels in isolated proteins from all hearts.
SUR1 was also detected in isolated WT atrial and ventricular proteins although, when loaded with the same total protein, we routinely observed a greater signal in atrium than ventricle (Figure 2D). This suggests that SUR1 is more strongly expressed in the atria than the ventricle, although this need not be a reflection of expression in myocytes per se. To examine this specifically, SUR1 distribution in isolated myocytes was examined by immunofluorescence. Little or no signal above background was observed in WT or SUR1–/– ventricular myocytes, although a strong signal was detected in SUR1-overexpressing transgenic ventricular myocytes (Figure 3A). In contrast, a strong membrane-localized SUR1 signal was detected in isolated WT atrial myocytes (Figure 3A), and SUR1 immunofluorescence was prominent in atrial tissue sections (Figure 3B). Consistent with the immunofluorescence data, and again indicating minimal or absent SUR1 expression in ventricular myocytes, no SUR1 signal was detected by Western blot when proteins were isolated from dissociated ventricular myocytes (Figure I in the online data supplement, available http://circres.ahajournals.org).
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SUR1 Is Not an Essential Component of Ventricular KATP Channel
The above results demonstrate that SUR1 is expressed in murine atrial myocytes and suggest that the subunit may be a prominent in atrial, but not ventricular, sarcolemmal KATP channels. To examine the role of SUR1 in functional expression of sarcolemmal channels, we measured total KATP current activated by complete metabolic inhibition (2.5 µg/mL oligomycin, plus substitution of glucose with 10 mmol/L 2-deoxyglucose) in isolated myocytes. Metabolic inhibition induced substantial KATP conductance in both WT and SUR1–/– ventricular myocytes (3.14±0.59 versus 3.00±0.47 nS/pF; n=4 [WT] and 6 [SUR1–/–], respectively; Figure 4). We additionally assessed ventricular KATP channel activity in isolated inside-out patch clamp experiments. Again, KATP current density was not significantly altered in SUR1–/– membranes (254.4±79.8 versus 159.6±37.7 pA/patch; n=9 [WT] and 15 [SUR1–/–] and ATP sensitivity of channels in patches from WT and SUR1–/– ventricular myocytes was similar (Figure 4B). Ventricular KATP from WT and SUR1–/– myocytes revealed no differences in pinacidil or MgADP sensitivity (supplemental Figure II). Taken together, the results demonstrate that SUR1 is not necessary for the functional expression of the ventricular KATP channel. Although they do not discount the possibility that SUR1 may be a nonessential component, they are consistent with the prevailing notion that the coassembly of SUR2A and Kir6.2 subunits generates the ventricular sarcolemmal KATP channel.
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SUR1 Is an Essential Component of the Atrial KATP Channel
In whole-cell conditions, with 0 or 500 µmol/L MgATP in the pipette, we frequently observed spontaneous activation of WT atrial KATP within 1 minute, even without application of metabolic inhibition or KCOs. Under similar conditions, no KATP activation was observed in SUR1–/– atrial myocytes. Diazoxide also activated substantial KATP (48.3±20.5 nS, n=8) in WT atrial myocytes perfused but no KATP in SUR1–/– myocytes (2.6±12.2 nS, n=5). The absence of sarcolemmal KATP in SUR1–/– atrial myocytes was confirmed in excised inside-out patches: large ATP-sensitive current was observed (Figure 5A) in WT atrial patches (47.55±10.48 pA/patch, n=12), but essentially no current was observed in SUR1–/– patches (4.67±3.05 pA/patch, n=14). Taken together, the data demonstrate that SUR1 is an essential component of the KATP channel in mouse atrial sarcolemma.
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Pharmacological Profile Analysis of Atrial and Ventricular KATP
The SUR subunit of the KATP channel is the binding site for inhibitory sulfonylureas and KCO drugs, and different SUR isoforms confer different pharmacological properties on the channel.1 Specifically, recombinant SUR1/Kir6.2 channels exhibit prominent sensitivity to the KCO diazoxide, whereas SUR2A/Kir6.2 channels are relatively insensitive.3,9,31–33 Similarly, pinacidil specifically activates SUR2A/Kir6.2 but not SUR1/Kir6.2 channels.31,34,35 Thus the molecular identity of the SUR subunit in a given KATP complex can be inferred from its pharmacological "fingerprint," and the disparate function of SUR1 in atrial and ventricular KATP (ie, SUR1 is required in the atria) suggests that the channels will have distinct fingerprints. To test this, diazoxide and pinacidil action were assessed in inside-out patches from atrial or ventricular myocytes and compared with the action on channels of defined composition, expressed heterologously in COSm6 cells (Figure 6). As expected, recombinant SUR1/Kir6.2 channels are more sensitive to diazoxide than recombinant SUR2A/Kir6.2 channels (Figure 6A). Consistent with a significant functional role for SUR1, KATP channels in atrial myocytes were also strongly activated by diazoxide but were insensitive to pinacidil. On the other hand, ventricular KATP and recombinant SUR2A/Kir6.2 channels were markedly stimulated by pinacidil but not diazoxide (Figure 6B). Together with the lack of effect of SUR1 knockout on ventricular KATP density (Figure 4), or functional properties (supplemental Figure II), the results are most consistent with the only significant SUR contributors to mouse atrial and ventricular KATP channels being SUR1 and SUR2A, respectively.
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Atrial and Ventricular KATP Channels Are Structurally Distinct
It is currently widely accepted that cardiac sarcolemmal KATP channels are formed by the coassembly of SUR2A and Kir6.2. KATP channel activity is essentially absent in isolated ventricular myocytes from both Kir6.2–/– and SUR2–/– animals, providing strong evidence that this is indeed the case in the ventricle.12,13,36 However, the present results incontrovertibly demonstrate that this is not so for atrial myocytes. Biochemical and functional data clearly show that SUR1 is strongly expressed in the WT atrium and that atrial sarcolemmal KATP requires SUR1 for functional channel expression. As a result of this regionally distinct channel structure, sarcolemmal KATP channels in mouse heart have very different pharmacological fingerprints. Like recombinant SUR1/Kir6.2 channels, atrial KATP is more sensitive to diazoxide than pinacidil, whereas ventricular KATP has the opposite KCO specificity, reiterating the phenotype of recombinant SUR2A-Kir6.2 channels in inside-out patch clamp experiments.
The present study is restricted to analysis in the mouse, the only species in which it is possible to make the critical comparison with the relevant gene knockouts. However, the notion that KATP channel structure varies in different heart regions is not entirely novel and several studies have demonstrated regional pharmacology in other species. KATP channels in cultured neonatal rat atrial and ventricular myocytes vary in their response to KCOs and metabolic inhibitors19,20 and antisense oligonucleotides directed against SUR1 have been shown to inhibit KATP current in cultured neonatal ventricular myocytes.18 In the present study, we have limited analysis to mouse tissue, because comparison between WT and SUR1–/– tissue is critical in allowing unequivocal delineation of the role of SUR1. To address the issue in larger animals (in which genetic knockout of SUR1 is not available), future studies must rely on pharmacology and biochemical studies. Although essential, these approaches will require careful interpretation, given the overlapping pharmacology of the different subunits.
Implications for Cardiac Arrhythmias, Ischemic Preconditioning, and Cardioprotection
When activated, KATP channels will shorten the action potential and reduce the effective refractory period, a significant risk factor for the development of reentrant arrhythmias like atrial fibrillation.37 To date, there have been very few studies addressing this possibility in humans, although a mutation in SUR2A has been associated with vein of Marshall adrenergic atrial fibrillation.38 The disparate structure of the atrial and ventricular KATP may have important implications for the maintenance of atrial fibrillation. Because channels composed of SUR1 and Kir6.2 turn on more readily15 (we see spontaneous activation of atrial but not ventricular KATP in whole-cell mode), it seems likely that the activation of SUR1-based atrial KATP during rapid pacing conditions of atrial fibrillation might act to stabilize the arrhythmia. In this regard, we have previously examined a series of transgenic mice overexpressing either SUR1 or SUR2A in the heart. Interestingly, we consistently observe delayed atrioventricular conduction only in mice that overexpress SUR1.22 When these SUR-overexpressing mice are crossed with animals additionally expressing ATP-insensitive Kir6.2 subunits, SUR2A-overexpressing animals remain essentially normal electrically, but SUR1-overexpressing animals now demonstrate a constellation of arrhythmias, including atrial fibrillation and sudden death.22
The phenomenon of ischemic preconditioning, in which a brief period of ischemia protects the heart from subsequent prolonged ischemia, has been observed in many species. Underlying mechanisms remain elusive, but pharmacological mimicry by KCOs indicates that KATP channel activation during the preconditioning phase may be critical. There has been a consensus that preconditioning results from activation of mitochondrial,39 not sarcolemmal, KATP channels.40–42 A key piece of evidence invoked to argue against a role for sarcolemmal (ie, SUR2A-based) KATP channels is that diazoxide can mimic preconditioning,43 and diazoxide action on SUR2A-based channels is clearly very weak.44 Early reports of an ATP-sensitive K channel in the mitochondria39 led to the notion that a "mitoKATP" might be the location at which diazoxide acts.40,45 Although electrophysiological characterization of the molecular entity purporting to be mitoKATP has been restricted to these few studies, many biochemical analyses of the volume and respiration of isolated mitochondria have confirmed that KATP drugs can have specific effects on mitochondrial membrane transport.46 Ardehali et al47 reported that a complex of at least 5 proteins, including succinate dehydrogenase, and a mitochondrial ABC protein (mABC1) forms a macromolecular complex in the mitochondrial inner membrane with KATP channel activity and overexpression or knockdown by small interfering RNA of this mABC1 protein in myocytes may lead to protection against oxidant stress or loss of cell viability.48 The present results show that SUR1 is in fact a component of the sarcolemmal channel in atrial myocytes. Neither SUR1 nor Kir6.2 has been identified in mitochondria49 and, interestingly, ischemic preconditioning is abolished in the Kir6.2 knockout mouse,50 as is diazoxide-induced preconditioning.51 Although the present data provide no argument against the existence of "mitoKATP," they nevertheless indicate that diazoxide will certainly have effects on atrial sarcolemmal KATP and that caution should be used in interpreting diazoxide action in the intact heart.
Conclusion
In summary, we demonstrate that SUR1 is an essential component of the atrial (but not ventricular) KATP channel in the adult mouse myocardium. Although it remains to be seen whether a similar distribution of SUR1 exists in other animals, including humans, this finding has important implications for cardiac KATP pharmacology, as well as for the potential role of KATP in pathophysiological arrhythmias and the response to ischemia.
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Acknowledgments |
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We are indebted to Ailing Tong for assistance with animal husbandry and to Drs Robert P. Mecham (Department of Cell Biology and Physiology, Washington University School of Medicine) and Show-Ling Shyng (Oregon Health Sciences University, Portland) for help with SUR1 antibody generation.
Sources of Funding
This work was supported by NIH grant HL95010 (to CGN).
Disclosures
None.
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Footnotes |
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Original received April 23, 2008; revision received October 15, 2008; accepted October 22, 2008.
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References |
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