Differential structure of atrial and ventricular K_ATP: atrial K_ATP channels require SUR1

Quantitative RT-PCR

Expression of SUR1 and SUR2A mRNA was examined using real time RT-PCR²³. Total RNA was isolated from cardiac atrial or ventricular tissue using TRIzol (Invitrogen) following manufacturer’s protocols. 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.). 75 ng of template RNA were used in all reactions. Following the RT reaction (45 min, 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 (C_t) was determined for each reaction. To control for variability in RNA quantity, the normalized value, ΔC_t, for each sample was calculated using the formula ΔC_t = C_t(SUR) – C_t(actin). Relative mRNA expression is reported as 2^−ΔCt * 1000.

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) following manufacturer protocols.

Protein isolation and analysis

Recombinant channel protein expressed in COSm6 cells was analyzed as described¹⁵. 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 mM sucrose, 10 mM 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 mM NaCl, 20 mM HEPES, 5 mM EDTA, 1% NP-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% PAGE and transferred to PVDF 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-X100. Following washing (3× 5 min), cells were incubated with anti-SUR1 antibody (1:300) and goat serum (1:1000) at room temperature. After washing (3× 5 min) 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 (mM: NaCl, 137; KCl, 5.4; NaH₂PO₄, 0.16; glucose, 10; MgCl₂, 0.5; HEPES, 5.0; NaHCO₃, 3.0; pH 7.3–7.4) with additions as described. Intracellular (pipette) solution contained (mM): KCl, 20; K-aspartate, 130; MgCl₂, 1; HEPES, 10; EGTA, 10; pH 7.3–7.4). Patch clamp electrodes (1–3 MΩ, 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 5–10 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 (V_hold = −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 MΩ) to minimize measurement errors. However, given the size of the K_ATP 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 (K_int) used in excised patch experiments had the following composition: 140 mM KCl, 1 mM EGTA, 10 mM HEPES, pH 7.3. Diazoxide and Pinacidil were stored as 100 mM stocks in DMSO. ATP and ADP (potassium salts) were stored as 100 mM stocks in K_int solution, with the pH of each stock solution adjusted to 7.3. All stock solutions were stored at −20 ºC, 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 v.9 software (Axon Inc.). Mean data are presented throughout the text as stimulated I_rel (current amplitude, normalized to the maximum K_ATP current per patch).

Data analysis

Data were analyzed using ClampFit and Microsoft Excel software. Results are presented as mean±SEM (standard error of the mean). Statistical tests and p-values (ANOVA, Tukey post-hoc test, where appropriate) are denoted in figure legends.

Regional SUR transcription in the mouse heart

Although its is widely accepted that Kir6.2 and SUR2A subunits generate the cardiac sarcolemmal K_ATP 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 K_ATP channels, in atria and ventricles of wild type (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 (Fig. 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 (Fig. 1B). Firstly, SUR2A transcript levels are considerably (∼10x) higher in the ventricle than atrium in WT animals, as also recently reported by Marrionneau et al²⁸. Secondly, SUR2A transcription and atrial/ventricular ratio are unaffected either by SUR1 knockout, or by SUR1 overexpression.

SUR1 protein expression and distribution in the mouse heart

Several studies have reported SUR1 protein in the heart^16;17 but none have utilized the critical SUR knockout tissue control. There are concerns regarding the specificity of anti-K_ATP subunit antibodies²⁹, and our experience with commercial anti-SUR1 antibodies suggested that non-specificity 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 (Fig. 2A). In addition, the antibody specifically recognizes SUR1 and no cross-reactivity is observed in samples from cells transfected with SUR2A or SUR2B (Fig. 2B).

Having confirmed specificity, proteins isolated from wild type and SUR1^−/− hearts were probed for SUR1. Significant core glycosylated (∼150 kDa³⁰) SUR1 is present in wild type, but not SUR1^−/−, hearts (Fig. 2C). In addition, using high sensitivity detection, higher order glycosylated proteins as well as a specific shorter protein ∼100 kDa are detected only in wild type hearts, and these same bands are all enhanced in hearts overexpressing SUR1 (SUR1-TG, Fig. 2C)²³. None of these bands are present in knockout hearts, with the exception of a very faint band at ∼150 kDa using high sensitivity detection, and a single major non-specific 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 (Fig. 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 (Fig. 3A). In contrast, a strong membrane-localized SUR1 signal was detected in isolated wild type atrial myocytes (Fig. 3A) and SUR1 immunofluorescence was prominent in atrial tissue sections (Fig. 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 (Online Fig. I).

SUR1 is not an essential component of ventricular K_ATP 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 K_ATP channels. To examine the role of SUR1 in functional expression of sarcolemmal channels, we measured total K_ATP current activated by complete metabolic inhibition (MI: 2.5 µg/ml oligomycin, plus substitution of glucose with 10 mM 2-deoxyglucose) in isolated myocytes. MI induced substantial K_ATP conductance in both WT and SUR1^−/− ventricular myocytes (3.14±0.59 vs. 3.00±0.47 nS/pF, n=4 (WT) and 6 (SUR1^−/−), respectively, Fig. 4). We additionally assessed ventricular K_ATP channel activity in isolated inside-out patch clamp experiments. Again, K_ATP current density was not significantly altered in SUR1^−/− membranes (254.4±79.8 vs. 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 (Fig. 4B). Ventricular K_ATP from WT and SUR1−/− myocytes revealed no differences in pinacidil- or MgADP-sensitivity (Online Fig. II). Taken together, the results demonstrate that SUR1 is not necessary for the functional expression of the ventricular K_ATP channel. While they do not discount the possibility that SUR1 may be a non-essential component, they are consistent with the prevailing notion that the coassembly of SUR2A and Kir6.2 subunits generates the ventricular sarcolemmal K_ATP channel.

SUR1 is an essential component of the atrial K_ATP channel

In whole-cell conditions, with zero or 500 µM MgATP in the pipette, we frequently observed spontaneous activation of wild type atrial K_ATP within 1 minute, even without application of metabolic inhibition (MI) or KCOs. Under similar conditions, no K_ATP activation was observed in SUR1^−/− atrial myocytes. Diazoxide activated substantial K_ATP (48.3 ± 20.5 nS, n = 8) in WT atrial myocytes perfused, but no K_ATP in SUR1^−/− myocytes (2.6 ± 12.2 nS, n = 5). The absence of sarcolemmal K_ATP in SUR1^−/− atrial myocytes was confirmed in excised inside-out patches: large ATP-sensitive current was observed (Fig. 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 K_ATP channel in mouse atrial sarcolemma.

Pharmacological profile analysis of atrial and ventricular K_ATP

The SUR subunit of the K_ATP channel is the binding site for inhibitory sulfonylureas and KCO drugs, and different SUR isoforms confer different pharamacological properties on the channel¹. Specifically, recombinant SUR1/Kir6.2 channels exhibit prominent sensitivity to the KCO diazoxide, while 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 K_ATP complex can be inferred from its pharmacological “fingerprint”, and the disparate function of SUR1 in atrial and ventricular K_ATP (i.e. 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 to the action on channels of defined composition, expressed heterologously in COSm6 cells (Fig. 6). As expected, recombinant SUR1/Kir6.2 channels are more sensitive to diazoxide than recombinant SUR2A/Kir6.2 channels (Fig. 6A). Consistent with a significant functional role for SUR1, K_ATP channels in atrial myocytes were also strongly activated by diazoxide, but were insensitive to pinacidil. On the other hand, ventricular K_ATP 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 K_ATP density (Fig. 4), or functional properties (Online Fig. II), the results are most consistent with the only significant SUR contributors to mouse atrial and ventricular K_ATP channels being SUR1 and SUR2A, respectively.

Atrial and ventricular K_ATP channels are structurally distinct

It is currently widely accepted that cardiac sarcolemmal K_ATP channels are formed by the co-assembly of SUR2A and Kir6.2. K_ATP 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 wild type atrium, and that atrial sarcolemmal K_ATP requires SUR1 for functional channel expression. As a result of this regionally distinct channel structure, sarcolemmal K_ATP channels in mouse heart have very different pharmacological fingerprints. Like recombinant SUR1/Kir6.2 channels, atrial K_ATP is more sensitive to diazoxide than pinacidil, while ventricular K_ATP 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 K_ATP channel structure varies in different heart regions is not entirely novel and several studies have demonstrated regional pharmacology in other species. K_ATP channels in cultured neonatal rat atrial and ventricular myocytes vary in their response to KCOs and metabolic inhibitors^19;20 and antisense oligonucleotides directed against SUR1 have been shown to inhibit K_ATP current in cultured neonatal ventricular myocytes¹⁸. In the present study we have limited analysis to mouse tissue, as 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. While 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, K_ATP channels will shorten the action potential and reduce the effective refractory period, a significant risk factor for the development of re-entrant arrhythmias like atrial fibrillation (AF)³⁷. To date, there have been very few studies addressing this possibility in man, although a mutation in SUR2A has been associated with vein of Marshall adrenergic atrial fibrillation³⁸. The disparate structure of the atrial and ventricular K_ATP may have important implications for the maintenance of AF. Because channels composed of SUR1 and Kir6.2 turn on more readily¹⁵ (we see spontaneous activation of atrial but not ventricular K_ATP in whole-cell mode), it seems likely that the activation of SUR1-based atrial K_ATP during rapid pacing conditions of AF 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 atrio-ventricular conduction only in mice that overexpress SUR1²². When these SUR-overexpressing mice are crossed with animals additionally expressing ATP-insensitive Kir6.2 subunits, SUR2A overexpressers remain essentially normal electrically, but SUR1-overexpressing animals now demonstrate a constellation of arrhythmias, including AF, and sudden death²².

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 K_ATP channel activation during the preconditioning phase may be critical. There has been a consensus that preconditioning results from activation of mitochondrial³⁹, not sarcolemmal, K_ATP channels^40–42. A key piece of evidence invoked to argue against a role for sarcolemmal (i.e, SUR2A–based) K_ATP channels is that diazoxide can mimic preconditioning⁴³, and diazoxide action on SUR2A–based channels is clearly very weak⁴⁴. Early reports of an ATP-sensitive K channel in the mitochondria³⁹ led to the notion that a ‘mitoK_ATP’ might be the location at which diazoxide acts^40;45. While electrophysiological characterization of the molecular entity purporting to be mitoK_ATP has been restricted to these few studies, many biochemical analyses of the volume and respiration of isolated mitochondria have confirmed that K_ATP drugs can have specific effects on mitochondrial membrane transport⁴⁶. Ardehali et al.⁴⁷ reported that a complex of at least five proteins, including succinate dehydrogenase, and a mitochondrial ABC protein (mABC1) forms a macromolecular complex in the mitochondrial inner membrane with K_ATP channel activity and overexpression or knockdown by siRNA of this mABC1 protein in myocytes may lead to protection against oxidant stress or loss of cell viability⁴⁸. The present results show that SUR1 is in fact a component of the sarcolemmal channel in atrial myocytes. Neither SUR1 nor Kir6.2 have been identified in mitochondria⁴⁹ and, interestingly, ischemic preconditioning is abolished in the Kir6.2 knockout mouse⁵⁰, as is diazoxide induced preconditioning⁵¹. While the present data provide no argument against the existence of ‘mitoK_ATP’, they nevertheless indicate that diazoxide will certainly have effects on atrial K_ATP, 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) K_ATP channel in the adult mouse myocardium. While it remains to be seen whether a similar distribution of SUR1 exists in other animals, including humans, this finding has important implications for the cardiac K_ATP pharmacology, as well as the potential role of K_ATP in pathophysiological arrhythmias and the response to ischemia.