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The mitochondrial ATP sensitive potassium channel (mKATP) is implicated in cardioprotection by ischemic preconditioning (IPC), but the molecular identity of the channel remains controversial. The validity of current methods to assay mKATP activity is disputed.
We sought to develop novel methods to assay mKATP activity and its regulation.
Using a thallium (Tl+) sensitive fluorophore, we developed a novel Tl+ flux based assay for mKATP activity, and used this assay probe several aspects of mKATP function. The following key observations were made: (i) Time-dependent run-down of mKATP activity was reversed by phosphatidylinositol-4,5-bisphosphate (PIP2). (ii) Dose responses of mKATP to nucleotides revealed a UDP EC50 of ~20 μmol/L and an ATP IC50 of ~5 μmol/L. (iii) The antidepressant fluoxetine (Prozac™) inhibited mKATP (IC50 2.4 μmol/L). Fluoxetine also blocked cardioprotection triggered by IPC, but did not block protection triggered by a mKATP independent stimulus. The related antidepressant zimelidine was without effect on either mKATP or IPC.
The Tl+ flux mKATP assay was validated by correlation with a classical mKATP channel osmotic swelling assay (R2 0.855). The pharmacologic profile of mKATP (response to ATP, UDP, PIP2, and fluoxetine) is consistent with that of an inward rectifying K+ channel (KIR) and is somewhat closer to that of the KIR6.2 than the KIR6.1 isoform. The effect of fluoxetine on mKATP-dependent cardioprotection has implications for the growing use of antidepressants in patients who may benefit from preconditioning.
The mitochondrial ATP sensitive potassium channel (mKATP) is thought to be essential for cardioprotection recruited by ischemic preconditioning (IPC),1, 2 but despite intense research the molecular identity of this channel remains unclear. The simplest thesis is that mKATP channels are derivative of surface KATP channels, and thus composed of inward rectifying K+ channels (KIR) and sulfonylurea receptors (SUR). The cardiomyocyte surface KATP channel is comprised of KIR6.2 and SUR2A isoforms,3 but efforts to conclusively assign these proteins to the cardiac mKATP have been unsuccessful to date.
Neither Kir6 nor SUR genes contain mitochondrial target sequences, and Kir6/SUR proteins are not found in mitochondrial proteome databases or prediction engines.4, 5 Furthermore, immune-based methods to detect KIR/SUR subunits in mitochondria are plagued by issues of antibody specificity6 and mitochondrial purity/contamination. Several of the key pharmacologic reagents used to study mKATP channels (e.g. the agonist diazoxide (DZX) and antagonist 5-hydroxydecanoate (5-HD)) are also known to exhibit off-target effects.7, 8 Targeted gene deletion in mice to identify the mKATP channel involved in IPC has proven futile, due to the confounding cardiovascular effects of knocking out KIR6 and SUR genes (Kcnj8, Kcnj11, Abcc8, and Abcc9) on surface KATP channel function. In general, KIR and SUR knockouts exhibit profound defects in glucose/insulin handling,9–12 which impacts the response to IPC.13
A recent study using custom-made antibodies and SUR knockout mice identified shortform splice variants of SUR2 in mitochondria.14 Furthermore, recent pharmacological evidence suggests that complex II of the respiratory chain (succinate dehydrogenase) may be a regulatory component of the mKATP channel.15, 16 However, both these findings leave the identity of the K+ channel-forming subunit of mKATP unknown. In this regard, mKATP is similar to other mitochondrial ion channels which exist at a phenomenological level but have not been molecularly identified (e.g., the mitochondrial Ca2+ uniporter).
A major obstacle in studying the mKATP channel has been the availability of a reliable assay. Most studies to date have utilized an isolated mitochondrial rapid swelling assay, in which K+ uptake into mitochondria is followed by osmotically-obliged water, leading to mild swelling that is assayed as light scattering in a spectrophotometer.17, 18 This assay has been criticized as irreproducible by some laboratories,19 with the precise timing of mitochondrial isolation appearing to be a critical factor.20
Studying the literature on surface KATP channels, two key biochemical properties that appeared to have been overlooked in the mKATP channel field were the permeability of surface KATP channels for the heavy metal thallium (Tl+),21 and the modulation of channel run-down by phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP2).22, 23 Herein, we developed a novel Tl+ fluorescence based assay for mKATP channel activity, and used this assay to show that the channel is subject to run-down that is reversed by PIP2. It is anticipated that both these discoveries will advance the study of this channel. Furthermore, the antidepressant fluoxetine (FLX), which is known to modulate KIR channels,24, 25 was found herein to block the mKATP channel and to block IPC, but FLX did not block mKATP-independent cardioprotection. The implications of these data for clinical use of FLX in cardiovascular disease patients are discussed.
Full experimental details are in the online supplement. Cardiac mitochondria were rapidly isolated from male Sprague-Dawley rat hearts by differential centrifugation in sucrose-based buffer as previously described.20 Protein was determined by the Folin-phenol method.26 Within 1.5 hr of mitochondrial isolation the activity of mKATP was monitored by the osmotic swelling assay as previously described.20
A novel fluorescence-based Tl+ flux assay for mKATP activity was also developed. The ionic radii of Tl+ (0.154 nm) and K+ (0.144 nm) are similar,27 and thus Tl+ is widely used as an analog to study membrane K+ transport.21, 27–30 The assay made use of the fluorescent indicator BTC-AM, which is better known as a ratiometric Ca2+ sensor, but is also sensitive to Tl+ with a distinct spectral response preventing signal overlap between these sensitivities. Mitochondria were loaded with BTC-AM during the isolation procedure and stored on ice until use. In the assay, 0.3 mg BTC-AM loaded mitochondria were added to a rapidly stirred cuvet containing 2 ml of chloride-free Tl+ assay buffer at 37°C. Tested compounds were present from the beginning of the assay, and baseline fluorescence was recorded for 10 s. prior to addition of TlSO4 (2 mmol/L final) via a syringe port. Fluorescence was monitored in a Varian Cary Eclipse spectrofluorometer (λex=488nm, λem525nm) and normalized to baseline. Full details including the concentrations and preparation methods for all reagents used in the assay, are in the online supplement.
Isolated rat heart perfusions (Langendorff) were performed as previously described.16 Following 20 min. equilibration, hearts were divided into 7 groups: (i) IR alone, comprising 20 min. vehicle (water or DMSO) infusion, 30 s. wash-out, 25 min. global ischemia, 120 min. reperfusion; (ii) FLX + IR, comprising 20 min. FLX infusion (5 μmol/L), 30 s. wash-out, then IR; (iii) IPC + IR, comprising 3 × 5 min. ischemia interspersed with 5 min. reperfusion, then IR; (iv) FLX + IPC + IR, comprising 5 min. FLX infusion (5 μmol/L), plus FLX infused throughout the 3 reperfusion phases of IPC (i.e. 20 min. total FLX delivery), 30 s. wash-out, then IR; (v) Zimelidine + IPC + IR. As above, replacing FLX with zimelidine (5 μmol/L). (vi) FCCP + IR, comprising 20 min. FCCP infusion (30 nmol/L 31, 32), then IR; vii) FCCP + FLX + IR, comprising 20 min. infusion of both FCCP (30 nmol/L) and FLX (5 μmol/L), then IR. Following reperfusion hearts were stained with tetrazolium chloride (TTC), imaged, and infarct size measured as previously described.16
In all experiments, each “N” was an independent heart perfusion or mitochondrial isolation from a single animal on one day. Statistical differences between groups were determined using ANOVA, with significance defined as p<0.05.
In seeking to develop an assay for mKATP channel activity that does not measure secondary effects such as water uptake (as is the case for the osmotic swelling assay), we discerned that the heavy metal thallium (Tl+) is widely used as a surrogate substrate to study K+ channel function.21, 27–30 A fluorescent probe that responds to [Tl+] is commercially available (FluxOR™, Invitrogen, Carlsbad CA), but careful analysis of the literature underlying this reagent revealed that the active component was BTC-AM, a more economical reagent.28–30 Thus, isolated mitochondria were loaded with BTC-AM as the basis for a Tl+ uptake assay of K+ channel activity.
Figure 1A shows the addition of Tl+ to BTC-AM loaded mitochondria resulted in increased fluorescence due to rapid Tl+ influx and the establishment of a new steady-state. The fluorescence increase was largely inhibited by ATP, consistent with Tl+ transport by a KATP channel. Furthermore, the effect of ATP could be overridden by the mKATP channel opener AA5, and this effect was in-turn blocked by the mKATP antagonist 5-HD. These data are quantified in Figure 1B, which also shows the effects of mKATP reagents DZX (agonist) and glyburide (antagonist). The ionophores valinomycin and nonactin, both of which transport Tl+,33 resulted in maximal Tl+ flux into mitochondria, and the mitochondrial uncoupler FCCP inhibited Tl+ uptake indicating a requirement for membrane potential. It was hypothesized that the steady state is likely due to balancing of Tl+ influx by its efflux through the K+/H+ exchanger (KHE). However, attempts to modulate KHE activity with the inhibitors DCCD and quinine were inconclusive (data not shown). Validation of the Tl+ assay for mKATP channel activity was also performed by a direct comparison with results from mKATP osmotic swelling assays run in parallel under a variety of open/closed conditions. Figure 1C shows that the 2 assays correlated well (r2 = 0.855).
A general property of KIR channels is their tendency to “run-down” over time, a phenomenon attributed to loss of the phospholipid PIP2 from a binding site on the channel.34 The mKATP channel (which is constitutively open in isolated mitochondria) also loses activity following mitochondrial isolation, which may underlie the reported poor reproducibility of mKATP channel activity measurements.19 Upon investigating the relationship between these phenomena, it was found that incubation of mitochondria on ice for 5 hrs. resulted in complete loss of mKATP channel activity, and that PIP2 addition restored channel activity (Figure 2). Furthermore, the full pharmacologic profile of mKATP channel activity (i.e. inhibition by ATP, activation by AA5, and re-inhibition by 5HD) was recovered in PIP2-treated aged mitochondria. The same concentrations of the PIP2 breakdown products inositol triphosphate (IP3), 1,2-dioctanoyl glycerol (DOG), or 1,2-dipalmitoyl glycerol (DPG) did not affect mKATP activity. The polyvalent cation neomycin, which is known to inhibit KIR channel activity by sequestering PIP2,35 was able to reverse the mKATP channel-restorative effects of PIP2. Identical results were obtained with the osmotic swelling mKATP channel assay (Figure S1). Overall these data suggest that the mKATP channel contains a PIP2 sensitive subunit, possibly a KIR channel. Consistent with this, both the Tl+ and swelling assays revealed that mKATP sensitivity to the nucleotides UDP and ATP (Figure S2) was closer to that of the KIR6.2 than the KIR6.1 isoform.36–39
Several classes of KIR channel are known to be inhibited by fluoxetine (Prozac™), an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class.24, 25 As shown in Figure S3, KIR6 channels (components of KATP channels) are an order of magnitude more sensitive to FLX than KIR4 channels, a well-known FLX target.40 Thus, we investigated the possibility that FLX might block mKATP channels. As shown in Figure 3, FLX blocked mKATP channel activity with an IC50 of 2.3 μmol/L, while a related SSRI zimelidine (ZM) did not. Identical results were obtained with the osmotic swelling mKATP assay (Figure S3). Furthermore, FLX blocked AA5- or DZX-mediated opening of mKATP (Figure S3).
Given the importance of the mKATP channel in IPC, we hypothesized that FLX may block IPC. Figure 3 shows that 5 μmol/L FLX completely blocked IPC-mediated cardioprotection in a rat perfused heart model of IR injury, while ZM was without effect. Notably FLX did not enhance baseline IR injury in this model, indicating that blockage of IPC was not due to an equal-but-opposite injurious effect, canceling out cardioprotection. Furthermore, FLX had no effect on cardioprotection mediated by FCCP (Figure S4), which occurs independent of the mKATP channel.31, 32
The major findings of this study are as follows: (i) Development of a novel Tl+ flux based assay for the mKATP channel; (ii) Time-dependent loss of mKATP channel activity is a genuine run-down phenomenon and is reversed by PIP2; (iii) FLX blocks both mKATP channel activity and IPC-mediated cardioprotection. This is the first demonstration of the modulation of a mitochondrial ion channel by PIP2, and the first identification of a mitochondrial ion channel target for FLX. Collectively, the data support the concept that mKATP contains a bona fide KIR channel. The effects of FLX on IPC may elucidate some of the reported negative impact of SSRI use on the outcome of cardiac surgery in humans.41
Work on the mKATP channel to date has relied on a variety of assays, many of which measure downstream effects of mitochondrial K+ uptake such as changes in respiration,42 matrix alkalinization,42 flavoprotein fluorescence,43 and swelling induced light scatter.42 Such methods are limited by the ability of other mitochondrial phenomena (e.g. electron transport chain activity, volume changes, membrane potential) to interfere with the measured parameters. Direct measurement of mitochondrial K+ fluxes using the potassium-binding fluorescent indicator (PBFI) is difficult because its Kd for K+ of ~8 mmol/L42 would result in saturation at typical intramitochondrial K+ levels (~150 mmol/L).44 Thus herein we chose to exploit another property of K+ channels, their ability to transport Tl+ as a surrogate for K+.21, 27 While Tl+ acetate has previously been used in swelling-based studies on mKATP45, this study is the first application of a Tl+ sensitive probe, BTC-AM,28–30 to study mKATP.
The kinetics of the Tl+ based mKATP channel assay are superior to those of the swelling based assay.16 Following Tl+ addition maximal fluorescence is attained within 2–4 s., compared to a time-lag of 20–30 s. for maximal signal intensity in the osmotic swelling assay. Unfortunately the high flux rate of Tl+ through K+ channels (~2x K+ flux 21), coupled with the relatively slow mixing time in the fluorescence cuvet, does not permit precise channel kinetics to be determined in this apparatus. Current best estimates for mKATP channel conductivity range from 10 to 300 pS.15, 46, 47
Another barrier to investigating the mKATP channel has been the rapid loss of channel activity over time in isolated mitochondrial preparations.20 Previous work showed that the purified mKATP channel runs-down in an electrophysiology setting and can be re-activated by very high concentrations of UDP.48 However, the cause of channel activity loss in intact mitochondria was unknown, and could easily be due to proteolytic degradation. The finding herein that time-dependent mKATP channel inactivation in intact mitochondria can be reversed by PIP2 indicates this is a genuine run-down phenomenon, which is a common property of KIR channels.49
KATP channels were the first channels identified to depend on phosphoinositides such as PIP2,22, 23 and this is the first study to identify a mitochondrial ion channel that responds to PIP2. Such regulation of mKATP channel activity by PIP2 may have implications for the function of this channel in IPC. PIP2 has been found in mitochondrial membranes, 50 but its endogenous source in mitochondria is unknown. Notably, the run-down of a planar lipid bilayer reconstituted mKATP was reversed by ATP/Mg2+,51 suggesting the mitochondrial high energy phosphate pool may be important in maintaining membrane lipid phosphorylation status. However, this phenomenon required very specific experimental conditions (i.e. addition and removal of ATP/Mg2+ to and from different sides of the membrane in a particular order), and attempts to reproduce this in isolated mitochondria were unsuccessful (data not shown). The role of lipid kinases (e.g., PI3K), phospholipases (e.g., PLC), and other components of the IP3/DAG signaling pathway in regulating mKATP is also unknown, but the involvement of such signaling components in IPC52, 53 suggests a potential novel pharmacological target (i.e., mitochondrial PIP2 turnover) to modulate preconditioning.
The Tl+ assay was also used to probe the response of mKATP to nucleotides (Figure S2). The mKATP EC50 for UDP (~20 μmol/L) was closer to that of KIR6.2 (~200 μmol/L) than KIR6.1 (~4 mmol/L), and the mKATP IC50 for ATP (~4.5 μmol/L) was also closer to that of KIR6.2 (~15 μmol/L) than KIR 6.1 (~350 μmol/L). While these data agree with previous studies on mKATP,48 a variety of labeling, electrophysiological and genetic studies across multiple species and tissues have suggested the presence of either KIR6.1, KIR6.2, both, or neither in mitochondria. 6, 10, 54–62 An overall consensus is that mKATP likely contains a KIR channel, but the definitive assignment of a particular KIR isoform is not yet possible.
The discovery that mKATP activity is blocked by FLX is also consistent with the consensus that mKATP contains a KIR. Fluoxetine has previously been shown to inhibit KIR channels, while related SSRIs (e.g. zimelidine) had no effect.24, 25 Our data (Figure S3) suggest that KIR6 channels may be the most sensitive to FLX of all KIR isoforms,24, 25, 40, 63 and in agreement with this the mKATP exhibits a strikingly low FLX IC50 of 2.4 μmol/L (Figure 3). Physiological concentrations of FLX are in the range of 1 – 20 μmol/L.63 The fact that FLX is a lipophilic cation (LogP 4.8),64 coupled with the highly membranous nature of mitochondria, may serve to concentrate FLX in the organelle. In a mitochondria-rich tissue such as myocardium, the mitochondrion may be a primary target for FLX.
The discovery that FLX can block IPC-mediated cardioprotection is both consistent with its effect on mKATP activity, and consistent with a critical role for mKATP in IPC signaling.1, 16, 65 The lack of effect of another SSRI, zimelidine, on either IPC or mKATP activity suggests that this effect is not mediated via the SSRI mode of action. The observation that FLX also blocks mKATP channel opening by the highly specific agonist AA5 also suggests a direct mKATP effect. Furthermore, the lack of effect of FLX on FCCP-mediated cardioprotection, which is completely independent of mKATP channels,31, 32 suggests that the protection-blocking effect of FLX is specific to mKATP channel-mediated protection, and does not extend to all modes of protection. The current lack of a molecular identity for the mKATP does not permit decisive knock-out experiments to verify whether the effects of FLX observed in the intact heart are mediated by mKATP.
In the US, antidepressants are the most commonly prescribed class of medication,66 with FLX alone prescribed >23 million times in 2008.67 Although SSRIs are known to negatively impact the outcome of cardiac surgery,41 they are widely prescribed to patients with acute coronary syndrome.68 Notably, while IPC elicits solid protection in animal models of IR injury, its application in humans is limited by confounding effects such as age,69 gender,70 diabetes,13 and other medications.71 To this list of medications FLX must now be added, with the implication that successful cardioprotection in humans may require FLX withdrawal. Furthermore, the mood enhancer lithium is also known to both increase cardiac PIP2 lelvels72 and to induce cardioprotection,73 suggesting that some of the protective effects of lithium previously attributed to GSK-3β inhibition73 may be mediated via the mKATP channel.
In summary, we have developed herein a novel assay for the mKATP channel, and used this assay to reveal novel sensitivities of the channel to phosphoinositides and antidepressants. It is anticipated that this assay may find widespread use in the mKATP field, leading ultimately to the identification of this important channel.
What is known?
What new information does this article contribute?
Summary of Novelty and Significance
Cardiac ischemia/reperfusion (IR) injury is an important worldwide morbidity factor. Strategies to protect the heart from IR injury (such as during heart attack) are limited, but one promising avenue is ischemic preconditioning (IPC). The mitochondrial ATPsensitive K+ channel (mKATP) has been suggested to mediate the protection afforded by IPC; however, the molecular identity of this channel is unknown, and its assay is also technically challenging, thus hindering drug-development efforts. Using a Tl+-sensitive fluorophore, a novel assay was developed herein to measure mKATP activity. Using this assay, we show that loss of mKATP channel activity over time is reversed by the lipid PIP2. These findings should greatly facilitate mKATP research, hopefully leading to a molecular identity. Furthermore, this is the first report of a PIP2 sensitive phenomenon in mitochondria; it may possibly relate to the mechanism of channel regulation in IPC itself. Finally, we found that the antidepressant fluoxetine (Prozac™) inhibited mKATP and also blocked the protective effects of IPC. Given the widespread use of fluoxetine in cardiac patients, this may have important implications for the potential application ofIPC in humans.
We thank Teresa A. Sherman (URMC) for technical assistance.
SOURCES OF FUNDING This work was funded by a pre-doctoral fellowship award to APW from the American Heart Association, Founders Affiliate (0815770D), and by grants from the US National Institutes of Health to PSB (RO1-HL071158), and PSB & KWN (RO1-GM-087483).
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