5-Hydroxydecanoate and coenzyme A are inhibitors of native sarcolemmal KATP channels in inside-out patches

Background

5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5?HD + CoA + ATP → 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K_ATP (sarcK_ATP) channel activity.

Methods

Single sarcK_ATP channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK_ATP channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A.

Results

5-HD inhibited (IC₅₀ ∼ 30 μM) K_ATP channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 μM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 μM coenzyme A (CoA) inhibited sarcK_ATP channels. Using giant inside-out patches, which are weakly sensitive to “contaminating” CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK_ATP channel activation by either metabolic inhibition or rilmakalim.

General significance

SarcK_ATP channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at “directly” inhibiting sarcK_ATP and mitoK_ATP channels. However, in intact cells, 5-HD fails to inhibit sarcK_ATP channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.     

Ligand-insensitive State of Cardiac ATP-sensitive K+ Channels : Basis for Channel Opening

The mechanism by which ATP-sensitive K⁺ (K_ATP) channels open in the presence of inhibitory concentrations of ATP remains unknown. Herein, using a four-state kinetic model, we found that the nucleotide diphosphate UDP directed cardiac K_ATP channels to operate within intraburst transitions. These transitions are not targeted by ATP, nor the structurally unrelated sulfonylurea glyburide, which inhibit channel opening by acting on interburst transitions. Therefore, the channel remained insensitive to ATP and glyburide in the presence of UDP. “Rundown” of channel activity decreased the efficacy with which UDP could direct and maintain the channel to operate within intraburst transitions. Under this condition, the channel was sensitive to inhibition by ATP and glyburide despite the presence of UDP. This behavior of the K_ATP channel could be accounted for by an allosteric model of ligand-channel interaction. Thus, the response of cardiac K_ATP channels towards inhibitory ligands is determined by the relative lifetime the channel spends in a ligand-sensitive versus -insensitive state. Interconversion between these two conformational states represents a novel basis for K_ATP channel opening in the presence of inhibitory concentrations of ATP in a cardiac cell.     

Leptin Regulates KATP Channel Trafficking in Pancreatic β-Cells by a Signaling Mechanism Involving AMP-activated Protein Kinase (AMPK) and cAMP-dependent Protein Kinase (PKA)

Pancreatic β-cells secrete insulin in response to metabolic and hormonal signals to maintain glucose homeostasis. Insulin secretion is under the control of ATP-sensitive potassium (K_ATP) channels that play key roles in setting β-cell membrane potential. Leptin, a hormone secreted by adipocytes, inhibits insulin secretion by increasing K_ATP channel conductance in β-cells. We investigated the mechanism by which leptin increases K_ATP channel conductance. We show that leptin causes a transient increase in surface expression of K_ATP channels without affecting channel gating properties. This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. The effect of leptin on K_ATP channels is dependent on the protein kinases AMP-activated protein kinase (AMPK) and PKA. Activation of AMPK or PKA mimics and inhibition of AMPK or PKA abrogates the effect of leptin. Leptin activates AMPK directly by increasing AMPK phosphorylation at threonine 172. Activation of PKA leads to increased channel surface expression even in the presence of AMPK inhibitors, suggesting AMPK lies upstream of PKA in the leptin signaling pathway. Leptin signaling also leads to F-actin depolymerization. Stabilization of F-actin pharmacologically occludes, whereas destabilization of F-actin simulates, the effect of leptin on K_ATP channel trafficking, indicating that leptin-induced actin reorganization underlies enhanced channel trafficking to the plasma membrane. Our study uncovers the signaling and cellular mechanism by which leptin regulates K_ATP channel trafficking to modulate β-cell function and insulin secretion.     

Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density

Pancreatic ATP-sensitive potassium (K_ATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed K_ATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that K_ATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases K_ATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native K_ATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated K_ATP channel trafficking may play a role in the regulation of insulin secretion.     

Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II

Cardiac ATP-sensitive potassium (K_ATP) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac K_ATP channels. We used real-time monitoring of K_ATP channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant K_ATP channel subunits to track the dynamics of K_ATP channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of K_ATP channels. This process required phosphorylation of threonine at 180 and 224 and an intact ³³⁰YSKF³³³ endocytosis motif of the K_ATP channel Kir6.2 pore-forming subunit. A molecular model of the μ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that μ2 docks by interaction with ³³⁰YSKF³³³ and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on μ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac K_ATP channel subunits. This mechanism couples the surface expression of cardiac K_ATP channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance.     

Mitochondria Present in Excised Patches From Pancreatic B-cells May Form Microcompartments With ATP-Dependent Potassium Channels

Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K_ATP) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or -ketoisocaproic acid (10 mM) reduced the open probability of K_ATP channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K_ATP channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K_ATP channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K_ATP channels thus forming functional microcompartments.     

Activation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitive potassium channels via a ROS/calmodulin/CaMKII signaling cascade

Background

Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (K_ATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.

Methods and Findings

Single-channel recordings of cardiac K_ATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type K_ATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H₂O₂ scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H₂O₂ also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of K_ATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.

Conclusion

The present study provides novel evidence that PKG exerts dual regulation of cardiac K_ATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H₂O₂ in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening K_ATP channels and contribute to cardiac protection against ischemia-reperfusion injury.     

Reduction in number of sarcolemmal KATP channels slows cardiac action potential duration shortening under hypoxia

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K_ATP) channels. K_ATP channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K_ATP channel subunit were compared with littermate controls. Evaluation of K_ATP channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80–85% reduction in cardiac K_ATP channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K_ATP channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.     

Carbamazepine inhibits ATP-sensitive potassium channel activity by disrupting channel response to MgADP

In pancreatic β-cells, K_ATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit K_ATP channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1. In addition, sulfonylureas have been shown to function as pharmacological chaperones to correct channel biogenesis and trafficking defects. Recently, we reported that carbamazepine, an anticonvulsant known to inhibit voltage-gated sodium channels, has profound effects on K_ATP channels. Like sulfonylureas, carbamazepine corrects trafficking defects in channels bearing mutations in the first transmembrane domain of SUR1. Moreover, carbamazepine inhibits the activity of K_ATP channels such that rescued mutant channels are unable to open when the intracellular ATP/ADP ratio is lowered by metabolic inhibition. Here, we investigated the mechanism by which carbamazepine inhibits K_ATP channel activity. We show that carbamazepine specifically blocks channel response to MgADP. This gating effect resembles that of sulfonylureas. Our results reveal striking similarities between carbamazepine and sulfonylureas in their effects on K_ATP channel biogenesis and gating and suggest that the 2 classes of drugs may act via a converging mechanism.     

Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism

ATP-sensitive potassium (K_ATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant K_ATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct K_ATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited K_ATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the K_ATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant K_ATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore K_ATP channel expression and function in a subset of congenital hyperinsulinism patients.     

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K_ATP channels directly associate with ankyrin-B in heart via the K_ATP channel α-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K_ATP channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K_ATP channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.     

Sarcolemmal ATP-sensitive potassium channels modulate skeletal muscle function under low-intensity workloads

ATP-sensitive potassium (K_ATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have K_ATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle–specific disruption of K_ATP channel function consume more energy than wild-type littermates. However, how K_ATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted K_ATP channel function. This workload was sufficient to induce K_ATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of K_ATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle K_ATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required.     

Hypothyroidism Decreases the ATP Sensitivity of KATP Channels from Rat Heart

The effects of thyroid status on the properties of ATP-sensitive potassium channels were investigated. Single-channel recordings were made using excised inside-out membrane patches from enzymatically dissociated ventricular myocytes from hearts of control and thyroidectomized rats and each group was studied with and without administration of thyroid hormone. In patches excised from hypothyroid myocytes the IC₅₀ for ATP inhibition of K_ATP channels was 110 μm. This value was 3-fold higher than the IC₅₀ in control myocytes (43 μm). Treatment of hypothyroid rats to restore physiological levels of thyroid hormone (tri-iodothyronine, T₃), resulted in a return to normal ATP-sensitivity (IC₅₀= 46 μm). In patches from animals rendered hyperthyroid, the IC₅₀ for ATP was 50 μm and this value was not significantly different from the control. There was no difference in the cooperativity of ATP-binding (Hill coefficient, n_H) among control (n_H= 2.2), hypothyroid (n_H= 2.1), T₃-treated (n_H= 2.0) and hyperthyroid groups (n_H= 2.4). The unitary conductance was unchanged and there was no apparent change in intraburst kinetics between examples of single K_ATP channels from control and hypothyroid rats. Action potentials recorded in myocytes from hypothyroid rats were significantly shortened by 50 μm levcromakalim, a K_ATP channel opener (P < 0.001) but unchanged in control myocytes. We conclude that hypothyroidism significantly decreased the ATP-sensitivity of K_ATP channels, whereas the induction of hyperthyroid conditions did not alter the ATP-sensitivity of these channels. Thus, hypothyroidism is likely to have important physiological consequences under circumstances in which K_ATP channels are activated, such as during ischemia. Received: 1 July 1997/Revised: 24 December 1997     

Effect of Adenosine and Intracellular GTP on KATP Channels of Mammalian Skeletal Muscle

We investigated the action of adenosine and GTP on K_ATP channels, using inside-out patch clamp recordings from dissociated single fibers of rat flexor digitorum brevis (FDB) skeletal muscle. In excised patches, K_ATP channels could be activated by a combination of an extracellular adenosine agonist and intracellular Mg²⁺-ATP and GTP or GTP-γ-S. The activation required hydrolyzable ATP and could be partially reversed with Mg²⁺, suggesting that it may involve a G-protein dependent phosphorylation of K_ATP channels. We found that K_ATP channels of the rat FDB could not be activated by Mg²⁺-ATP alone or by Mg²⁺-ATP in the presence of extracellular adenosine. Patches whose channel activity had been `rundown' by Ca²⁺ could not be recovered by adenosine, GTP or Mg²⁺-ATP. K_ATP channels activated by adenosine receptor agonists had a similar ATP sensitivity to those under control conditions; but adenosine appears to be able to switch these K_ATP channels from an inactive to an active mode. Received: 29 December 1995/Revised: 22 March 1996     

Exercise responsive genes measured in peripheral blood of women with Chronic Fatigue Syndrome and matched control subjects

Background

Electrophysiological data suggest that cardiac K_ATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other K_ATP channel subunits) is poorly defined. We examined the localization of each of the K_ATP channel subunits in the mouse and rat heart.

Results

Immunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular K_ATP channels.

Conclusions

Collectively, our data demonstrate unique cellular and subcellular K_ATP channel subunit expression patterns in the heart. These results suggest distinct roles for K_ATP channel subunits in diverse cardiac structures.     

Evidence for Direct Physical Association between a K+ Channel (Kir6.2) and an ATP-Binding Cassette Protein (SUR1) Which Affects Cellular Distribution and Kinetic Behavior of an ATP-Sensitive K+ Channel

Structurally unique among ion channels, ATP-sensitive K⁺ (K_ATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K⁺ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2ΔC37). Kir6.2ΔC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2ΔC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 exhibited single-channel activity characteristic of pancreatic K_ATP channels. Kir6.2ΔC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K⁺ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a K_ATP channel.     

Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

K_ATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P₂ and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing K_ATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles.This article is part of the themed issue ‘Evolution brings Ca²⁺ and ATP together to control life and death’.     

Carbamazepine inhibits ATP-sensitive potassium channel activity by disrupting channel response to MgADP

In pancreatic β-cells, K_ATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit K_ATP channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1. In addition, sulfonylureas have been shown to function as pharmacological chaperones to correct channel biogenesis and trafficking defects. Recently, we reported that carbamazepine, an anticonvulsant known to inhibit voltage-gated sodium channels, has profound effects on K_ATP channels. Like sulfonylureas, carbamazepine corrects trafficking defects in channels bearing mutations in the first transmembrane domain of SUR1. Moreover, carbamazepine inhibits the activity of K_ATP channels such that rescued mutant channels are unable to open when the intracellular ATP/ADP ratio is lowered by metabolic inhibition. Here, we investigated the mechanism by which carbamazepine inhibits K_ATP channel activity. We show that carbamazepine specifically blocks channel response to MgADP. This gating effect resembles that of sulfonylureas. Our results reveal striking similarities between carbamazepine and sulfonylureas in their effects on K_ATP channel biogenesis and gating and suggest that the 2 classes of drugs may act via a converging mechanism.