Original articleAMP-activated protein kinase connects cellular energy metabolism to KATP channel function☆
Graphical abstract
Highlights
► During metabolic impairment, a change in the ATP:ADP ratio stimulates KATP channels opening. ► The ATP:AMP ratio is an acquisitively sensitive indicator of alterations in the metabolic status. ► We show that AMP-activated protein kinase (AMPK) activity promotes KATP channel opening. ► AMPK physically interacts with KATP channel subunits, suggestive of local signaling. ► Thus, small changes in AMP may trigger KATP channel availability under ischemic conditions.
Introduction
ATP-sensitive K+ (KATP) channels are found in most tissue beds, including the heart, skeletal and smooth muscle, brain, kidney and pancreatic β-cells [1]. Although the various subtypes of KATP channels differ from each other in terms of their gating properties and pharmacological sensitivities, a common property is their regulation by intracellular nucleotides. The cellular ATP:ADP ratio is considered to be the prime regulator of KATP channels in cardiac muscle and the pancreatic β-cell [2]. As such, KATP channels function to couple intracellular metabolic events to membrane excitability and cellular effector responses. The cloning of the molecular subunits of KATP channels have revealed them to be hetero-octameric complexes, consisting of four pore-forming inward rectifier subunits (Kir6.1 or Kir6.2) and four regulatory subunits (SUR1 or SUR2) [2]. Since the discovery of KATP channels over two decades ago, much work has gone into the delineation of mechanisms that regulate their activity, including their modulation by nucleotides, cellular metabolic events, pharmacological agents and regulation by signaling pathways. Despite these advances, the triggers for KATP channel opening during myocardial ischemia and metabolic impairment are not fully understood.
AMP-activated protein kinase (AMPK) represents the mammalian form of the core component of a kinase cascade that is conserved between fungi, plants, and animals [3]. When activated, AMPK switches off ATP consuming pathways (e.g. biosynthetic pathways) while switching on a variety of pathways to enhance ATP production and cell survival. The latter includes increased β-oxidation of free fatty acids, increased formation of creatine phosphate, and enhanced membrane glucose transport by membrane translocation of GLUT-4 [3]. AMPK is a heterotrimeric protein composed of a catalytic α-subunit and regulatory β and γ subunits, which are important for protein stability and substrate specificity. Each subunit has two or more different isoforms [3]. The α1 subunit is widely expressed, while the α2 subunit is predominantly found in liver, heart and skeletal muscle [4]. AMPK is activated through Thr172 phosphorylation by one or more upstream kinases (AMPKK) and allosterically by increases in the AMP:ATP and creatine:phosphocreatine ratios [3], [5]. AMPK has previously been proposed to regulate KATP channels trafficking in a cellular model of hypoxia-induced “ischemic preconditioning” [6], but there are no reports that AMPK directly affects cardiac KATP channel function. Our data demonstrate that rat ventricular KATP channel activity is regulated by AMPK, and that this kinase therefore directly connects alterations in cellular energy metabolism (i.e. the ATP:AMP ratio) with the cardiac KATP channel function. Furthermore, the AMPK α-subunit associates with KATP channel subunits, suggesting that AMPK may be a local signaling component of the KATP channel macromolecular complex.
Section snippets
Preparation of single ventricular myocytes
Ventricular myocytes were enzymatically isolated from male Sprague–Dawley rats (~ 200 g). Hearts were rapidly excised after pentobarbital overdose (60 mg/kg), rinsed with ice-cold Tyrode's solution (in mM: NaCl 137, KCl 5.4, HEPES 10, MgCl2 1, NaH2PO4 0.33, CaCl2 1.8; glucose 10; pH 7.4), cannulated and retrogradedly perfused with oxygenated Tyrode's solution for 3–5 min at 37 °C. The perfusate was switched to nominally Ca2+-free Tyrode's buffer for 5 min, followed by perfusion for 11–13 min with the
Results
We recently characterized the cardiac KATP channel macromolecular complex using proteomic approaches and found the enzymes of the glycolytic pathway to be important associated proteins [8]. These mass spectrometry experiments also identified 5′-AMP-activated protein kinase (AMPK) catalytic α subunit in an immunoprecipitate obtained with an anti-Kir6.2 antibody (data not shown). Experiments were therefore performed to investigate the functional relevance of this observation.
Physiological actions of AMPK and its effect on KATP channels
Biochemically, AMPK has been identified over 2 decades ago as an ultra-sensitive sensor of cellular stress. Even small rises in cellular AMP levels activate AMPK, which results in ATP consumption pathways to be inhibited and ATP synthesis pathways to be activated [3]. The activity of AMPK is increased during muscle contraction, hypoxia, ischemia, heat shock, a decrease in pH, inhibition of glycolysis and by uncouplers of oxidative phosphorylation [21], [22]. In addition to acting as a fuel
Disclosures
None.
Acknowledgments
These studies were supported by the National Institutes of Health (HL064838 and HL085820), the American Heart Association (Established Investigator Award to WAC) and in part by the New York Masonic Seventh District Association, Inc.
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2019, Forensic Science InternationalCitation Excerpt :Typical for when measuring KATP channels with this method, channel activity was present immediately after patch excision into a bath solution devoid of ATP, and channel activity was rapidly blocked when applying 1 mM ‘cytosolic’ ATP by using a rapid solution exchange system (Fig. 2). For these studies, we have generated a human SUR2A construct, which resulted in smaller patch currents compared to the rat SUR2A construct that we have used in past studies [35,36]. For wild-type Kir6.2/SUR2A, 26 of 38 excised patches exhibited none, or only a few, active KATP channels under ATP-free conditions, whereas 12/38 patches exhibited robust currents with six or more active channels.
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2019, Advances in Protein Chemistry and Structural BiologyCitation Excerpt :One of the signaling molecules that targets KATP channels is the AMPK (Kudo et al., 1996). KATP channels are hetero-octameric complexes expressed in most of the tissues, including the heart, brain, kidney, skeletal and smooth muscle, and pancreatic β-cells (Budas, Jovanovic, Crawford, & Jovanovic, 2004; Lim et al., 2009; Yoshida et al., 2012). These channels are activated both by decreasing cellular ATP levels and increasing ADP levels during energy starvation.
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This article is part of a Special Issue entitled “Local Signaling in Myocytes”.