ABSTRACT
BACKGROUND: Opening of cardiac ATP-sensitive potassium channels (K(ATP) channels) is a well-characterized protective mechanism against ischemia and reperfusion injury. Evidence exists for an involvement of both sarcolemmal and mitochondrial K(ATP) channels in such protection. Classically, cardiac sarcolemmal K(ATP) channels are thought to be composed of Kir6.2 (inward-rectifier potassium channel 6.2) and SUR2A (sulfonylurea receptor type 2A) subunits; however, the evidence is strong that SUR1 (sulfonylurea receptor type 1) subunits are also expressed in the heart and that they may have a functional role. The aim of this study, therefore, was to examine the role of SUR1 in myocardial infarction. METHODS AND RESULTS: We subjected mice lacking SUR1 subunits to in vivo myocardial ischemia/reperfusion injury. Interestingly, the SUR1-null mice were markedly protected against the ischemic insult, displaying a reduced infarct size and preservation of left ventricular function, which suggests a role for this K(ATP) channel subunit in cardiovascular function during conditions of stress. CONCLUSIONS: SUR1 subunits have a high sensitivity toward many sulfonylureas and certain K(ATP) channel-opening drugs. Their potential role during ischemic events should therefore be considered both in the interpretation of experimental data with pharmacological agents and in the clinical arena when the cardiovascular outcome of patients treated with antidiabetic sulfonylureas is being considered.
Subject(s)
ATP-Binding Cassette Transporters/physiology , KATP Channels/physiology , Multidrug Resistance-Associated Proteins/physiology , Myocardial Infarction/genetics , Myocardial Reperfusion Injury/genetics , Ventricular Dysfunction, Left/genetics , ATP-Binding Cassette Transporters/biosynthesis , ATP-Binding Cassette Transporters/drug effects , ATP-Binding Cassette Transporters/genetics , Animals , Diabetes Complications/physiopathology , Fibrosis , Hypoglycemic Agents/pharmacology , KATP Channels/chemistry , KATP Channels/drug effects , Mice , Mice, Knockout , Multidrug Resistance-Associated Proteins/deficiency , Multidrug Resistance-Associated Proteins/drug effects , Multidrug Resistance-Associated Proteins/genetics , Myocardial Infarction/complications , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/physiopathology , Myocarditis/etiology , Myocarditis/genetics , Myocarditis/prevention & control , Myocytes, Cardiac/physiology , Patch-Clamp Techniques , Potassium Channels/biosynthesis , Potassium Channels/genetics , Potassium Channels, Inwardly Rectifying/biosynthesis , Potassium Channels, Inwardly Rectifying/genetics , Receptors, Drug/biosynthesis , Receptors, Drug/genetics , Sulfonylurea Compounds/pharmacology , Sulfonylurea Receptors , Ventricular Dysfunction, Left/etiology , Ventricular Dysfunction, Left/physiopathology , Ventricular Dysfunction, Left/prevention & controlABSTRACT
The regulation of ATP-sensitive potassium (K(ATP)) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K(ATP) channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K(ATP) channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K(ATP) channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K(ATP) channel complex) causes channel closure.
Subject(s)
Gene Expression Regulation , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/chemistry , Pyruvate Kinase/chemistry , Triose-Phosphate Isomerase/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Animals , Bacteria/metabolism , COS Cells , Cells, Cultured , Chlorocebus aethiops , DNA/metabolism , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Electrophysiology , Glycolysis , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Heart Ventricles/metabolism , Hypoxia , Immunoglobulin G/chemistry , Immunoprecipitation , Kinetics , Mice , Microscopy, Fluorescence , Muscle Cells/metabolism , Mutation , Myocardium/metabolism , Patch-Clamp Techniques , Potassium/chemistry , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/metabolism , Protein Binding , Protein Structure, Tertiary , Pyruvate Kinase/metabolism , Rats , Rats, Sprague-Dawley , Transfection , Two-Hybrid System TechniquesABSTRACT
9,10-Epoxy-12-octadecenoic acid (EOA), a metabolite of linoleic acid, causes cardiac arrest in dogs. Other metabolites of linoleic acid also have toxic effects. This study investigates the mechanism of action of four of these compounds on cardiac Na(+) current (I(Na)). The whole-cell patch-clamp technique was used to investigate the effects of EOA, 9,10-dihydroxy-12-octadecenoic acid (DHOA), and their corresponding methyl esters (9,10-epoxy-12-octadecenoic methyl ester, EOM; and 9,10-dihydroxy-12-octadecenoic methyl ester, DHOM) on I(Na) in isolated adult rat ventricular myocytes. Extracellular application of each compound elicited a concentration-dependent inhibition of I(Na). The dose-response curve yielded 50% inhibition concentrations of 301 +/- 117 microM for DHOA, 41 +/- 6 microM for DHOM, 34 +/- 5 microM for EOA, and 160 +/- 41 microM for EOM. Although there was no effect on activation, 50 microM DHOM, EOA, and EOM significantly hyperpolarized the steady-state inactivation curve by approximately -6 mV. Furthermore, EOM significantly increased the slope of the steady-state inactivation curve. These compounds also seemed to stabilize the inactivated state because the time for recovery from inactivation was significantly slowed from a control value of 12.9 +/- 0.5 ms to 30.5 +/- 3.3, 31.4 +/- 1.4, and 20.5 +/- 1.0 ms by 50 microM DHOM, EOA, and EOM, respectively. These compounds have multiple actions on Na(+) channels and that despite their structural similarities their actions differ from each other. The steady-state block of I(Na) suggests that either the pore is being blocked or the channels are prevented from gating to the open state. In addition, these compounds stabilize the inactivated state and promote increased population of a slower inactivated state.
