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Rationale : Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium (mitoK ATP ) channel but its composition is not fully understood. Objective : We hypothesized that the mitoK ATP channel contains a sulfonylurea receptor (SUR)2 regulatory subunit and aimed to identify the molecular structure. Methods and Results : Western blot analysis in cardiac mitochondria detected a 55-kDa mitochondrial SUR2 (mitoSUR2) short form, 2 additional short forms (28 and 68 kDa), and a 130-kDa long form. RACE (Rapid Amplification of cDNA Ends) identified a 1.5-Kb transcript, which was generated by a nonconventional intraexonic splicing (IES) event within the 4th and 29th exons of the SUR2 mRNA. The translated product matched the predicted size of the 55-kDa short form. In a knockout mouse (SUR2KO), in which the SUR2 gene was disrupted, the 130-kDa mitoSUR2 was absent, but the short forms remained expressed. Diazoxide failed to induce increased fluorescence of flavoprotein oxidation in SUR2KO cells, indicating that the diazoxide-sensitive mitoK ATP channel activity was associated with 130-kDa–based channels. However, SUR2KO mice displayed similar infarct sizes to preconditioned wild type, suggesting a protective role for the remaining short form-based channels. Heterologous coexpression of the SUR2 IES variant and Kir6.2 in a K + transport mutant Escherichia coli strain permitted improved cell growth under acidic pH conditions. The SUR2 IES variant was localized to mitochondria, and removal of a predicted mitochondrial targeting sequence allowed surface expression and detection of an ATP-sensitive current when coexpressed with Kir6.2. Conclusions : We identify a novel SUR2 IES variant in cardiac mitochondria and provide evidence that the variant-based channel can form an ATP-sensitive conductance and may contribute to cardioprotection.

ATP-sensitive K + channels (K ATP ) couple intermediary metabolism to cellular activity, and may play a role in the autoregulation of vascular tones. Such a regulation requires cellular mechanisms for sensing O 2 , CO 2 , and pH. Our recent studies have shown that the pancreatic K ATP isoform (Kir6.2/SUR1) is regulated by CO 2 /pH. To identify the vascular K ATP isoform(s) and elucidate its response to hypercapnic acidosis, we performed these studies on vascular smooth myocytes (VSMs). Whole-cell and single-channel currents were studied on VSMs acutely dissociated from mesenteric arteries and HEK293 cells expressing Kir6.1/SUR2B. Hypercapnic acidosis activated an inward rectifier current that was K + -selective and sensitive to levcromakalim and glibenclamide with unitary conductance of ≈35pS. The maximal activation occurred at pH 6.5 to 6.8, and the current was inhibited at pH 6.2 to 5.9. The cloned Kir6.1/SUR2B channel responded to hypercapnia and intracellular acidification in an almost identical pattern to the VSM current. In situ hybridization histochemistry revealed expression of Kir6.1/SUR2B mRNAs in mesenteric arteries. Hypercapnia produced vasodilation of the isolated and perfused mesenteric arteries. Pharmacological interference of the K ATP channels greatly eliminated the hypercapnic vasodilation. These results thus indicate that the Kir6.1/SUR2B channel is a critical player in the regulation of vascular tones during hypercapnic acidosis.

Abstract —The ATP-sensitive K + (K ATP ) channels are composed of the pore-forming K + channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle–type K ATP channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-K ATP channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of K ATP channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on K ATP channels containing different SURs.

Abstract —The functional and pharmacological properties of ATP-sensitive K + (K ATP ) channels were studied in primary cultured neonatal rat atrial appendage cardiomyocytes. Activation of a whole-cell inward rectifying K + current depended on the pipette ATP concentration and correlated with a membrane hyperpolarization close to the K + equilibrium potential. The K ATP current could be activated either spontaneously or by a hypotonic stretch of the membrane induced by lowering the osmolality of the bathing solution from 290 to 260 mOsm/kg H 2 O or by the K + channel openers diazoxide and cromakalim with EC 50 ≈1 and 10 nmol/L, respectively. The activated atrial K ATP current was highly sensitive to glyburide, with an IC 50 of 1.22±0.15 nmol/L. Recorded in inside-out patches, the neonatal atrial K ATP channel displayed a conductance of 58.0±2.2 pS and opened in bursts of 133.8±20.4 ms duration, with an open time duration of 1.40±0.10 ms and a close time duration of 0.66±0.04 ms for negative potentials. The channel had a half-maximal open probability at 0.1 mmol/L ATP, was activated by 100 μmol/L diazoxide, and was inhibited by glyburide, with an IC 50 in the nanomolar range. Thus, pending further tests at low concentrations of K ATP channel openers, the single-channel data confirm the results obtained with whole-cell recordings. The neonatal atrial appendage K ATP channel thus shows a unique functional and pharmacological profile resembling the pancreatic β-cell channel for its high affinity for glyburide and diazoxide and for its conductance, but also resembling the ventricular channel subtype for its high affinity for cromakalim, its burst duration, and its sensitivity to ATP. Reverse transcriptase–polymerase chain reaction experiments showed the expression of Kir6.1, Kir6.2, SUR1A, SUR1B, SUR2A, and SUR2B subunits, a finding supporting the hypothesis that the neonatal atrial K ATP channel corresponds to a novel heteromultimeric association of K ATP channel subunits.

See related articles, pages 1458–1465 The discovery of ATP-sensitive K+ currents (KATP) offered an ideal molecular mechanism for coupling energy availability to excitability, function, and ATP consumption in myocardium,1 as well as other excitable tissues such as pancreatic β cells,2 wherein these channels regulate excitation–secretion coupling and are targets for antidiabetic drugs like sulfonylureas.3 With the cloning of the genes underlying KATP channels, it became clear that these channels were comprised of heteromultimers of 4 inwardly rectifying K+ channels (Kir6.1 or Kir6.2) and 4 ATP-binding cassette sulfonylurea receptors (SUR1 or SUR2).4 Importantly, KATP channels with different molecular constituents show distinct functional and regulatory properties that correlate with the response of KATP channels in various excitable cells, presumably to meet the diverse, and often unique, energy-sensing requirements between different tissues. For example, KATP channels are formed almost exclusively by Kir6.2 and SUR1 genes in pancreatic β cells,4 where these channels use the relative MgADP/MgATP ratio to tightly couple subplasmamembrane glucose levels with insulin release by modulating membrane potential and thereby calcium influx.5 By contrast, sarcolemmal KATP channels in cardiomyocytes (as well as skeletal muscle) have been shown to be comprised primarily of coassembled Kir6.2 and SUR2A proteins.6 Although the role of cardiac sarcolemmal KATP channels in cardiomyocytes is incompletely understood, cardiac sarcolemmal KATP channels appear to coordinate energy consumption with energy availability during periods of stress induced by ischemia, changes in workload, or adrenergic stimulation by modulating the action potential profile.7–9 In addition, sarcolemmal KATP channels also participate in the phenomenon of ischemic …

The isoform-specific structure of the ATP-sensitive potassium (K ATP ) channel endows it with differential fundamental properties, including physiological activation and pharmacology. Numerous studies have convincingly demonstrated that the pore-forming Kir6.2 ( KCNJ11 ) and regulatory SUR2A ( ABCC9 ) subunits are essential elements of the sarcolemmal K ATP channel in cardiac ventricular myocytes. Using a novel antibody directed against the COOH terminus of SUR1 ( ABCC8 ), we show that this K ATP subunit is also expressed in mouse myocardium and is the dominant SUR isoform in the atrium. This suggests differential sarcolemmal K ATP composition in atria and ventricles, and, to test this, K ATP currents were measured in isolated atrial and ventricular myocytes from wild-type and SUR1 −/− animals. K ATP conductance is essentially abolished in SUR1 −/− atrial myocytes but is normal in SUR1 −/− ventricular myocytes. Furthermore, pharmacological properties of wild-type atrial K ATP match closely the properties of heterologously expressed SUR1/Kir6.2 channels, whereas ventricular K ATP properties match those of heterologously expressed SUR2A/Kir6.2 channels. Collectively, the data demonstrate a previously unappreciated K ATP channel heterogeneity: SUR1 is an essential component of atrial, but not ventricular, K ATP channels. Differential molecular make-up of the 2 channels underlies differential pharmacology, with important implications when considering sulfonylurea therapy or dissecting the role of cardiac K ATP pharmacologically, as well as for understanding of the role of diazoxide in preconditioning.

ATP-binding cassette (ABC) proteins are highly conserved and widely expressed throughout nature and found in all organisms, both prokaryotic and eukaryotic. They mediate myriad critical cellular processes, from nutrient import to toxin efflux using the energy derived from ATP hydrolysis. Most ABC proteins mediate transport of substances across lipid membranes. However, there are atypical ABC proteins that mediate other processes. These include, but are not limited to, DNA repair (bacterial MutS), ion transport (cystic fibrosis transmembrane receptor), and mRNA trafficking (yeast Elf1p). The sulfonylurea receptor (SUR) is another atypical ABC protein that regulates activity of the potassium ATP channel (K ATP ). K ATP is widely expressed in nearly all tissues of higher organisms and couples cellular energy status to membrane potential. K ATP is particularly important in the regulation of insulin secretion from pancreatic β-cells and in regulating action potential duration in muscle cells. SUR is indispensable for normal channel function, and mutations in genes encoding SURs increase the susceptibility to diabetes, myocardial infarction, and heart failure. Here, we review the structure and function of ABC proteins and discuss SUR, its regulation of the K ATP channel, and its role in cardiovascular disease.

The activation of ATP-sensitive K + channels by protein kinase A in vascular smooth muscle is an important component of the action of vasodilators. In this study, we examine the molecular mechanisms of regulation of the cloned equivalent of this channel comprising the sulfonylurea receptor 2B and the inward rectifier 6.1 subunit (SUR2B/Kir6.1). Specifically, we focus on whether the channel is directly phosphorylated and the sites at which this occurs in the protein complex. We identify one site in Kir6.1 (S385) and two sites in SUR2B (T633 and S1465) using a combination of biochemical and functional assays. Our work supports a model in which multiple sites in the channel complex have to be phosphorylated before activation occurs.

ATP-sensitive K + (K ATP ) channels are composed of sulfonylurea receptors (SURs) and inwardly rectifying Kir6.2-channels. The C-terminal 42 amino acid residues (C42) of SURs are responsible for ADP-induced differential activation of K ATP channels in SUR-subtypes. By examining ADP-effect on K ATP channels containing various chimeras of SUR2A and SUR2B, we identified a segment of 7 residues at central portion of C42 critical for this phenomenon. A 3-D structure model of the region containing the second nucleotide-binding domain (NBD2) of SUR and C42 was developed based on the structure of HisP, a nucleotide-binding protein forming the bacterial Histidine transporter complex. In the model, the polar and charged residues in the critical segment located within a distance that allows their electrostatic interaction with Arg1344 at the Walker-A loop of NBD2. Therefore, the interaction might be involved in the control of ADP-induced differential activation of SUR2-subtype K ATP channels.

Abstract —ATP-sensitive potassium (K ATP ) channels in striated myocytes are heteromultimers of K IR 6.2, a weak potassium inward rectifier, plus SUR2A, a low-affinity sulfonylurea receptor. We have cloned human K IR 6.2 (huK IR 6.2) and a huSUR2A that corresponds to the major, full-length splice variant identified by polymerase chain reaction analysis of human cardiac poly A + mRNA. ATP- and glibenclamide-sensitive K + channels were produced when both subunits were coexpressed in COSm6 and Chinese hamster ovary cells lacking endogenous K ATP channels, but not when huSUR2A or huK IR 6.2 were transfected alone. Recombinant channels activated by metabolic inhibition in cell-attached configuration or in inside-out patches with ATP-free internal solution were compared with sarcolemmal K ATP channels in human ventricular cells. The single-channel conductance of ≈80 pS measured at −40 mV in quasi-symmetrical ≈150 mmol/L K + solutions, the intraburst kinetics that were dependent on K + driving force, and the weak inward rectification were indistinguishable for both channels. Similar to the native channels, huSUR2A/huK IR 6.2 recombinant channels were inhibited by ATP at quasi-physiological free Mg 2+ (≈0.7 mmol/L) or in the absence of Mg 2+ , with an apparent IC 50 of ≈20 μmol/L and a pseudo-Hill coefficient of ≈1. They were “refreshed” by MgATP and stimulated by ADP in the presence of Mg 2+ when inhibited by ATP. The huSUR2A/huK IR 6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg 2+ but were insensitive to diazoxide. The results suggest that reconstituted huSUR2A/huK IR 6.2 channels represent K ATP channels in sarcolemma of human cardiomyocytes and are an adequate experimental model with which to examine structure-function relationships, molecular physiology, and pharmacology of these channels from human heart.

Abstract —A defining property of ATP-sensitive K + (K ATP ) channels is inhibition by sulfonylurea drugs, yet the response of cardiac K ATP channels toward sulfonylureas during myocardial ischemia is not consistent. Altered channel sensitivity toward sulfonylureas has, in part, been ascribed to antagonism by cytosolic nucleotide diphosphates, although the mechanism of interaction remains unclear. Herein, in inside-out patches excised from cardiomyocytes, we observed a dual response of K ATP channels toward the sulfonylurea drug, glyburide, in the presence of cytosolic UDP. Specifically, glyburide failed to inhibit spontaneous K ATP channel activity in the presence of UDP but inhibited UDP-induced channel activity after rundown of spontaneous channel openings. Such behavior of K ATP channels cannot be explained by differences in the level of channel activity or by UDP-induced displacement of glyburide. Rather, the dual response toward the sulfonylurea could be attributed to a property of K ATP channels to switch between operative conditions (spontaneous versus UDP-induced) each associated with a distinct responsiveness toward ligands. Conversion of post-rundown K ATP channels to the spontaneously operative channel condition, by Mg-ATP, restored the ability of UDP to antagonize the inhibitory action of glyburide lost after rundown, suggesting that the response of the channel to glyburide is phosphorylation dependent. The existence of distinct operative conditions of cardiac K ATP channels could be the basis for the inconsistent response of the channel toward sulfonylurea drugs and should be considered when sulfonylureas are used to implicate the opening of K ATP channels in the myocardium.