Physiological and pathophysiological roles of ATP-sensitive K+ channels

ATP-sensitive potassium (K(ATP)) channels are present in many tissues, including pancreatic islet cells, heart, skeletal muscle, vascular smooth muscle, and brain, in which they couple the cell metabolic state to its membrane potential, playing a crucial role in various cellular functions. The K(ATP) channel is a hetero-octamer comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2). Kir6.x belongs to the inward rectifier K(+) channel family; SUR belongs to the ATP-binding cassette protein superfamily. Heterologous expression of differing combinations of Kir6.1 or Kir6.2 and SUR1 or SUR2 variant (SUR2A or SUR2B) reconstitute different types of K(ATP) channels with distinct electrophysiological properties and nucleotide and pharmacological sensitivities corresponding to the various K(ATP) channels in native tissues. The physiological and pathophysiological roles of K(ATP) channels have been studied primarily using K(ATP) channel blockers and K(+) channel openers, but there is no direct evidence on the role of the K(ATP) channels in many important cellular responses. In addition to the analyses of naturally occurring mutations of the genes in humans, determination of the phenotypes of mice generated by genetic manipulation has been successful in clarifying the function of various gene products. Recently, various genetically engineered mice, including mice lacking K(ATP) channels (knockout mice) and mice expressing various mutant K(ATP) channels (transgenic mice), have been generated. In this review, we focus on the physiological and pathophysiological roles of K(ATP) channels learned from genetic manipulation of mice and naturally occurring mutations in humans.     

Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits

ATP-sensitive K(+) (K(ATP)) channels, composed of inward rectifier K(+) (Kir)6.x and sulfonylurea receptor (SUR)x subunits, are expressed on cellular plasma membranes. We demonstrate an essential role for SUR2 subunits in trafficking K(ATP) channels to an intracellular vesicular compartment. Transfection of Kir6.x/SUR2 subunits into a variety of cell lines (including h9c2 cardiac cells and human coronary artery smooth muscle cells) resulted in trafficking to endosomal/lysosomal compartments, as assessed by immunofluorescence microscopy. By contrast, SUR1/Kir6.x channels efficiently localized to the plasmalemma. The channel turnover rate was similar with SUR1 or SUR2, suggesting that the expression of Kir6/SUR2 proteins in lysosomes is not associated with increased degradation. Surface labeling of hemagglutinin-tagged channels demonstrated that SUR2-containing channels dynamically cycle between endosomal and plasmalemmal compartments. In addition, Kir6.2 and SUR2 subunits were found in both endosomal and sarcolemmal membrane fractions isolated from rat hearts. The balance of these K(ATP) channel subunits shifted to the sarcolemmal membrane fraction after the induction of ischemia. The K(ATP) channel current density was also increased in rat ventricular myocytes isolated from hearts rendered ischemic before cell isolation without corresponding changes in subunit mRNA expression. We conclude that an intracellular pool of SUR2-containing K(ATP) channels exists that is derived by endocytosis from the plasma membrane. In cardiac myocytes, this pool can potentially play a cardioprotective role by serving as a reservoir for modulating surface K(ATP) channel density under stress conditions, such as myocardial ischemia.     

Establishment and application of in vitro membrane potential assays in cell lines with endogenous or recombinant expression of ATP-sensitive potassium channels (Kir6.2/SUR1) using a fluorescent probe kit

The flow of current through the adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP)) of the isoform Kir6.2/SUR1 regulates the resting membrane potential in the pancreatic beta-cell. In combination with the cellular glucose metabolism, it is an important minute-to-minute regulator of insulin secretion and whole-body glucose homeostasis. The same K(ATP) isoform is further reported to be present in glucagon-secreting alpha-cells, intestinal L-cells, and glucose-responsive neurons in the hypothalamus. All in all, this makes Kir6.2/SUR1 an interesting drug target. Using a commercially available fluorescent membrane potential probe kit and a conventional 96-well fluorescence plate reader, the authors have developed and established qualitative membrane potential assays used to screen for potassium channel closers (KCCs) and openers (KCOs) in insulin- and glucagon-secreting cell lines as well as in cells with recombinant expression of the human Kir6.2/SUR1 channel complex. Both glucose- and KCC-induced depolarization could be demonstrated. The magnitudes of these responses and KCO-induced repolarization at high glucose displayed some variation between the different cell lines but a similar rank order of test compounds. Some cell types required the presence of a KCC, such as tolbutamide, to display significant effects of KCOs. The authors find that robust and reliable functional in vitro assays compatible with medium-throughput screening and high-throughput screening can be developed as a base for finding new, more potent, and isoform-selective KCCs and KCOs.     

The first nucleotide binding domain of the sulfonylurea receptor 2A contains regulatory elements and is folded and functions as an independent module

The sulfonylurea receptor 2A (SUR2A) is an ATP-binding cassette (ABC) protein that forms the regulatory subunit of ATP-sensitive potassium (K(ATP)) channels in the heart. ATP binding and hydrolysis at the SUR2A nucleotide binding domains (NBDs) control gating of K(ATP) channels, and mutations in the NBDs that affect ATP hydrolysis and cellular trafficking cause cardiovascular disorders. To date, there is limited information on the SUR2A NBDs and the effects of disease-causing mutations on their structure and interactions. Structural and biophysical studies of NBDs, especially from eukaryotic ABC proteins like SUR2A, have been hindered by low solubility of the isolated domains. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the precise definition of the domain boundaries. Putative boundaries of SUR2A NBD1 were identified by structure-based sequence alignments and subsequently tested by exploring the solubility of SUR2A NBD1 constructs with different N and C termini. We have determined boundaries of SUR2A NBD1 that allow for soluble heterologous expression of the protein, producing a folded domain with ATP binding activity. Surprisingly, our alignment and screening data indicate that SUR2A NBD1 contains two putative, previously unidentified, regulatory elements: a large insert within the β-sheet subdomain and a C-terminal extension. Our approach, which combines the use of structure-based sequence alignments and predictions of disordered regions combined with biochemical and biophysical studies, may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins.     

Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B

ATP-sensitive potassium (K(ATP)) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle K(ATP) channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state. To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of K(ATP) channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido-[alpha-(32)P]ATP or 8-azido-[gamma-(32)P]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg(2+) dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A. This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of K(ATP) channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic K(ATP) channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs.     

黄芫花提取物对V79细胞和WB肝细胞的生物...：1....

A Chinese herb, wikstroemia Chamaedaphen (WC) extract, recently has been shown to be a potential tumor promoting agent on uterine cervical carcinoma induced by HSV-2 or MCA in mice. To determine whether the tumor promoting effects of WC extract were mediated through inhibition of gap junctional intercellular communication (GJIC) with relation to cellular growth, experiments were conducted on Chinese hamster V79 cells and rat WB liver cells by utilization of SLDT method for GJIC detection and cell growth curve examination, 3H-TdR incorporation, mitotic index (MI) and Flow Cytometry (FCM) methods. TPA was used for comparative purpose. WC extract inhibited GJIC and stimulated cell growth in a dose (2-200 micrograms/ml) and time (0-72 hr)-dependent manner in both cell lines. Both WC extract and TPA treatments increased V79 cell growth rate. The average cell doubling-time was decreased from 36.5 hr in control V79 cells to 28.2 hr in WC extract (10 micrograms/ml) and 20.9 hr in TPA (50 ng/ml) treatment by the 3rd day. Stimulating effect of both drugs on DNA synthesis of V79 cells was demonstrated. The results of FCM and MI indicated that the cell number of M-phase cells was increased after drug treatment. It is suggested that (1) tumor promoting effect of WC extract might be mediated through inhibition of GJIC: (2) inhibition of GJIC is closely correlated with increased cell growth rate and entry of cell division cycle.     

Localization of sulfonylurea receptor subunits, SUR2A and SUR2B, in rat heart.

To understand the possible functions and subcellular localizations of sulfonylurea receptors (SURs) in cardiac muscle, polyclonal anti-SUR2A and anti-SUR2B antisera were raised. Immunoblots revealed both SUR2A and SUR2B expression in mitochondrial fractions of rat heart and other cellular fractions such as microsomes and cell membranes. Immunostaining detected ubiquitous expression of both SUR2A and SUR2B in rat heart in the atria, ventricles, interatrial and interventricular septa, and smooth muscles and endothelia of the coronary arteries. Electron microscopy revealed SUR2A immunoreactivity in the cell membrane, endoplasmic reticulum (ER), and mitochondria. SUR2B immunoreactivity was mainly localized in the mitochondria as well as in the ER and cell membrane. Thus, SUR2A and SUR2B are not only the regulatory subunits of sarcolemmal K(ATP) channels but may also function as regulatory subunits in mitochondrial K(ATP) channels and play important roles in cardioprotection.     

Assembly limits the pharmacological complexity of ATP-sensitive potassium channels

ATP-sensitive potassium channels (K(ATP) channels) are formed from an octameric complex of an inwardly rectifying K(+) channel (Kir6.1, Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, and SUR2B). In this study we have attempted to address the question of whether SUR heteromultimers can form using a combination of biochemical and electrophysiological approaches. We have constructed monoclonal stable lines in HEK293 cells co-expressing Kir6.2 with SUR1 and SUR2A. Using coimmunoprecipitation analysis with SUR isotype-specific antibodies two biochemical populations are distinguished, one containing SUR1 and the other SUR2A. It is not possible to detect immune complexes containing both SUR1 and SUR2A. Functional studies were undertaken and whole cell membrane currents were studied using the patch clamp. Concentrations of sulfonylureas and potassium channel openers were determined that selectively inhibited or activated SUR1/Kir6.2 and SUR2A/Kir6.2. In the cell line expressing SUR1/SUR2AKir6.2 we were unable to demonstrate a population of channels with unique pharmacological properties. Thus we conclude from these studies that heteromultimeric channel complexes containing both SUR1 and SUR2A are not formed, suggesting an incompatibility between different SUR subtypes. This incompatibility limits the pharmacological complexity of K(ATP) channels that may be observed in native tissues.     

M-LDH serves as a sarcolemmal K(ATP) channel subunit essential for cell protection against ischemia

ATP-sensitive K(+) (K(ATP)) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K(+) channel core, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of channel gating. Here, we have shown that the muscle form (M-LDH), but not heart form (H-LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal K(ATP) channel by interacting with the Kir6.2 subunit via its N-terminus and with the SUR2A subunit via its C-terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M-LDH in the channel protein complex was required for opening of K(ATP) channels during ischemia and ischemia-resistant cellular phenotype. We conclude that M-LDH is an integral part of the sarcolemmal K(ATP) channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the K(ATP) channels activity that is essential for cell protection against ischemia.     

Glucagon-like peptide-1 inhibits pancreatic ATP-sensitive potassium channels via a protein kinase A- and ADP-dependent mechanism

Glucagon-like peptide-1 (GLP-1) elicits a glucose-dependent insulin secretory effect via elevation of cAMP and activation of protein kinase A (PKA). GLP-1-mediated closure of ATP-sensitive potassium (K(ATP)) channels is involved in this process, although the mechanism of action of PKA on the K(ATP) channels is not fully understood. K(ATP) channel currents and membrane potentials were measured from insulin-secreting INS-1 cells and recombinant beta-cell K(ATP) channels. 20 nM GLP-1 depolarized INS-1 cells significantly by 6.68 +/- 1.29 mV. GLP-1 reduced recombinant K(ATP) channel currents by 54.1 +/- 6.9% in mammalian cells coexpressing SUR1, Kir6.2, and GLP-1 receptor clones. In the presence of 0.2 mM ATP, the catalytic subunit of PKA (cPKA, 20 nM) had no effect on SUR1/Kir6.2 activity in inside-out patches. However, the stimulatory effects of 0.2 mM ADP on SUR1/Kir6.2 currents were reduced by 26.7 +/- 2.9% (P < 0.05) in the presence of cPKA. cPKA increased SUR1/Kir6.2 currents by 201.2 +/- 20.8% (P < 0.05) with 0.5 mM ADP present. The point mutation S1448A in the ADP-sensing region of SUR1 removed the modulatory effects of cPKA. Our results indicate that PKA-mediated phosphorylation of S1448 in the SUR1 subunit leads to K(ATP) channel closure via an ADP-dependent mechanism. The marked alteration of the PKA-mediated effects at different ADP levels may provide a cellular mechanism for the glucose-sensitivity of GLP-1.     

The tolbutamide site of SUR1 and a mechanism for its functional coupling to K(ATP) channel closure.

Micromolar concentrations of tolbutamide will inhibit (SUR1/K(IR)6. 2)(4) channels in pancreatic beta-cells, but not (SUR2A/K(IR)6.2)(4) channels in cardiomyocytes. Inhibition does not require Mg(2+) or nucleotides and is enhanced by intracellular nucleotides. Using chimeras between SUR1 and SUR2A, we show that transmembrane domains 12-17 (TMD12-17) are required for high-affinity tolbutamide inhibition of K(ATP) channels. Deletions demonstrate involvement of the cytoplasmic N-terminus of K(IR)6.2 in coupling sulfonylurea-binding with SUR1 to the stabilization of an interburst closed configuration of the channel. The increased efficacy of tolbutamide by nucleotides results from an impairment of their stimulatory action on SUR1 which unmasks their inhibitory effects. The mechanism of inhibition of beta-cell K(ATP) channels by sulfonylureas during treatment of non-insulin-dependent diabetes mellitus thus involves two components, drug-binding and conformational changes within SUR1 which are coupled to the pore subunit through its N-terminus and the disruption of nucleotide-dependent stimulatory effects of the regulatory subunit on the pore. These findings uncover a molecular basis for an inhibitory influence of SUR1, an ATP-binding cassette (ABC) protein, on K(IR)6.2, a ion channel subunit.     

Interaction of vanadate with the cloned beta cell K(ATP) channel.

Vanadate is used as a tool to trap magnesium nucleotides in the catalytic site of ATPases. However, it has also been reported to activate ATP-sensitive potassium (K(ATP)) channels in the absence of nucleotides. K(ATP) channels comprise Kir6.2 and sulfonylurea receptor subunits (SUR1 in pancreatic beta cells, SUR2A in cardiac and skeletal muscle, and SUR2B in smooth muscle). We explored the effect of vanadate (2 mM), in the absence and presence of magnesium nucleotides, on different types of cloned K(ATP) channels expressed in Xenopus oocytes. Currents were recorded from inside-out patches. Vanadate inhibited Kir6.2/SUR1 currents by approximately 50% but rapidly activated Kir6.2/SUR2A ( approximately 4-fold) and Kir6. 2/SUR2B ( approximately 2-fold) currents. Mutations in SUR that abolish channel activation by magnesium nucleotides did not prevent the effects of vanadate. Studies with chimeric SUR indicate that the first six transmembrane domains account for the difference in both the kinetics and the vanadate response of Kir6.2/SUR1 and Kir6. 2/SUR2A. Boiling the vanadate solution, which removes the decavanadate polymers, largely abolished both stimulatory and inhibitory actions of vanadate. Our results demonstrate that decavanadate modulates K(ATP) channel activity via the SUR subunit, that this modulation varies with the type of SUR, that it differs from that produced by magnesium nucleotides, and that it involves transmembrane domains 1-6 of SUR.     

Evidence for presence of ATP-sensitive K+ channels in rat colonic smooth muscle cells.

Coexpression of sulfonylurea receptor (SUR) and inward-rectifying K+ channel (Kir6.1 or 6.2) subunit yields ATP-sensitive K+ (K(ATP)) channels. Three subtypes of SUR have been cloned: pancreatic (SUR1), cardiac (SUR2A), and vascular smooth muscle (SUR2B). The distinct responses to K+ channel openers (KCOs) produced in different tissues may depend on the SUR isoform of K(ATP) channel. Therefore, we investigated the effects of pinacidil and diazoxide, two KCOs, on K(ATP) currents in intestinal smooth muscle cells of the rat colon (circular layer) using whole-cell voltage clamp. Pinacidil stimulated a time-independent K+ current evoked by various test potentials from a holding potential of -70 mV. The reversal potential of the stimulated current was about -75 mV, which is close to the equilibrium potential for K+ (E(K)). Both pinacidil and diazoxide dose-dependently stimulated K+ currents (evoked by ramp pulses), with EC50 values of 1.3 and 34.2 microM, respectively. The stimulated current was completely reversed by glybenclamide (3 microM). Since the EC50 values are close to those reported for vascular smooth muscle (VSM) cells, the SUR subtype may be similar to that in VSM cells, and could form the functional K(ATP) channel in rat colonic smooth muscle cells.     

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

The ATP-sensitive K(+) (K(ATP)) channels in both sarcolemmal (sarcK(ATP)) and mitochondrial inner membrane (mitoK(ATP)) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK(ATP) contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoK(ATP) remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K(ATP) into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K(ATP) channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing K(ATP) channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K(ATP) channels in mitochondria was significantly increased by PMA, as shown by K(ATP)-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K(ATP) channels was blocked by a selective PKCepsilon inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K(ATP) channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCepsilon. PKC isoform-regulated mitochondrial import of K(ATP) channels may have significant implication in cardioprotection of IPC.     

Pharmacological modulation of K(ATP) channels

Pharmacological modulation of ATP-sensitive K+ (K(ATP)) channels is used in the treatment of a number of clinical conditions, including type 2 diabetes and angina. The sulphonylureas and related drugs, which are used to treat type 2 diabetes, stimulate insulin secretion by closing K(ATP) channels in pancreatic beta-cells. Agents used to treat angina, by contrast, act by opening K(ATP) channels in vascular smooth and cardiac muscle. Both the therapeutic K(ATP) channel inhibitors and the K(ATP) channel openers target the sulphonylurea receptor (SUR) subunit of the K(ATP) channel, which exists in several isoforms expressed in different tissues (SUR1 in pancreatic beta-cells, SUR2A in cardiac muscle and SUR2B in vascular smooth muscle). The tissue-specific action of drugs that target the K(ATP) channel is attributed to the properties of these different SUR subtypes. In this review, we discuss the molecular basis of tissue-specific drug action, and its implications for clinical practice.     

The effect of acrylonitrile on gap junctional intercellular communication in rat astrocytes

Rats chronically exposed to acrylonitrile (ACN) have shown a dose-dependent increase in the incidence of astrocytomas in the brain. The mechanism(s) by which ACN induces cancer in rodents has not been established. ACN does not appear to be directly genotoxic in the brain and thus a nongenotoxic mode of action has been proposed. Inhibition of gap junctional intercellular communication (GJIC) has been shown to be a property of many nongenotoxic carcinogens. The present study examined the effects of ACN on GJIC in a rat astrocyte transformed cell line, DI TNC1 cells (a target cell for ACN carcinogenicity) and primary cultured hepatocytes (a nontarget cell for ACN carcinogenicity). ACN inhibited GJIC in rat astrocytes in a dose-dependent manner. Inhibition of GJIC was observed following 2 h treatment with 0.10 mmol/L and 1.00 mmol/L ACN. However, in primary cultured hepatocytes, ACN exposed did not result in inhibition of GJIC even after 48 h of continued treatment. In the astrocytes, GJIC inhibition plateaued after 4 h of treatment and remained blocked throughout the entire experimental period examined. Inhibition of GJIC in DI TNC1 cells was reversed by removal of ACN from the culture medium after 4 or 24 h of treatment. Cotreatment of astrocytes with vitamin E reduced the effect of ACN-induced inhibition of GJIC. Similarly, inhibition of GJIC was prevented by treatment with 2-oxothiazolidine-4-carboxylic acid (OTC), a precursor of glutathione synthesis. Decreasing cellular glutathione by treatment with buthionine sulfoxamine alone (without ACN) did not affect GJIC in astrocytes. Collectively, these results demonstrate that treatment with ACN caused a selective inhibition of GJIC in rat DI TNC1 astrocytes (the target cell type), but not in rat hepatocytes (a nontarget tissue). Inhibition of GJIC in astrocytes was reversed by treatment with antioxidants and suggests a potential role for oxidative stress in ACN-induced carcinogenesis.     

Resveratrol binds to the sulfonylurea receptor (SUR) and induces apoptosis in a SUR subtype-specific manner

Sulfonylurea receptors (SURs) constitute the regulatory subunits of ATP-sensitive K+ channels (K(ATP) channels). SUR binds nucleotides and synthetic K(ATP) channel modulators, e.g. the antidiabetic sulfonylurea glibenclamide, which acts as a channel blocker. However, knowledge about naturally occurring ligands of SUR is very limited. In this study, we show that the plant phenolic compound trans-resveratrol can bind to SUR and displace binding of glibenclamide. Electrophysiological measurements revealed that resveratrol is a blocker of pancreatic SUR1/K(IR)6.2 K(ATP) channels. We further demonstrate that, like glibenclamide, resveratrol induces enhanced apoptosis. This was shown by analyzing different apoptotic parameters (cell detachment, nuclear condensation and fragmentation, and activities of different caspase enzymes). The observed apoptotic effect was specific to cells expressing the SUR1 isoform and was not mediated by the electrical activity of K(ATP) channels, as it was observed in human embryonic kidney 293 cells expressing SUR1 alone. Enhanced susceptibility to resveratrol was not observed in pancreatic beta-cells from SUR1 knock-out mice or in cells expressing the isoform SUR2A or SUR2B or the mutant SUR1(M1289T). Resveratrol was much more potent than glibenclamide in inducing SUR1-specific apoptosis. Treatment with etoposide, a classical inducer of apoptosis, did not result in SUR isoform-specific apoptosis. In conclusion, resveratrol is a natural SUR ligand that can induce apoptosis in a SUR isoform-specific manner. Considering the tissue-specific expression patterns of SUR isoforms and the possible effects of SUR mutations on susceptibility to apoptosis, these observations could be important for diabetes and/or cancer research.     

Vasodilation induced by oxygen/glucose deprivation is attenuated in cerebral arteries of SUR2 null mice

Physiological functions of arterial smooth muscle cell ATP-sensitive K(+) (K(ATP)) channels, which are composed of inwardly rectifying K(+) channel 6.1 and sulfonylurea receptor (SUR)-2 subunits, during metabolic inhibition are unresolved. In the present study, we used a genetic model to investigate the physiological functions of SUR2-containing K(ATP) channels in mediating vasodilation to hypoxia, oxygen and glucose deprivation (OGD) or metabolic inhibition, and functional recovery following these insults. Data indicate that SUR2B is the only SUR isoform expressed in murine cerebral artery smooth muscle cells. Pressurized SUR2 wild-type (SUR2(wt)) and SUR2 null (SUR2(nl)) mouse cerebral arteries developed similar levels of myogenic tone and dilated similarly to hypoxia (<10 mmHg Po(2)). In contrast, vasodilation induced by pinacidil, a K(ATP) channel opener, was ～71% smaller in SUR2(nl) arteries. Human cerebral arteries also expressed SUR2B, developed myogenic tone, and dilated in response to hypoxia and pinacidil. OGD, oligomycin B (a mitochondrial ATP synthase blocker), and CCCP (a mitochondrial uncoupler) all induced vasodilations that were ～39-61% smaller in SUR2(nl) than in SUR2(wt) arteries. The restoration of oxygen and glucose following OGD or removal of oligomycin B and CCCP resulted in partial recovery of tone in both SUR2(wt) and SUR2(nl) cerebral arteries. However, SUR(nl) arteries regained ～60-82% more tone than did SUR2(wt) arteries. These data indicate that SUR2-containing K(ATP) channels are functional molecular targets for OGD, but not hypoxic, vasodilation in cerebral arteries. In addition, OGD activation of SUR2-containing K(ATP) channels may contribute to postischemic loss of myogenic tone.     

The stereoenantiomers of a pinacidil analog open or close cloned ATP-sensitive K+ channels

ATP-dependent K(+) channels (K(ATP) channels) are composed of pore-forming subunits Kir6.x and sulfonylurea receptors (SURs). Cyanoguanidines such as pinacidil and P1075 bind to SUR and enhance MgATP binding to and hydrolysis by SUR, thereby opening K(ATP) channels. In the vasculature, openers of K(ATP) channels produce vasorelaxation. Some novel cyanoguanidines, however, selectively reverse opener-induced vasorelaxation, suggesting that they might be K(ATP) channel blockers. Here we have analyzed the interaction of the enantiomers of a racemic cyanoguanidine blocker, PNU-94750, with Kir6.2/SUR channels. In patch clamp experiments, the R-enantiomer (PNU-96293) inhibited Kir6.2/SUR2 channels (IC(50) approximately 50 nm in the whole cell configuration), whereas the S-enantiomer (PNU-96179) was a weak opener. Radioligand binding studies showed that the R-enantiomer was more potent and that it was negatively allosterically coupled to MgATP binding, whereas the S-enantiomer was weaker and positively coupled. Binding experiments also suggested that both enantiomers bound to the P1075 site of SUR. This is the first report to show that the enantiomers of a K(ATP) channel modulator affect channel activity and coupling to MgATP binding in opposite directions and that these opposite effects are apparently mediated by binding to the same (opener) site of SUR.