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.     

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.     

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.     

Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca2+ homeostasis under metabolic stress.

Metabolic injury is a complex process affecting various tissues, with intracellular Ca2+ loading recognized as a common precipitating event leading to cell death. We have recently observed that cells overexpressing recombinant ATP-sensitive K+ (KATP) channel subunits may acquire resistance against metabolic stress. To examine whether, under metabolic challenge, intracellular Ca2+ homeostasis can be maintained by an activator of channel proteins, we delivered Kir6.2 and SUR2A genes, which encode KATP channel subunits, into a somatic cell line lacking native KATP channels. Hypoxia-reoxygenation was simulated by application and removal of the mitochondrial poison 2,4 dinitrophenol. Under such metabolic stress, Ca2+ loading was induced by Ca2+ influx during hypoxia and release of Ca2+ from intracellular stores during reoxygenation. Delivery of Kir6.2/SUR2A genes, in conjunction with the KATP channel activator pinacidil, prevented intracellular Ca2+ loading irrespective of whether the channel opener was applied throughout the duration of hypoxia-reoxygenation or transiently during the hypoxic or reoxygenation stage. In all stages of injury, the effect of pinacidil was inhibited by the selective antagonist of KATP channel, 5-hydroxydecanoate. The present study provides evidence that combined use of gene delivery and pharmacological targeting of recombinant proteins can handle intracellular Ca2+ homeostasis under hypoxia-reoxygenation irrespective of the stage of the metabolic insult.     

ATP modulation of ATP-sensitive potassium channel ATP sensitivity varies with the type of SUR subunit

ATP-sensitive potassium (K(ATP)) channels comprise Kir and SUR subunits. Using recombinant K(ATP) channels expressed in Xenopus oocytes, we observed that MgATP (100 microm) block of Kir6.2/SUR2A currents gradually declined with time, whereas inhibition of Kir6.2/SUR1 or Kir6.2DeltaC36 currents did not change. The decline in Kir6.2/SUR2A ATP sensitivity was not observed in Mg(2+) free solution and was blocked by the phosphatidylinositol (PI) 3-kinase inhibitors LY 294002 (10 microm) and wortmannin (100 microm), and by neomycin (100 microm). These results suggest that a MgATP-dependent synthesis of membrane phospholipids produces a secondary decrease in the ATP sensitivity of Kir6.2/SUR2A. Direct application of the phospholipids PI 4,5-bisphosphate and PI 3,4,5-trisphosphate in the presence of 100 microm MgATP activated all three types of channel, but the response was faster for Kir6.2/SUR2A. Chimeric studies indicate that the different responses of Kir6.2/SUR2A and Kir6.2/SUR1 are mediated by the first six transmembrane domains of SUR. The MgATP-dependent loss of ATP sensitivity of Kir6.2/SUR2A was enhanced by the actin filament disrupter cytochalasin and blocked by phalloidin (which stabilizes the cytoskeleton). Phalloidin did not block the effect of PI 3,4,5-trisphosphate. This suggests that MgATP may cause disruption of the cytoskeleton, leading to enhanced membrane phospholipid levels (or better targeting to the K(ATP) channel) and thus to decreased channel ATP sensitivity.     

新型钾通道开放剂对心血管ATP-敏感性钾通道基因表达的调节作用

目的：研究脂肪胺类的新型钾通道开放剂（KCO）埃他卡林（Ipt）和氰胍类的KCO吡那地尔（Pin）对大鼠心血管ATP-敏感性钾通道（KATP）的亚基SUR1、SUR2、Kir6．1和Kir6．2等在mRNA水平的调节作用。方法：SD大鼠给药1周后处死并取组织,提取总RNA,利用反转录-聚合酶链式反应（RT-PCR）研究以上基因在mRNA水平的改变。结果：与正常对照相比,心脏组织中,Ipt和Pin对KATP的4个亚基在mRNA水平均无显著影响;主动脉平滑肌上,Ipt对4个亚基的mRNA表达无显著影响,但Pin可显著上调SUR2的mRNA表达;尾动脉平滑肌上,Ipt对Kit6．1／Kit6．2、Pin对SUR2／Kir6．1均有显著下调的作用。结论：心肌、大动脉平滑肌和小动脉平滑肌KATP基因表达的调控不同,Ipt选择性调节小动脉平滑肌Kit6．1／Kit6．2;Ipt对心血管KATP基因表达的调节作用不同于Pin。     

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.     

Disopyramide block of K(ATP) channels is mediated by the pore-forming subunit

The class Ia antiarrhythmic agent disopyramide blocks native ATP-sensitive K+ (K(ATP)) channels at micromolar concentrations. The K(ATP) channel is a complex of a pore-forming inwardly rectifying K+ channel (Kir6.2) and a sulfonylurea receptor (SUR). The aim of the present study was to further localize the site of action of disopyramide. We have used a C-terminal truncated form of Kir6.2 (Kir6.2delta26), which--in contrast to Kir6.2--expresses independently of SUR. Kir6.2delta26 channels were expressed in African green monkey kidney COS-7 cells, and enhanced green fluorescent protein (EGFP) cDNA was used as a reporter gene. EGFP fluorescence was visualized by a laser scanning confocal microscope. Disopyramide applied to the cytoplasmic membrane surface of inside-out patches inhibited Kir6.2delta26 channels half-maximally at 7.1 microM (at pH 7.15). Lowering the intracellular pH to 6.5 potentiated the inhibition of Kir6.2delta26 channels by disopyramide. These observations suggest that disopyramide directly blocks the pore-forming Kir6.2 subunit, in particular at reduced intracellular pH values that occur under cardiac ischaemia.     

ATP-sensitive potassium channel: a novel target for protection against UV-induced human skin cell damage

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of K(ATP) channels are functionally expressed in HaCaT cells and both non-selective K(ATP) channel opener pinacidil and mitoK(ATP) (mitochondrial K(ATP)) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective K(ATP) channel blocker glibenclamide and selective mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). Also, activation of K(ATP) channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoK(ATP) is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that K(ATP) openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging.     

Structure-function relationships in the beta-cell K(ATP) channel

The ATP-sensitive potassium (K(ATP)) channel plays a key role in controlling beta-cell membrane potential and insulin secretion. The channels are composed of two subunits, Kir6.2, which forms the channel pore, and SUR1, which contains binding sites for nucleotides and sulphonylureas and acts as a channel regulator. Our current studies are aimed at delineating the molecular interactions involved in assembly and ligand binding by K(ATP) channel proteins. We have employed a complementation approach in which SUR1 half-molecules are co-expressed in insect cells using a baculovirus system. Together with data from truncated SUR1 molecules and a fusion protein in which SUR1 is linked to Kir6.2, we have interpreted our findings in terms of a model for the structure of the K(ATP) channel. The main features of the model are: (i) the C-terminal end of SUR1 is close to the N-terminus of Kir6.2; (ii) the two nucleotide binding domains (NBDs) of SUR1--NBD1 and NBD2--are in proximity; (iii) transmembrane helix 12 of SUR1 is orientated in such a way that it can make contact with Kir6.2; (iv) formation of the glibenclamide binding site requires that the two cytosolic loops (CLs) CL3 and CL8 are located close to each other; (v) there are homomeric interactions between the NBD1 domains of neighbouring subunits. We suggest that binding of glibenclamide leads to conformational changes in CL3 and CL8 leading to rearrangement of transmembrane helices. These effects are transmitted to Kir6.2 to result in channel closure.     

PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation.

ATP-sensitive potassium (K(ATP)) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K(ATP)) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate K(ATP) channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K(ATP) channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.     

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K(+) channels by G-protein betagamma-subunits

To determine the interaction site(s) of ATP-sensitive K(+) (K(ATP)) channels for G-proteins, sulfonylurea receptor (SUR2A or SUR1) and pore-forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS-7. Intracellular application of the G-protein betagamma2-subunits (G(betagamma)(2)) caused a reduction of ATP-induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. G(betagamma)(2) bound in vitro to both intracellular (loop-NBD) and C-terminal segments of SUR2A, each containing a nucleotide-binding domain (NBD). Furthermore, a single amino acid substitution in the loop-NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the G(betagamma)(2)-dependent alteration of the channel activities. These findings provide evidence that G(betagamma) modulates K(ATP) channels through a direct interaction with the loop-NBD of SUR.     

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.     

Insulin down-regulates cardioprotective SUR2A in the heart-derived H9c2 cells: A possible explanation for some adverse effects of insulin therapy

Some recent studies associated insulin therapy with negative cardiovascular events and shorter lifespan. SUR2A, a K_ATP channel subunit, regulate cardioprotection and cardiac ageing. Here, we have tested whether glucose and insulin regulate expression of SUR2A/K_ATP channel subunits and resistance to metabolic stress in heart H9c2 cells. Absence of glucose in culture media decreased SUR2A mRNA, while mRNAs of Kir6.2, Kir6.1, SUR1 and IES SUR2B were increased. 2-deoxyglucose (50 mM) decreased mRNAs of SUR2A, SUR2B and SUR1, did not affect IES SUR2A and IES SUR2B mRNAs and increased Kir6.2 mRNA. No glucose and 2-deoxyglucose (50 mM) decreased resistance to an inhibitor of oxidative phosphorylation, DNP (10 mM). 50 mM glucose did not alter K_ATP channel subunits nor cellular resistance to DNP (10 mM). Insulin (20 ng/ml) in both physiological and high glucose (50 mM) down-regulated SUR2A while upregulating Kir6.1 and Kir6.2 (in high glucose only). Insulin (20 ng/ml) in physiological and high glucose decreased cell survival in DNP (10 mM). As opposed to Kir6.2, infection with SUR2A resulted in titre-dependent cytoprotection. We conclude that insulin decreases resistance to metabolic stress in H9c2 cells by decreasing SUR2A expression. Lower cardiac SUR2A levels underlie increased myocardial susceptibility to metabolic stress and shorter lifespan.     

Expression of ATP sensitive K+ channel subunit Kir6.1 in rat kidney

ATP-sensitive K+ (K(ATP)) channels in kidney are considered to play roles in regulating membrane potential during the change in intracellular ATP concentration. They are composed of channel subunits (Kir6.1, Kir6.2), which are members of the inwardly rectifying K+ channel family, and sulphonylurea receptors (SUR1, SUR2A and SUR2B), which belong to the ATP-binding cassette superfamily. In the present study, we have investigated the expression and localization of Kir6.1 in rat kidney with Western blot analysis, immunohistochemistry, in situ hybridization histochemistry, and immunoelectron microscopy. Western blot analysis showed that Kir6.1 was expressed in the mitochondria and microsome fractions of rat kidney and very weakly in the membrane fractions. Immunohistochemistry revealed that Kir6.1 was widely distributed in renal tubular epithelial cells, glomerular mesangial cells, and smooth muscles of blood vessels. In immunoelectron microscopy, Kir6.1 is mainly localized in the mitochondria, endoplasmic reticulum (ER), and very weakly in cell membranes. Thus, Kir6.1 is contained in the kidney and may be a candidate of mitochondrial K(ATP) channels.     

Characterization of low-affinity binding sites for glibenclamide on the Kir6.2 subunit of the beta-cell KATP channel

The ATP-sensitive K+ channel, an octameric complex of two structurally unrelated types of subunits, SUR1 and Kir6.2, plays a central role in the physiological regulation of insulin secretion. The sulfonylurea glibenclamide, which trigger insulin secretion by blocking the ATP-sensitive K+ channel, interacts with both high and low affinity binding sites present on beta-cells. The high affinity binding site has been localized on SUR1 but the molecular nature of the low affinity site is still uncertain. In this study, we analyzed the pharmacology of glibenclamide in a transformed COS-7 cell line expressing the rat Kir6.2 cDNA and compared with that of the MIN6 beta cell line expressing natively both the Kir6.2 and the SUR1 subunits. Binding studies and Scatchard analysis revealed the presence of a single class of low affinity binding sites for glibenclamide on the COS/Kir6.2 cells with characteristics similar to that observed for the low affinity site of the MIN6 beta cells.     

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.     

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.     

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.