A role of the sulfonylurea receptor 1 in endocytic trafficking of ATP-sensitive potassium channels

The ATP-sensitive potassium (K(ATP) ) channel consisting of sulfonylurea receptor 1 (SUR1) and inward-rectifier potassium channel 6.2 (Kir6.2) has a well-established role in insulin secretion. Mutations in either subunit can lead to disease due to aberrant channel gating, altered channel density at the cell surface or a combination of both. Endocytic trafficking of channels at the plasma membrane is one way to influence surface channel numbers. It has been previously reported that channel endocytosis is dependent on a tyrosine-based motif in Kir6.2, while SUR1 alone is unable to internalize. In this study, we followed endocytic trafficking of surface channels in real time by live-cell imaging of channel subunits tagged with an extracellular minimal α-bungarotoxin-binding peptide labeled with a fluorescent dye. We show that SUR1 undergoes endocytosis independent of Kir6.2. Moreover, mutations in the putative endocytosis motif of Kir6.2, Y330C, Y330A and F333I are unable to prevent channel endocytosis. These findings challenge the notion that Kir6.2 bears the sole endocytic signal for K(ATP) channels and support a role of SUR1 in this trafficking process.     

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic cells where targets ATP-sensitive K⁺ (K_ATP) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K_ATP channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic KATP channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K_ATP channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K_ATP channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.     

Dynamic sensitivity of ATP-sensitive K(+) channels to ATP

ATP and MgADP regulate K(ATP) channel activity and hence potentially couple cellular metabolism to membrane electrical activity in various cell types. Using recombinant K(ATP) channels that lack sensitivity to MgADP, expressed in COSm6 cells, we demonstrate that similar on-cell activity can be observed with widely varying apparent submembrane [ATP] ([ATP](sub)). Metabolic inhibition leads to a biphasic change in the channel activity; activity first increases, presumably in response to a fast decrease in [ATP](sub), and then declines. The secondary decrease in channel activity reflects a marked increase in ATP sensitivity and is correlated with a fall in polyphosphoinositides (PPIs), including phosphatidylinositol 4,5-bisphosphate, probed using equilibrium labeling of cells with [(3)H]myo-inositol. Both ATP sensitivity and PPIs rapidly recover following removal of metabolic inhibition, and in both cases recovery is blocked by wortmannin. These data are consistent with metabolism having a dual effect on K(ATP) channel activity: rapid activation of channels because of relief of ATP inhibition and much slower reduction of channel activity mediated by a fall in PPIs. These two mechanisms constitute a feedback system that will tend to render K(ATP) channel activity transiently responsive to a change in [ATP](sub) over a wide range of steady state concentrations.     

Sulfonylureas correct trafficking defects of ATP-sensitive potassium channels caused by mutations in the sulfonylurea receptor

The pancreatic ATP-sensitive potassium (K(ATP)) channel, a complex of four sulfonylurea receptor 1 (SUR1) and four potassium channel Kir6.2 subunits, regulates insulin secretion by linking metabolic changes to beta-cell membrane potential. Sulfonylureas inhibit K(ATP) channel activities by binding to SUR1 and are widely used to treat type II diabetes. We report here that sulfonylureas also function as chemical chaperones to rescue K(ATP) channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. By contrast, diazoxide, a potassium channel opener that also binds SUR1, had no effect on surface expression of either mutant. Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional K(ATP) channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain K(ATP) channel trafficking mutations.     

Tandem function of nucleotide binding domains confers competence to sulfonylurea receptor in gating ATP-sensitive K+ channels

Fundamental to the metabolic sensor function of ATP-sensitive K(+) (K(ATP)) channels is the sulfonylurea receptor. This ATP-binding cassette protein, which contains nucleotide binding domains (NBD1 and NBD2) with conserved Walker motifs, regulates the ATP sensitivity of the pore-forming Kir6.2 subunit. Although NBD2 hydrolyzes ATP, a property essential in K(ATP) channel gating, the role of NBD1, which has limited catalytic activity, if at all, remains less understood. Here, we provide functional evidence that cooperative interaction, rather than the independent contribution of each NBD, is critical for K(ATP) channel regulation. Gating of cardiac K(ATP) channels by distinct conformations in the NBD2 ATPase cycle, induced by gamma-phosphate analogs, was disrupted by point mutation not only of the Walker motif in NBD2 but also in NBD1. Cooling membrane patches to decelerate the intrinsic ATPase activity counteracted ATP-induced K(ATP) channel inhibition, an effect that mimicked stabilization of the MgADP-bound posthydrolytic state at NBD2 by the gamma-phosphate analog orthovanadate. Temperature-induced channel activation was abolished by mutations that either prevent stabilization of MgADP at NBD2 or ATP at NBD1. These findings provide a paradigm of K(ATP) channel gating based on integration of both NBDs into a functional unit within the multimeric channel complex.     

Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes

We investigated the regulation ofATP-sensitive K⁺ (K_ATP) currents in murinecolonic myocytes with patch-clamp techniques. Pinacidil(10⁵ M) activated inward currents in the presence of highexternal K⁺ (90 mM) at a holding potential of 80 mV indialyzed cells. Glibenclamide (10⁵ M) suppressedpinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10⁷ M) inhibited pinacidil-activated current.4--Phorbol ester (5 × 10⁷ M), an inactive formof PDBu, had no effect on pinacidil-activated current. In cell-attachedpatches, the open probability of K_ATP channels wasincreased by pinacidil, and PDBu suppressed openings ofK_ATP channels. When cells were pretreated withchelerythrine (10⁶ M) or calphostin C (10⁷M), inhibition of the pinacidil-activated whole cell currents by PDBuwas significantly reduced. In cells studied with the perforated patchtechnique, PDBu also inhibited pinacidil-activated current, and thisinhibition was reduced by chelerythrine (10⁶ M).Acetylcholine (ACh; 10⁵ M) inhibited pinacidil-activatedcurrents, and preincubation of cells with calphostin C(10⁷ M) decreased the effect of ACh. Cells dialyzed withprotein kinase C -isoform (PKC) antibody had normal responses topinacidil, but the effects of PDBu and ACh on K_ATP wereblocked in these cells. Immunofluorescence and Western blots showedexpression of PKC in intact muscles and isolated smooth muscle cellsof the murine proximal colon. These data suggest that PKC regulates K_ATP in colonic muscle cells and that the effects of ACh onK_ATP are largely mediated by PKC. PKC appears to be themajor isozyme that regulates K_ATP in murine colonic myocytes.

     

Effects of lomefloxacin and norfloxacin on pancreatic beta-cell ATP-sensitive K(+) channels

In patients administered lomefloxacin alterations in blood glucose concentrations have been observed in some cases and lomefloxacin has previously been shown to augment insulin release from rat pancreatic islets at micromolar concentrations. The aim of the present study was to compare the effects of two structurally related fluoroquinolones, lomefloxacin and norfloxacin, on ATP-sensitive K(+) (K(ATP)) currents from the clonal insulinoma cell line RINm5F using the whole-cell configuration of the patch-clamp technique. The application of lomefloxacin concentration-dependently blocked K(ATP) currents from RINm5F cells with a half-maximally inhibitory concentration of 81 microM, whereas the application of norfloxacin (at concentrations up to 300 microM) had only minor effects on K(ATP) currents. Block of pancreatic beta-cell K(ATP) currents could be mediated by interaction of lomefloxacin either with the regulatory subunit (SUR1) or with the pore-forming subunit (Kir6.2). We favour the latter hypothesis, since some fluoroquinolones have recently been shown to block the pore-forming subunit of the cardiac rapid delayed rectifier K(+) current I(Kr) (which is encoded by HERG (human ether-a-go-go-related gene)). Thus, as demonstrated for cardiac HERG channels in previous studies and for pancreatic beta-cell K(ATP) channels in the present study, fluoroquinolones differ markedly in their potencies to inhibit K(+) channel activity.     

Compromised ATP binding as a mechanism of phosphoinositide modulation of ATP-sensitive K+ channels

Inhibition of ATP-sensitive K(+) (K(ATP)) channels by ATP, a process presumably initiated by binding of ATP to the pore-forming subunit, Kir6.2, is reduced in the presence of phosphoinositides (PPIs). Previous studies led to the hypothesis that PPIs compromise ATP binding. Here, this hypothesis was tested using purified Kir6.2. We show that PPIs bind purified Kir6.2 in an isomer-specific manner, that biotinylated ATP analogs photoaffinity label purified Kir6.2, and that this labeling is weakened in the presence of PPIs. Patch-clamp measurements confirmed that these ATP analogs inhibited Kir6.2 channels, and that PPIs decreased the level of inhibition. These results indicate that interaction of PPIs with Kir6.2 impedes ATP-binding activity. The PPI regulation of ATP binding revealed in this study provides a putative molecular mechanism that is potentially pivotal to the nucleotide sensitivity of K(ATP) channels.     

Block of cardiac ATP-sensitive K(+) channels reduces hydroxyl radicals in the rat myocardium

The present study examined whether opening of an ATP-sensitive K(+) (K(ATP)) channel can induce hydroxyl free radical ((*)OH) generation in the rat myocardium. Sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused directly through a microdialysis probe to detect the generation of (*)OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA). Induction of cromakalim (100 microM), a K(ATP) channel opener, through the microdialysis probe significantly increased the level of 2,3-DHBA. Another K(ATP) channel opener, nicorandil, also increased the level of 2,3-DHBA. When iron(II) was administered to cromakalim-pretreated animals, a marked elevation of DHBA was observed, compared with iron(II) only-treated animals. A positive linear correlation between iron(II) and formation of (*)OH, trapped as DHBA in the dialysate, was shown (r(2) = 0.988). When corresponding experiments were performed with nicorandil-treated animals, a positive linear correlation between iron(II) and DHBA in the dialysate was shown (r(2) = 0.988). However, the presence of glibenclamide (1-50 microM) decreased the cromakalim-induced 2,3-DHBA formation in a concentration-dependent manner (IC(50) = 9.1 microM). 5-Hydroxydecanoate (5-HD; 100 microM), another K(ATP) channel antagonist, also decreased cromakalim-induced (*)OH formation. The IC(50) value for 5-HD against cromakalim-evoked increase in 2,3-DHBA was 107.2 microM. In the presence of glibenclamide (10 microM), the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, the normal elevation of 2,3-DHBA in the heart dialysate was not observed in animals pretreated with glibenclamide (10 microM). When corresponding experiments were performed with 5-HD (100 microM) pretreated animals, the same results were obtained. These results suggest that opening of cardiac K(ATP) channels may cause (*)OH generation.     

ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors

This study describes a novel mechanism of regulation of the high-affinity Na(+)-dependent adenosine transporter (CNT2) via the activation of A(1) adenosine receptors (A(1)R). This regulation is mediated by the activation of ATP-sensitive K(+) (K(ATP)) channels. The high-affinity Na(+)-dependent adenosine transporter CNT2 and A(1)R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (-)-N(6)-(2-phenylisopropyl)adenosine is fully inhibited by K(ATP) channel blockers and mimicked by a K(ATP) channel opener. A(1)R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A(1)R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A(1)R, CNT2, and K(ATP) channels. These results also suggest that the activation of K(ATP) channels under metabolic stress can be mediated by the activation of A(1)R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for K(ATP) channels.     

Structural similarities between glutamate receptor channels and K(+) channels examined by scanning mutagenesis

The pores of glutamate receptors and K(+) channels share sequence homology, suggesting a conserved secondary structure. Scanning mutagenesis with substitution of alanine and tryptophan in GluR6 channels was performed based on the structure of KcsA. Our assay used disruption of voltage-dependent polyamine block to test for changes in the packing of pore-forming regions. Alanine scanning from D567 to R603 revealed reduced rectification resulting from channel block in two regions. A periodic pattern from F575 to M589 aligned with the pore helix in KcsA, whereas a cluster of sensitive positions around Q590, a site regulated by RNA editing, mapped to the selectivity filter in KcsA. Tryptophan scanning from D567 to R603 revealed similar patterns, but with a complete disruption of spermine block for 7 out of the 37 positions and a pM dissociation constant for Q590W. Molecular modeling with KcsA coordinates showed that GluR6 pore helix mutants disrupting polyamine block pack against M1 and M2, and are not exposed in the ion channel pore. In the selectivity filter, tryptophan creates an aromatic cage consistent with the pM dissociation constant for Q590W. A scan with glutamate substitution was used to map the cytoplasmic entrance to the pore based on charge neutralization experiments, which established that E594 was uniquely required for high affinity polyamine block. In E594Q mutants, introduction of glutamate at positions S593-L600 restored polyamine block at positions corresponding to surface-exposed residues in KcsA. Our results reinforce proposals that the pore region of glutamate receptors contains a helix and pore loop analogous to that found in K(+) channels. At the cytoplasmic entrance of the channel, a negatively charged amino acid, located in an extended loop with solvent-exposed side chains, is required for high affinity polyamine block and probably attracts cations via a through space electrostatic mechanism.     

Modulation of the trafficking efficiency and functional properties of ATP-sensitive potassium channels through a single amino acid in the sulfonylurea receptor

Mutations in the sulfonylurea receptor 1 (SUR1), a subunit of ATP-sensitive potassium (K(ATP)) channels, cause familial hyperinsulinism. One such mutation, deletion of phenylalanine 1388 (DeltaPhe-1388), leads to defects in both trafficking and MgADP response of K(ATP) channels. Here we investigated the biochemical features of Phe-1388 that control the proper trafficking and function of K(ATP) channels by substituting the residue with all other 19 amino acids. Whereas surface expression is largely dependent on hydrophobicity, channel response to MgADP is governed by multiple factors and involves the detailed architecture of the amino acid side chain. Thus, structural features in SUR1 required for proper channel function are distinct from those required for correct protein trafficking. Remarkably, replacing Phe-1388 by leucine profoundly alters the physiological and pharmacological properties of the channel. The F1388L-SUR1 channel has increased sensitivity to MgADP and metabolic inhibition, decreased sensitivity to glibenclamide, and responds to both diazoxide and pinacidil. Because this conservative amino acid substitution occurs in the SUR2A and SUR2B isoforms, the mutation provides a mechanism by which functional diversities in K(ATP) channels are generated.     

Dual phosphorylations underlie modulation of unitary KCNQ K(+) channels by Src tyrosine kinase

Src tyrosine kinase suppresses KCNQ (M-type) K(+) channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed.     

Endogenous cannabinoid receptor agonist anandamide induces peripheral antinociception by activation of ATP-sensitive K+ channels

AimsThe effects of several potassium (K⁺) channel blockers were studied to determine which K⁺ channels are involved in peripheral antinociception induced by the cannabinoid receptor agonist, anandamide.Main methodsHyperalgesia was induced by subcutaneous injection of 250 μg carrageenan into the plantar surface of the hind paw of rats. The extent of hyperalgesia was measured using a paw pressure test 3 h following carrageenan injection. The weight in grams (g) that elicited a nociceptive response, paw flexion, during the paw pressure test was used as the nociceptive response threshold.Key findingsDoses of 50, 75, and 100 ng of anandamide elicited a dose-dependent antinociceptive effect. Following a 100 ng dose of anandamide no antinociception was observed in the paw that was contralateral to the anandamide injection site, which shows that anandamide has a peripheral site of action. Pretreatment with 20, 40 and 80 μg AM251, a CB₁ receptor antagonist, caused a dose-dependent decrease in anandamide-induced antinociception, suggesting that the CB₁ receptor is directly involved in anandamide effect. Treatment with 40, 80 and 160 μg glibenclamide, an ATP-sensitive K⁺ channel blocker, caused a dose-dependent reversal of anandamide-induced peripheral antinociception. Treatment with other K⁺ channel antagonists, tetraethylammonium (30 μg), paxilline (10 μg) and dequalinium (50 μg), had no effect on the induction of peripheral antinociception by anandamide.SignificanceThis study provides evidence that the peripheral antinociceptive effect of the cannabinoid receptor agonist, anandamide, is primarily caused by activation of ATP-sensitive K⁺ channels and does not involve other potassium channels.     

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.     

Syntaxin-1A interacts with distinct domains within nucleotide-binding folds of sulfonylurea receptor 1 to inhibit beta-cell ATP-sensitive potassium channels

The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W(A)), signature sequence linker, and Walker B (W(B)), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W(A) and W(B) regions of both NBFs. Patch clamp recordings on INS-1 and primary rat β-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W(B) (not NBF1-W(A)), NBF2-W(A), and NBF2-W(B). The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W(A) (K719M) or NBF2-W(A) (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K(ATP) channels. These data suggest that NBF1-W(A) (Lys-719) and NBF2-W(A) (Lys-1385) are critical for Syn-1A-K(ATP) channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W(A) motifs and sites adjacent to W(B) motifs of NBF1 and NBF2 but transduces its inhibitory actions on K(ATP) channel activity via some but not all of these NBF domains.     

AMP-activated protein kinase mediates preconditioning in cardiomyocytes by regulating activity and trafficking of sarcolemmal ATP-sensitive K(+) channels

Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that is complex and yet to be fully defined. AMP-activated kinase (AMPK) is activated in cells under conditions associated with ATP depletion and increased AMP/ATP ratio. In the present study, we have taken advantage of a cardiac phenotype overexpressing a dominant negative form of the alpha2 subunit of AMPK to analyze the role, if any, that AMPK plays in preconditioning the heart. We have found that myocardial preconditioning activates AMPK in wild type, but not transgenic mice. Cardiac cells from transgenic mice could not be preconditioned, as opposed to cells from the wild type. The cytoprotective effect of AMPK was not related to the effect that preconditioning has on mitochondrial membrane potential as revealed by JC-1, a mitochondrial membrane potential-sensitive dye, and laser confocal microscopy. In contrast, experiments with di-8-ANEPPS, a sarcolemmal-potential sensitive dye, has demonstrated that intact AMPK activity is required for preconditioning-induced shortening of the action membrane potential. The preconditioning-induced activation of sarcolemmal K(ATP) channels was observed in wild type, but not in transgenic mice. HMR 1098, a selective inhibitor of sarcolemmal K(ATP) channels opening, inhibited preconditioning-induced shortening of action membrane potential as well as cardioprotection afforded by AMPK. Immunoprecipitation followed by Western blotting has shown that AMPK is essential for preconditioning-induced recruitment of sarcolemmal K(ATP) channels. Based on the obtained results, we conclude that AMPK mediates preconditioning in cardiac cells by regulating the activity and recruitment of sarcolemmal K(ATP) channels without being a part of signaling pathway that regulates mitochondrial membrane potential.     

Single cardiac outwardly rectifying K+ channels modulated by protein kinase A and a G-protein

Elementary K⁺ currents were recorded at 19 °C in cell-attached and in inside-out patches excised from neonatal rat heart myocytes. An outwardly rectifying K⁺ channel which prevented Na⁺ ions from permeating could be detected in about 10% of the patches attaining (at 5 mmol/l external K⁺ and between – 20 mV and + 20 mV) a unitary conductance of 66 +- 3.9 pS. K _{(outw.-rect.)} ⁺ channels have one open and at least two closed states. Open probability and _open rose steeply on shifting the membrane potential in the positive direction, thereby tending to saturate. Open probability (at –7 mV) was as low as 3 ± 1% but increased several-fold on exposing the cytoplasmic surface to Mg-ATP (100 mol/l) without a concomitant change of _open. No channel activation occurred in response to ATP in the absence of cytoplasmic Mg^–+. The cytoplasmic administration of the catalytic subunit of protein kinase A (120–150 /ml) or GTP--S (100 mol/l) caused a similar channel activation. GDP--S (100 mol/l) was also tested and found to be ineffective in this respect. This suggests that cardiac K _{(outw.-rect.)} ⁺ channels are metabolically modulated by both cAMP-dependent phosphorylation and a G-protein. Offprint requests to: M. Kohlhardt     

Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis

The ability of glycolysis, oxidative phosphorylation, the creatine kinase system, and exogenous ATP to suppress ATP-sensitive K+ channels and prevent cell shortening were compared in patch-clamped single guinea pig ventricular myocytes. In cell-attached patches on myocytes permeabilized at one end with saponin, ATP-sensitive K+ channels were activated by removing ATP from the bath, and could be closed equally well by exogenous ATP or substrates for endogenous ATP production by glycolysis (with the mitochondrial inhibitor FCCP present), mitochondrial oxidative phosphorylation, or the creatine kinase system. In the presence of an exogenous ATP-consuming system, however, glycolytic substrates (with FCCP present) were superior to substrates for either oxidative phosphorylation or the creatine kinase system at suppressing ATP-sensitive K+ channels. All three groups of substrates were equally effective at preventing cell shortening. In 6 of 38 excised inside-out membrane patches, ATP-sensitive K+ channels activated by removing ATP from the bath were suppressed by a complete set of substrates for the ATP-producing steps of glycolysis but not by individual glycolytic substrates, which is consistent with the presence of key glycolytic enzymes located near the channels in these patches. Under whole-cell voltage-clamp conditions, inclusion of 15 mM ATP in the patch electrode solution dialyzing the interior of the cell did not prevent activation of the ATP-sensitive K+ current under control conditions or during exposure to complete metabolic inhibition. In isolated arterially perfused rabbit interventricular septa, selective inhibition of glycolysis caused an immediate increase in 42K+ efflux rate, which was prevented by 100 microM glyburide, a known blocker of ATP-sensitive K+ channels. These observations suggest that key glycolytic enzymes are associated with cardiac. ATP-sensitive K+ channels and under conditions in which intracellular competition for ATP is high (e.g., in beating heart) that act as a preferential source of ATP for these channels.