An adenylate kinase is involved in K<Subscript>ATP</Subscript> channel regulation of mouse pancreatic beta cells

Aims/hypothesis In a previous study, we demonstrated that a creatine kinase (CK) modulates K_ATP channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether K_ATP channel regulation in beta cells is determined by a metabolic interaction between adenylate kinase (AK) and CK. Methods Single channel activity was measured with the patch–clamp technique in the inside-out (i/o) and open-cell attached (oca) configuration. Results The ATP sensitivity of K_ATP channels was higher in i/o patches than in permeabilised beta cells (oca). One reason for this observation could be that the local ATP:ADP ratio in the proximity of the channels is determined by factors not active in i/o patches. AMP (0.1 mmol/l) clearly increased open channel probability in the presence of ATP (0.125 mmol/l) in permeabilised cells but not in excised patches. This suggests that AK-catalysed ADP production in the vicinity of the channels is involved in K_ATP channel regulation. The observation that the stimulatory effect of AMP on K_ATP channels was prevented by the AK inhibitor P ¹,P ⁵-di(adenosine-5′)pentaphosphate (Ap₅A; 20 μmol/l) and abolished in the presence of the non-metabolisable ATP analogue adenosine 5′-(β,γ-imido)triphosphate tetralithium salt (AMP-PNP; 0.12 mmol/l) strengthens this idea. In beta cells from AK1 knockout mice, the effect of AMP was less pronounced, though not completely suppressed. The increase in K_ATP channel activity induced by AMP in the presence of ATP was outweighed by phosphocreatine (1 mmol/l). We suggest that this is due to an elevation of the ATP concentration by CK. Conclusions/interpretation We propose that phosphotransfer events mediated by AK and CK play an important role in determining the effective concentrations of ATP and ADP in the microenvironment of pancreatic beta cell K_ATP channels. Thus, these enzymes determine the open probability of K_ATP channels and eventually the actual rate of insulin secretion.     

Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: indications for a long-chain acyl-CoA ester binding motif

Aims/hypothesis The pancreatic beta cell ATP-sensitive potassium (K_ATP) channel, composed of the pore-forming α subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory β subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K_ATP channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. Materials and methods Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. Results Oocytes expressing Kir6.2ΔC26 K332A repeatedly generated K⁺currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 μmol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2ΔC26), Kir6.2ΔC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2ΔC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K_ATP channel and Kir6.2ΔC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. Conclusions/interpretation These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.     

Potent and selective activation of the pancreatic beta-cell type KATP channel by two novel diazoxide analogues

Aims/hypothesis We investigated the pharmacological properties of two novel ATP sensitive potassium (K_ATP) channel openers, 6-Chloro-3-isopropylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC 55-0118) and 6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414), on the cloned cardiac (Kir6.2/SUR2A), smooth muscle (Kir6.2/SUR2B) and pancreatic beta cell (Kir6.2/SUR1) types of K_ATP channel.Methods We studied the effects of these compounds on whole-cell currents through cloned K_ATP channels expressed in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes.Results In HEK 293 cells, NNC 55-0118 and NN414 activated Kir6.2/SUR1 currents with EC₅₀ values of 0.33 µmol/l and 0.45 µmol/l, respectively, compared with that of 31 µmol/l for diazoxide. Neither compound activated Kir6.2/SUR2A or Kir6.2/SUR2B channels expressed in oocytes, nor did they activate Kir6.2 expressed in the absence of SUR. Current activation was dependent on the presence of intracellular MgATP, but was not supported by MgADP.Conclusion/interpretation Both NNC 55-0118 and NN414 selectively stimulate the pancreatic beta-cell type of K_ATP channel with a higher potency than diazoxide, by interaction with the SUR1 subunit. The high selectivity and efficacy of the compounds could prove useful for treatment of disease states where inhibition of insulin secretion is beneficial.Abbreviations K_ATP channel ATP-sensitive potassium channel - SUR sulphonylurea receptor - KCO K⁺ channel opener - Kir inwardly rectifying K⁺ channel - TEVC two electrode voltage clamp - HEK293 cell Human Embryonic Kidney 293 cell     

Gliclazide produces high-affinity block of KATP channels in mouse isolated pancreatic beta cells but not rat heart or arterial smooth muscle cells

Aims/hypothesis: Sulphonylureas stimulate insulin secretion by closing ATP-sensitive potassium (K_ATP) channels in the pancreatic beta-cell membrane. K_ATP channels are also found in other tissues, including heart and smooth muscle, where they link cellular metabolism to electrical activity. The sulphonylurea gliclazide blocks recombinant beta-cell K_ATP channels (Kir6.2/SUR1) but not heart (Kir6.2/SUR2A) or smooth muscle (Kir6.2/SUR2B) K_ATP channels with high potency. In this study, we examined the specificity of gliclazide for the native (as opposed to recombinant) K_ATP channels in beta cells, heart and smooth muscle. Methods: The action of the drug was studied by whole-cell current recordings of native K_ATP channels in isolated pancreatic beta-cells and myocytes from heart and smooth muscle. Results: Gliclazide blocked whole-cell beta-cell K_ATP currents with an IC ₅₀ of 184 ± 30 nmol/l (n = 6–10) but was much less effective in cardiac and smooth muscle (IC ₅₀s of 19.5 ± 5.4 μmol/l (n = 6–12) and 37.9 ± 1.0 μmol/l (n = 5–10), respectively). In all three tissues, the action of the drug on whole-cell K_ATP currents was rapidly reversible. In inside-out patches on beta-cells, gliclazide (1 μmol/l) produced a maximum of 66 ± 13 % inhibition (n = 5), compared with more than 98 % block in the whole-cell configuration. Conclusion/interpretation: Gliclazide is a high-potency sulphonylurea which shows specificity for the pancreatic beta-cell K_ATP channel over heart and smooth muscle. In this respect, it differs from glibenclamide. The difference in the maximal block observed in the excised patch and whole-cell recordings from beta-cells, may be due to the absence of intracellular Mg-nucleotides in the excised patch experiments. [Diabetologia (2001) 44: 1019–1025] Received: 21 March 2001 and in revised form: 30 April 2001     

Characterisation of new K<Subscript>ATP</Subscript>-channel mutations associated with congenital hyperinsulinism in the Finnish population

Aims/hypothesis ATP-sensitive potassium (K_ATP) channels are crucial for the regulation of insulin secretion from pancreatic beta cells and mutations in either the Kir6.2 or SUR1 subunit of this channel can cause congenital hyperinsulinism (CHI). The aim of this study was to analyse the functional consequences of four CHI mutations (A1457T, V1550D and L1551V in SUR1, and K67N in Kir6.2) recently identified in the Finnish population.Methods Wild type or mutant Kir6.2 and SUR1 subunits were coexpressed in Xenopus oocytes. The functional properties of the channels were examined by measuring currents in intact oocytes or giant inside-out membrane patches. Surface expression was measured by enzyme-linked immunosorbance assay, using HA-epitope-tagged subunits.Results Two mutations (A1457T and V1550D) prevented trafficking of the channel to the plasma membrane. The L1551V mutation reduced surface expression 40-fold, and caused loss of MgADP and diazoxide activation. Both these factors will contribute to the lack of K_ATP current activation observed in response to metabolic inhibition in intact oocytes. The L1551V mutation also increased the channel open probability, thereby producing a reduction in ATP-sensitivity (from 10 µmol/l to 120 µmol/l). The fourth mutation (K67N mutation in Kir6.2) did not affect surface expression nor alter the properties of K_ATP channels in excised patches, but resulted in a reduced K_ATP current amplitude in intact cells on metabolic inhibition, through an unidentified mechanism.Conclusion/interpretation The four CHI mutations disrupted K_ATP channel activity by different mechanisms. Our results are discussed in relation to the CHI phenotype observed in patients with these mutations.Abbreviations CHI Congenital hyperinsulinism - HA haemagluttinin - K_ATP channel ATP-sensitive potassium channel - Po open probability - PIP₂ phosphatidyl inositol bis-phosphate - RLU relative light units - SUR sulphonylurea receptor - UTR untranslated region An erratum to this article can be found at     

Effect of repaglinide on cloned beta cell, cardiac and smooth muscle types of ATP-sensitive potassium channels

Aims/hypothesis. The carbamoylbenzoic acid derivative repaglinide is a potent short-acting insulin secretagogue that acts by closing ATP-sensitive potassium (K_ATP) channels in the plasma membrane of the pancreatic beta cell. In this paper we investigated the specificity of repaglinide for three types of cloned (K_ATP) channel composed of the inwardly rectifying potassium channel Kir6.2 and either the sulphonylurea receptor SUR1, SUR2A or SUR2B, corresponding to the beta cell, cardiac and either smooth muscle types of K_ATP channel, respectively. Methods. The action of the drug was studied by whole-cell current recordings of K_ATP channels expressed either in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes for detailed analysis of repaglinide action. Results. The drug blocked all three types of K_ATP channel with similar potency, by interacting with a low-affinity site on the pore-forming subunit of the channel (Kir6.2: half-maximal inhibition 230 μmol/l) and with a high-affinity site on the regulatory subunit, the sulphonylurea receptor (SUR: half-maximal inhibition 2–8 nmol/l). There was no difference in potency between channels containing SUR1, SUR2A or SUR2B. MgADP potentiated the inhibitory effect of repaglinide on Kir6.2/SUR1 and (to a lesser extent) Kir6.2/SUR2B, but not on Kir6.2/SUR2A. Conclusion/interpretation. Repaglinide interacts with a site common to all three types of sulphonylurea receptor leading to inhibition of the K_ATP channel. The fact that MgADP potentiated this effect in the case of the beta cell, but not cardiac, type of channel could help explain why the drug shows no adverse cardiovascular side-effects in vivo. [Diabetologia (2001) 44: 747–756] Received: 13 December 2000 and in revised form: 14 February 2001     

Functional Communication Between Cardiac ATPSensitive K+ Channel and Na/K ATPase

K_ATP Channel and Na/K ATPase. Introduction: Functional interaction between K_ATP channel and Na/K ATPase was studied in single guinea pig ventricular myocytes because both membrane molecules are known to he involved in ischemic episodes. Methods and Results: K_ATP channel currents were recorded at 36°C by using whole cell, cell attached, inside-out, and open cell-attached modes of patch clamp techniques on enzymatically isolated ventricular myocytes. In the whole cell mode, ouabain (1 μM) reversibly inhibited the K_ATP currents induced by metabolic stress (ATP-free pipette solution and 1 mM NaCN), but not those activated by cromakalim (100 μM), a K_ATP channel opener. In the cell-attached mode, ouabain concentration dependently inhibited K_ATP, channel opening induced by metabolic suppression (5.5 μM 2-deoxyglucose and 1 mM CN). Half-inhibition concentration for ouabain was 21.0 ± 5.5 nM and the Hill coefficient was 0.8 ± 0.1 (n = 26). However, ouabain did not have an effect on the channel activity induced by cromakalim (100 μM). In the inside-out mode, ouabain applied to the internal side of membrane did not affect the channel. In the open cell-attached mode made by preincubation with streptolysin-0 (0.08 U/mL), the K_ATP channels were not activated by the metabolic inhibitors but were by reducing extracellular ATP concentrations, because subsarcolenimal ATP concentration could he controlled through tiny membrane holes. The channels thus activated were not suppressed by ouabain. Conclusion: The inhibition of Na/K ATPase by ouahain appeared to block the K_ATP channels by accumulating subsarcolemmal ATP caused by a decrease of the transition from ATP to ADP. In the presence of ischemic episodes, the administration of digitalis compounds may affect the opening of K_ATP channels, which is primarily protective against the development of irreversible myocardial damage.     

The ATP- and tolbutamide-sensitivity of the ATP-sensitive K-channel from human pancreatic B cells

Summary The ATP- and sulphonylurea-sensitivity of the ATP-sensitive K-channel was measured in human pancreatic B cells. In inside-out patches, half-maximal inhibition of channel activity was produced by 10 mol/l ATP (with 2 mM Mg²⁺) and ATP-inhibition was partially antagonised by ADP. A significantly lower sensitivity to ATP was found in whole-cell recordings. Tolbutamide inhibited whole-cell ATP-sensitive K-currents half-maximally at 18 mol/l; the sensitivity to tolbutamide was somewhat less in the inside-out patch. Ca-activated K-channels were unaffected by tolbutamide (10 mmol/l). These results resemble those found for rodent B cells and suggest that sulphonylureas exert their therapeutic effects in Type 2 (non-insulin dependent) diabetes by inhibition of the ATP-sensitive K-channel.     

Leptin-stimulated KATP channel trafficking

Insulin secretion from pancreatic β-cells is initiated by the closure of ATP-sensitive K⁺ channels (K_ATP) in response to high concentrations of glucose, and this action of glucose is counteracted by the hormone leptin, an adipokine that signals through the Ob-R_b receptor to increase K_ATP channel activity. Despite intensive investigations, the molecular basis for K_ATP channel regulation remains uncertain, particularly from the standpoint of whether fluctuations in plasma membrane K_ATP channel content underlie alterations of K_ATP channel activity in response to glucose or leptin. Surprisingly, newly published findings reveal that leptin stimulates AMP-activated protein kinase (AMPK) in order to promote trafficking of K_ATP channels from cytosolic vesicles to the plasma membrane of β-cells. This action of leptin is mimicked by low concentrations of glucose that also activate AMPK and that inhibit insulin secretion. Thus, a new paradigm for β-cell stimulus-secretion coupling is suggested in which leptin exerts a tonic inhibitory effect on β-cell excitability by virtue of its ability to increase plasma membrane K_ATP channel density and whole-cell K_ATP channel current. One important issue that remains unresolved is whether high concentrations of glucose suppress AMPK activity in order to shift the balance of membrane cycling so that K_ATP channel endocytosis predominates over vesicular K_ATP channel insertion into the plasma membrane. If so, high concentrations of glucose might transiently reduce K_ATP channel density/current, thereby favoring β-cell depolarization and insulin secretion. Such an AMPK-dependent action of glucose would complement its established ability to generate an increase of ATP/ADP concentration ratio that directly closes K_ATP channels in the plasma membrane.     

Disopyramide and its metabolite enhance insulin release from clonal pancreatic beta-cells by blocking K(ATP) channels

In an insulin-secreting pancreatic -cell line (MIN6), insulin release was caused by disopyramide, an antiarrhythmic drug with Na-channel blocking action, and its main metabolite mono-isopropyl disopyramide (MIP). Insulin secretion, measured as immunoreactive insulin (IRI), was accelerated to 265.7% of the control by disopyramide and to 184.4% by MIP, with half-effective concentrations (EC₅₀) of 30.9 ± 1.5 M and 92.4 ± 2.2 M. We tested the possibility that these drugs induce insulin release by inhibiting ATP-sensitive K⁺ (K_ATP) channels of MIN6 cells. In the cell-attached or ATP-free inside-out mode with patch membranes on MIN6 cells, K-selective channels were recorded with unitary conductance of 70.5 ± 3.5 pS (150 mM external K⁺ ions at room temperature). The channels were concluded to be MIN6-K_ATP channels because they were closed by extracellular high glucose (11.0 mM) or glibenclamide (200 nM) and were reversibly activated by diazoxide (50 M). In the inside-out patch mode, they were inhibited by micromolar ATP. In both cell-attached and insideout mode, disopyramide and MIP inhibited single MIN6-K_ATP channels. In the inside-out mode, they produced a dose-dependent inhibition of channel activity: the half-blocking concentrations (IC⁵⁰) were 4.8 ± 0.2 M for disopyramide and 40.4 ± 3.1 M for MIP. It was therefore concluded that both agents exert insulinotrphic effect through the inhibition of membrane K_ATP channels in MIN6 cells.     

Differential sensitivity of beta-cell and extrapancreatic KATP channels to gliclazide

Aims/hypothesis. To investigate the tissue specificity of gliclazide for cloned beta-cell, cardiac and smooth muscle ATP-sensitive K-channels (K_ATP channels). These channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart, SUR2B in smooth muscle). Methods. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes, and channel activity was measured by recording macroscopic currents in giant inside-out membrane patches. Gliclazide was added to the intracellular membrane surface. Results. We reported previously that Kir6.2-SUR1 currents are blocked at two sites by tolbutamide: a high-affinity site on SUR1 and a low-affinity site on Kir6.2. We now show that gliclazide also inhibits beta-cell K_ATP channels at two sites: a high-affinity site, which is half-maximally blocked (K _i) at 50 ± 7 nmol/l (n = 8) and a low-affinity site with a K _i of 3.0 ± 0.6 mmol/l (n = 4). The high-affinity site on SUR1 was thus about 40-fold more sensitive to gliclazide than to tolbutamide (K _i∼ 2 μmol/l). Cloned cardiac and smooth muscle K_ATP channels did not show high-affinity block by gliclazide. Kir6.2-SUR2A currents exhibited a single low-affinity site with a K _i of 0.8 ± 0.1 mmol/l (n = 5), which is likely to reside on the Kir6.2 subunit. Conclusion/interpretation. Our results show that gliclazide is a sulphonylurea with high affinity and strong selectivity for the beta-cell type of K_ATP channel. [Diabetologia (1999) 42: 845–848] Received: 20 January 1999 and in final revised form: 4 March 1999     

Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in the ATP:ADP ratio in rat pancreatic islets

Aims/hypothesis The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion.Methods Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K⁺ channel (K_ATP-channel) conductance.Results We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca²⁺-induced exocytosis and K_ATP-channel conductance in single cells, were unaffected. Insulin secretion induced by α-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K_ATP-independent actions of fuel secretagogues are operational, as well as the first and K_ATP-dependent phase.Conclusions/interpretation Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.     

Tolbutamide potentiates the volume-regulated anion channel current in rat pancreatic beta cells

Aims/hypothesis Hypoglycaemic sulphonylureas are thought to stimulate insulin release by binding to a sulphonylurea receptor, closing K_ATP channels and inducing electrical activity. However, the fact that these drugs stimulate insulin release at high glucose concentrations where K_ATP channels are closed suggests additional ionic actions. The aim of this study was to test the hypothesis that sulphonylureas influence the current of the glucose- and volume-regulated anion channel.Methods Electrical and ion-channel activity were recorded in isolated rat beta cells using the patch-clamp technique. ⁸⁶Rb⁺ efflux was measured using intact islets. Beta cell volume was measured using a video-imaging technique.Results In the absence of glucose, tolbutamide (100 µmol/l) transiently depolarised the cells. In the presence of glucose (5 mmol/l), tolbutamide evoked a sustained period of electrical activity, whilst at 10 mmol/l glucose, the drug evoked a pronounced silent depolarisation. In the absence of glucose, tolbutamide inhibited ⁸⁶Rb⁺ efflux. However, at 10 mmol/l glucose, tolbutamide induced a transient stimulation of efflux. Tolbutamide potentiated the whole-cell volume-regulated anion conductance in a glucose-dependent manner with an EC₅₀ of 85 µmol/l. In single channel recordings, tolbutamide increased the channel-open probability. Tolbutamide caused beta cell swelling in the presence of glucose, but not in its absence.Conclusions/interpretation Tolbutamide can induce beta cell electrical activity by potentiating the glucose- and volume-regulated anion channel current. This effect is probably not due to a direct effect of the drug on the channel, but could be secondary to a metabolic action in the beta cell.     

Selectivity of repaglinide and glibenclamide for the pancreatic over the cardiovascular KATP channels

Aims/hypothesis Sulfonylureas and glinides close beta cell ATP-sensitive K⁺ (K_ATP) channels to increase insulin release; the concomitant closure of cardiovascular K_ATP channels, however, leads to complications in patients with cardiac ischaemia. The insulinotrope repaglinide is successful in therapy, but has been reported to inhibit the recombinant K_ATP channels of beta cells, cardiocytes and non-vascular smooth muscle cells with similar potencies, suggesting that the (patho-)physiological role of the cardiovascular K_ATP channels may be overstated. We therefore re-examined repaglinide’s potency at and affinity for the recombinant pancreatic, myocardial and vascular K_ATP channels in comparison with glibenclamide.Methods K_ATP channel subunits (i.e. inwardly rectifying K⁺ channels [Kir6.x] and sulfonylurea receptors [SURx]) were expressed in intact human embryonic kidney cells and assayed in whole-cell patch-clamp and [³H]glibenclamide binding experiments at 37°C.Results Repaglinide and glibenclamide, respectively, were ≥30 and ≥1,000 times more potent in closing the pancreatic than the cardiovascular channels and they did not lead to complete inhibition of the myocardial channel. Binding assays showed that the selectivity of glibenclamide was essentially based on high affinity for the pancreatic SUR, whereas binding of repaglinide to the SUR subtypes was rather non-selective. After coexpression with Kir6.x to form the assembled channels, however, the affinity of the pancreatic channel for repaglinide was increased 130-fold, an effect much larger than with the cardiovascular channels. This selective effect of coexpression depended on the piperidino substituent in repaglinide.Conclusions/interpretation Repaglinide and glibenclamide show higher potency and efficacy in inhibiting the pancreatic than the cardiovascular K_ATP channels, thus supporting their clinical use.The first two authors listed contributed equally to this work.     

Pancreatic β-cell K<Subscript>ATP</Subscript> channels: Hypoglycaemia and hyperglycaemia

The pancreatic β-cell ATP-sensitive K⁺ channel (K_ATP channel) plays a critical role in glucose homeostasis by linking glucose metabolism to electrical excitability and insulin secretion. Changes in the intracellular ratio of ATP/ADP mediate the metabolic regulation of channel activity. The β-cell K_ATP channel is a hetero-octameric complex composed of two types of subunits: four inward-rectifying potassium channel pore-forming (Kir6.2) subunits and four high-affinity sulfonylurea receptor 1 (SUR1) subunits. Kir6.2 and SUR1 are encoded by the genes KCNJ11 and ABCC8, respectively. Mutations in these genes can result in congenital hyperinsulinism and permanent neonatal diabetes. This review highlights the important role of the β-cell K_ATP channel in glucose physiology and provides an introduction to some of the other review articles in this special edition of the Reviews in Endocrine and Metabolic Disorders.     

Vascular ATP-Dependent Potassium Channels, Nitric Oxide, and Human Forearm Reactive Hyperemia

Vascular ATP-dependent potassium (K⁺ _ATP) channels open and contribute to reactive hyperemia (RH) in animals. The contribution of K⁺ _ATP channels to ischemic vasolidation during RH and interactions with endothelium-derived nitric oxide have not been well characterized in human subjects. RH blood flow responses (mL/dL) following 5 minutes of cuff occlusion were measured using strain-gauge plethysmography in 22 normal human subjects age 42 ± 2 years. Measurements were obtained at baseline and following intra-arterial administration of the K⁺ _ATP channel closer glibenclamide, the nitric oxide synthase inhibitor L-N-monomethyl arginine (L-NMMA), or both drugs simultaneously. Glibenclamide (100 µg/min) did not change basal flow (2.7 ± 0.3 to 2.7 ± 0.3 mL/min/dL), but L-NMMA (8 µmol/min) and combined glibenclamide and L-NMMA significantly (p < 0.05) decreased basal flow (3.0 ± 0.5 to 2.0 ± 0.2 and 3.3 ± 0.5 to 2.5 ± 0.3, respectively). Glibenclamide significantly (p < 0.01) decreased RH flow (18.2 ± 1.3 to 14.8 ± 1.3) and excess flow (5.3 ± 1.2 to 1.3 ± 1.3). L-NMMA significantly (p < 0.05) decreased RH flow (21.2 ± 1.8 to 18.9 ± 1.9) and tended to decrease excess flow (6.1 ± 2.2 to 3.9 ± 2.5). Combined drug infusion significantly (p < 0.1) decreased RH flow (21.6 ± 2.2 to 18.0 ± 2.4) and excess flow (6.3 ± 1.6 to 1.6 ± 1.6), with reductions in RH and excess flow similar to those following glibenclamide infusion alone. We conclude that forearm vascular K⁺ _ATP channels are closed at baseline. They open and contribute to RH vasodilation. The addition of nitric oxide inhibition to K⁺ _ATP channel blockade does not result in additive or synergistic inhibition of RH.     

Infarct Size Limitation by Bradykinin Receptor Activation Is Mediated by the Mitochondrial But Not by the Sarcolemmal KATP Channel

Earlier studies have shown that activation of bradykinin B₂ receptor triggers protein kinase C (PKC)-mediated cardioprotective mechanism in ischemic preconditioning (PC). In the present study, we examined whether the effector in this B₂-receptor triggered pathway of PC is the ATP sensitive potassium (K_ATP) channel in the mitochondria (mito-K_ATP channel) or K_ATP channel in the sarcolemma (sarc-K_ATP channel). Isolated rabbit hearts were perfused with modified Krebs-Henseleit buffer in a Langendorff mode, and regional myocardial ischemia was induced by occluding a left coronary artery for 30 min and then reperfusing for 2 hours. Infarct size was determined by triphenyltetrazolium chloride staining and expressed as a percentage of area at risk (% IS/AR). Infusion of bradykinin (500 nmol/L) for 15 min prior to ischemia significantly reduced % IS/AR from 37.4 ± 2.9 (SE) of the untreated controls to 12.0 ± 3.3%. This protective effect of bradykinin was completely abolished by coinfusion of 5-hydroxydecanoate (5-HD, 50 mol/L), a selective mito-K_ATP channel blocker (% IS/AR = 44.2 ± 6.4). In contrast, a high dose of HMR1098 (20 mol/L), which is a newly developed sarc-K_ATP channel selective blocker with IC₅₀ of 0.6 mol/L, failed to modify the infarct size limitation by preischemic infusion of bradykinin (% IS/AR = 11.7 ± 3.4). Neither 5-HD nor HMR1098 alone modified infarct size (% IS/AR = 37.8 ± 3.8 and 35.1 ± 6.2, respectively). These results suggest that opening of the mito-K_ATP channel but not the sarc-K_ATP channel is involved in infarct size limitation by a mechanism triggered by bradykinin B₂ receptor activation.     

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel

ATP-sensitive K⁺ (K_ATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of K_ATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the K_ATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. K_ATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the K_ATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.     

Sulphonylurea action revisited: the post-cloning era

Hypoglycaemic agents such as sulphonylureas and the newer group of "glinides" stimulate insulin secretion by closing ATP-sensitive potassium (K_ATP) channels in pancreatic beta cells, but have varying cross-reactivity with related channels in extrapancreatic tissues such as heart, vascular smooth and skeletal muscle. Experiments on the structure-function relationships of recombinant K_ATP channels and the phenotypes of mice deficient in different K_ATP channel subunits have provided important insights into the mechanisms underlying sulphonylurea selectivity, and the potential consequences of K_ATP channel blockade outside the pancreatic beta cell. The different pharmacological properties of K_ATP channels from beta cells compared with those from cardiac, smooth and skeletal muscle, are accounted for by the expression of alternative types of sulphonylurea receptor, with non-identical drug binding sites. The sulphonylureas and glinides are found to fall into two groups: one exhibiting selectivity for beta cell sulphonylurea receptors (SUR1), and the other blocking cardiovascular and skeletal muscle sulphonylurea receptors (SUR2) with potencies similar to their action on SUR1. In seeking potential side effects of K_ATP channel inhibitors in humans, it is essential to take these drug differences into account, along with the probability (suggested by the studies on K_ATP channel knockout mice) that the effects of extrapancreatic K_ATP channel inhibition might be either subtle or rare. Further studies are still required before a final decision can be made on whether non-selective agents are appropriate for the therapy of Type 2 diabetes.Abbreviations K_ATP channel ATP sensitive potassium channel - Kir inwardly-rectifying potassium channel - SUR sulphonylurea receptor - TMD transmembrane domain - NBD nucleotide binding domain - CL cytoplasmic linker - CHI congenital hyperinsulinism     

Glucose modulation of ATP-sensitive K-currents in wild-type, homozygous and heterozygous glucokinase knock-out mice

Summary One type of maturity-onset diabetes of the young (MODY2) is caused by mutations in the glucokinase gene, a key glycolytic enzyme in the beta cell and liver. Glucose fails to stimulate insulin secretion in mice in which the glucokinase gene has been selectively knocked out in the beta cell. We tested the hypothesis that this effect results from defective metabolic regulation of beta cell ATP-sensitive potassium (K_ATP) channels. Glucose had little effect on K_ATP currents in homozygous (-/-) mice but inhibited K_ATP currents in wild-type (+/+) and heterozygous ( + /-) mice with EC₅₀ of 3.2 mM and 5.5 mM, respectively, in newborn animals, and of 4.7 mM and 9.9 mM, respectively, in 1.5-year-old mice. Glucose (20 mmol/l) did not affect the resting membrane potential of -/- beta cells but depolarised wild-type and + /- beta cells and induced electrical activity. In contrast, 20 mmol/l ketoisocaproic acid or 0.5 mmol/l tolbutamide depolarised all three types of beta-cell. These results support the idea that defective glycolytic metabolism, produced by a loss (-/- mice) or reduction ( + /- mice) of glucokinase activity, leads to defective K_ATP channel regulation and thereby to the selective loss, or reduction, of glucose-induced insulin secretion. [Diabetologia (1998) 41: 654–659] Received: 22 October 1997 and in revised form 9 January 1998