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     

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     

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     

HMR 1098 is not an SUR isotype specific inhibitor of heterologous or sarcolemmal K ATP channels

Murine ventricular and atrial ATP-sensitive potassium (K_ATP) channels contain different sulfonylurea receptors (ventricular K_ATP channels are Kir6.2/SUR2A complexes, while atrial K_ATP channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) K_ATP channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular K_ATP channels over atrial K_ATP channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as ⁸⁶Rb⁺ efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated K_ATP currents were effectively inhibited by 10 μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated K_ATP currents were inhibited by HMR 1098 at a high concentration (100 μM) but not at a low concentration (10 μM). Consistent with this finding, HMR 1098 inhibits ⁸⁶Rb⁺ effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed K_ATP channels.     

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.     

Hyperinsulinism in mice with heterozygous loss of KATP channels

Aims/hypothesis ATP-sensitive K⁺ (K_ATP) channels couple glucose metabolism to insulin secretion in pancreatic beta cells. In humans, loss-of-function mutations of beta cell K_ATP subunits (SUR1, encoded by the gene ABCC8, or Kir6.2, encoded by the gene KCNJ11) cause congenital hyperinsulinaemia. Mice with dominant-negative reduction of beta cell K_ATP (Kir6.2[AAA]) exhibit hyperinsulinism, whereas mice with zero K_ATP (Kir6.2^−/−) show transient hyperinsulinaemia as neonates, but are glucose-intolerant as adults. Thus, we propose that partial loss of beta cell K_ATP in vivo causes insulin hypersecretion, but complete absence may cause insulin secretory failure. Materials and methods Heterozygous Kir6.2^+/− and SUR1^+/− animals were generated by backcrossing from knockout animals. Glucose tolerance in intact animals was determined following i.p. loading. Glucose-stimulated insulin secretion (GSIS), islet K_ATP conductance and glucose dependence of intracellular Ca²⁺ were assessed in isolated islets. Results In both of the mechanistically distinct models of reduced K_ATP (Kir6.2^+/− and SUR1^+/−), K_ATP density is reduced by ∼60%. While both Kir6.2^−/− and SUR1^−/− mice are glucose-intolerant and have reduced glucose-stimulated insulin secretion, heterozygous Kir6.2^+/− and SUR1^+/− mice show enhanced glucose tolerance and increased GSIS, paralleled by a left-shift in glucose dependence of intracellular Ca²⁺ oscillations. Conclusions/interpretation The results confirm that incomplete loss of beta cell K_ATP in vivo underlies a hyperinsulinaemic phenotype, whereas complete loss of K_ATP underlies eventual secretory failure.     

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     

Phentolamine block of KATP channels is mediated by Kir6.2

The ATP-sensitive K⁺-channel (K_ATP channel) plays a key role in insulin secretion from pancreatic β cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca²⁺ entry and insulin release. The β cell K_ATP channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K⁺-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit K_ATP channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. K_ATP channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac K_ATP channel.     

Vascular KATP channel structural dynamics reveal regulatory mechanism by Mg-nucleotides

Vascular tone is dependent on smooth muscle K_ATP channels comprising pore-forming Kir6.1 and regulatory SUR2B subunits, in which mutations cause Cantú syndrome. Unique among K_ATP isoforms, they lack spontaneous activity and require Mg-nucleotides for activation. Structural mechanisms underlying these properties are unknown. Here, we determined cryogenic electron microscopy structures of vascular K_ATP channels bound to inhibitory ATP and glibenclamide, which differ informatively from similarly determined pancreatic K_ATP channel isoform (Kir6.2/SUR1). Unlike SUR1, SUR2B subunits adopt distinct rotational “propeller” and “quatrefoil” geometries surrounding their Kir6.1 core. The glutamate/aspartate-rich linker connecting the two halves of the SUR-ABC core is observed in a quatrefoil-like conformation. Molecular dynamics simulations reveal MgADP-dependent dynamic tripartite interactions between this linker, SUR2B, and Kir6.1. The structures captured implicate a progression of intermediate states between MgADP-free inactivated, and MgADP-bound activated conformations wherein the glutamate/aspartate-rich linker participates as mobile autoinhibitory domain, suggesting a conformational pathway toward K_ATP channel activation.

Dynamic regulation of K⁺ channel gating is a primary point of control for processes governed by electrical excitability. ATP-sensitive potassium (K_ATP) channels, regulated by intracellular ATP to ADP ratios, transduce metabolic changes into electrical signals to govern many physiological processes (1). They are uniquely evolved hetero-octameric complexes comprising four pore-forming inwardly rectifying potassium channel subunits, Kir6.x, and four regulatory sulfonylurea receptors, SURx, nontransporting members of the ABCC subfamily of ABC transporters (2). Various Kir6.x/SURx combinations generate channel isoforms with distinct tissue distribution and function (3, 4). Kir6.2/SUR1 channels are expressed in pancreatic β cells and control glucose-stimulated insulin secretion. Kir6.2/SUR2A channels are the predominant isoform in myocardium, while Kir6.1/SUR2B channels are the major isoform found in vascular smooth muscle. SUR2A and 2B are two splice variants of ABCC9 that differ in their C-terminal 42 amino acids (aa). In vascular smooth muscle, K_ATP activation leads to membrane hyperpolarization and vasodilation (5), while inhibition or deletion causes membrane depolarization, vasoconstriction, and hypertension (5–8). Mutations in the vascular K_ATP channel genes (KCNJ8 and ABCC9) cause Cantú syndrome (9–11), a severe pleiotropic systemic hypotension disorder including hypertrichosis, osteochondrodysplasia, and cardiomegaly (12).K_ATP channel gating by intracellular ATP and ADP involves allosteric sites on both subunits. ATP binding to Kir6.x inhibits the channel. SURx, through induced dimerization of the paired nucleotide binding domains (NBDs), requiring MgADP bound to NBD2 and MgATP bound to noncatalytic NBD1, activates the channel (1, 4, 13). Like all Kir channels, opening further requires PIP₂ bound to Kir6.x (14–16). Despite these commonalities, vascular Kir6.1/SUR2B K_ATP channels have distinct biophysical properties, nucleotide sensitivities, and pharmacology that differentiate them from other isoforms (17–19). First, vascular K_ATP channel unitary conductance is half that of Kir6.2-containing channels. Second, vascular channels lack spontaneous activity, only opening in the presence of NBD-dimerizing Mg-dinucleotides/trinucleotides; in contrast, pancreatic or cardiac channels containing Kir6.2 open spontaneously in the absence of ATP. Third, once activated, vascular K_ATP channels are relatively insensitive to ATP inhibition, requiring mM concentrations to observe an effect, while their pancreatic or cardiac counterparts are blocked by ATP at μM concentrations. Lastly, the antidiabetic sulfonylurea drug glibenclamide (Glib), which inhibits SUR1-containing pancreatic channels with high affinity, is ∼10-fold less potent toward the vascular and cardiac channels containing SUR2. Glib has been shown to reverse defects from gain-of-function Cantú mutations in mice (20). However, clinical application in Cantú patients is hindered by hypoglycemia from inhibition of pancreatic channels (21). Structural mechanisms underlying unique biophysical, physiological, and pharmacological properties among K_ATP channels are unknown.Here, we report cryogenic electron microscopy (cryoEM) structures for the vascular K_ATP channel, Kir6.1/SUR2B, in the presence of ATP and Glib. The structures show conformations not previously seen in pancreatic K_ATP channels prepared under the same condition (22–24). First, unlike in Kir6.2, Kir6.1 cytoplasmic domains (CDs) were displaced from the membrane too far to interact with PIP₂ for channel opening. Second, unlike pancreatic channels, which have a predominant propeller-shaped conformation when bound to ATP and Glib (22, 24), vascular K_ATP channels held four distinct conformations, two resembling propellers and two quatrefoils, marked by varying degrees of rotation of SUR2B toward the core Kir6.1 tetramer. Importantly, a long segment of SUR not previously resolved in any K_ATP structures, linking NBD1 and transmembrane domain 2 (TMD2), was revealed within vascular K_ATP structures to mediate the cytosolic interface between SUR2B and Kir6.1. In particular, the linker’s unique 15 glutamate/aspartate residues termed the ED-domain (25) established a nexus of interactions engaging SUR2B-NBD2 with Kir6.1 C-terminal domain (CTD). Molecular dynamics (MD) simulations showed MgADP binding to NBD2 was accompanied by substantial reconfiguration at this nexus, revealing the ED-domain provides a mobile autoinhibitory interaction that guards the transition of SUR2B from MgADP-free inactivated state to MgADP-bound activated state. Together, our findings point to a structural pathway through which SUR regulates Kir6 channel gating.     

Cardiac sulfonylurea receptor short form-based channels confer a glibenclamide-insensitive KATP activity

The cardiac sarcolemmal ATP-sensitive potassium channel (K_ATP) consists of a Kir6.2 pore and an SUR2 regulatory subunit, which is an ATP-binding cassette (ABC) transporter. K_ATP channels have been proposed to play protective roles during ischemic preconditioning. An SUR2 mutant mouse was previously generated by disrupting the first nucleotide-binding domain (NBD1), where a glibenclamide action site was located. In the mutant ventricular myocytes, a non-conventional glibenclamide-insensitive (10 μM), ATP-sensitive current (I_KATPn) was detected in 33% of single-channel recordings with an average amplitude of 12.3 ± 5.4 pA per patch, an IC₅₀ to ATP inhibition at 10 μM and a mean burst duration at 20.6 ± 1.8 ms. Newly designed SUR2 isoform- or variant-specific antibodies identified novel SUR2 short forms in the sizes of 28 and 68 kDa in addition to a 150-kDa long form in the sarcolemmal membrane of wild-type (WT) heart. We hypothesized that channels constituted by these short forms that lack NBD1 confer I_KATPn. The absence of the long form in the mutant corresponded to loss of the conventional glibenclamide-sensitive K_ATP currents (I_KATP) in isolated cardiomyocytes and vascular smooth muscle cells but the SUR2 short forms remained intact. Nested exonic RT-PCR in the mutant indicated that the short forms lacked NBD1 but contained NBD2. The SUR2 short forms co-immunoprecipitated with Kir6.1 or Kir6.2 suggesting that the short forms may function as hemi-transporters reported in other eukaryotic ABC transporter subgroups. Our results indicate that different K_ATP compositions may co-exist in cardiac sarcolemmal membrane.     

Pharmacological Profile of U‐37883A,a Channel Blocker of Smooth Muscle‐Type ATP‐Sensitive K+ Channels

U‐37883A (PNU‐37883A, guanidine; 4‐morpholinecarboximidine‐N‐1‐adamantyl‐N′‐cyclohexyl hydrochloride) was originally developed as a potential diuretic with specific binding in kidney and vascular smooth muscle rather than in brain or pancreatic β cells. U‐37883A inhibits ATP‐sensitive K⁺ channels (K_ATP channels) in vascular smooth muscle at submicromolar concentrations whilst even at high concentrations (≥10 μM) it has no inhibitory effect at pancreatic, cardiac or skeletal K_ATP channels. Thus, it is generally thought that U‐37883A is a selective inhibitor of vascular smooth muscle K_ATP channels. Approximately one decade ago, K_ATP channels were cloned and found to consist of at least two subunits: an inwardly‐rectifying K⁺ channel six family (K_ir6.x; K_ir6.1 and K_ir6.2) which forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR.x; SUR1, SUR2A, and SUR2B) that accounts for several pharmacological properties. It is generally believed that different combinations of K_ir6.x and SUR.x determine the molecular properties of K_ATP channels. Thus, K_ir6.2/SUR1 channel represents the pancreatic β‐cell K_ATP channel, K_ir6.2/SUR2A channel is thought to represent the cardiac K_ATP channel, whereas K_ir6.1/SUR2B channel is likely to represent the vascular smooth muscle K_ATP channel. Recent molecular studies have shown that U‐37883A selectively suppresses the activity of recombinant K_ATP channels which contain K_ir6.1 subunits in the channel pore unit. It was thus thought that U‐37883A was a selective pharmacological tool which could be used to investigate the activity of vascular smooth muscle K_ATP channels. However, due to its multiple pharmacological actions on several ion channels and poor tissue selectivity, U‐37883A should not be viewed as a selective blocker of smooth muscle K_ATP channels.     

Tissue-specific effects of sulfonylureas: lessons from studies of cloned K(ATP) channels

Sulfonylureas stimulate insulin secretion in type-2 diabetic patients by blocking ATP-sensitive (K(ATP)) potassium channels in the pancreatic beta-cell membrane. This effect is mediated by the binding of the drug to the sulfonylurea receptor (SUR) subunit of the channel. K(ATP) channels are also present in other tissues, but often contain different types of SUR subunits (e.g., SUR1 in beta-cells, SUR2A in heart, SUR2B in smooth muscle). The sensitivity of these different types of K(ATP) channels to sulfonylureas is variable: gliclazide and tolbutamide block the beta-cell, but not the cardiac or smooth muscle, types of K(ATP) channel. In contrast, glibenclamide blocks all three types of channel with similar affinity. The reversibility of the drugs also varies, with tolbutamide and gliclazide being reversible on all three types of K(ATP) channel, while glibenclamide is reversible on cardiac, but not beta-cell, K(ATP) channels. This review summarizes current knowledge of how sulfonylureas act on the different types of K(ATP) channel found in beta-cells and in extrapancreatic tissues, and discusses the implications of these findings for their use as therapeutic agents.     

Coronary spasm and acute myocardial infarction due to a mutation (V734I) in the nucleotide binding domain 1 of ABCC9

Background

Alterations in coronary vasomotor tone may participate in the pathogenesis of acute myocardial infarction (AMI). Vascular ATP-sensitive K⁺ (K_ATP) channels, formed by Kir6.x/SUR2B, are key regulators of coronary tone and mutations in cardiac (Kir6.2/SUR2A) K_ATP channels result in heart disease. Here we explore the pathophysiological mechanism of a rare mutation (V734I) found in exon 17 of the ABCC9 gene, estimated to cause a 6.4-fold higher risk of AMI before the age of 60.

Methods and results

Eleven patients carrying the mutation were identified; they presented AMI of vasospastic origin associated with increased plasma levels of endothelin-1 and increased leukocyte ROCK activity. The effects of the mutation on the functional properties of the two splice variants of ABCC9 (SUR2A and SUR2B) were studied using patch-clamp electrophysiology. The mutation reduced the sensitivity to MgATP inhibition of Kir6.2/SUR2B channels but not of Kir6.2/SUR2A and Kir6.1/SUR2B channels. Furthermore, the stimulatory effects of MgNDP (MgADP, MgGDP and MgUDP) were unaltered in mutant Kir6.2/SUR2A and Kir6.1/SUR2B channels. In contrast, mutant channels composed of Kir6.2 and SUR2B were less sensitive to MgNDP activation, assessed in the presence of MgATP. The antianginal drug nicorandil activated Kir6.2/SUR2B–V734I channels, thus substituting for the loss of MgNDP stimulation, suggesting that this drug could be of therapeutic use in the treatment of AMI associated with V734I.

Conclusions

The 734I allele in ABCC9 may influence susceptibility to AMI by impairing the response of vascular, but not cardiac, K_ATP channels to intracellular nucleotides. This is the first human mutation in an ion channel gene to be implicated in AMI.     

Gliclazide modified release: a critical review of pharmacodynamic,metabolic, and vasoprotective effects

Gliclazide modified release (MR) is a new formulation of the drug gliclazide and is given once daily. The specifically designed hydrophilic matrix of gliclazide MR leads to a progressive drug release that parallels the 24-hour glycemic profile in type 2 diabetic patients. Development studies showed a sustained efficacy over 2 years coupled with a very good acceptability. Gliclazide MR acts selectively on adenosine triphosphate-dependent potassium (K_ATP) channels of the pancreatic β cell. No interaction with cardiovascular K_ATP channels has been shown, indicating that the drug can be safely used in patients with ischemic heart disease. In addition, gliclazide MR shows the ability to inhibit key mechanisms in diabetic angiopathy, independently of glycemic control.     

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.     

Ageing-induced decline in physical endurance in mice is associated with decrease in cardiac SUR2A and increase in cardiac susceptibility to metabolic stress: therapeutic prospects for up-regulation of SUR2A

Ageing is characterized by decline in physical endurance which has been suggested to be partly due to diminished functional and adaptive reserve capacity of the heart. Ageing is associated with decrease in numbers of sarcolemmal ATP-sensitive K⁺ (K_ATP) channels, but whether this has anything to do with ageing-induced decline in physical endurance is yet to be determined. We have previously shown that the numbers of sarcolemmal K_ATP channels are controlled by the level of expression of SUR2A, a K_ATP channel regulatory subunit. Here, we have found that ageing decreases the level of SUR2A mRNA in the heart without affecting expression of pore-forming K_ATP channel subunits, Kir6.1 and Kir6.2. This effect of ageing was associated with decrease in levels of fully-assembled sarcolemmal K_ATP channels. At the same time, ageing was associated with decreased physical endurance. In order to determine whether increased expression of SUR2A would counteract ageing-induced decrease in physical endurance, we have taken advantage of mice which SUR2A levels are regulated by more efficient CMV promoter. These mice had increased resistance of cardiomyocytes to metabolic stress/hypoxia and increased physical endurance when compared to the wild type. In transgenic mice, ageing did not affect the level of SUR2A mRNA in the heart and the level of fully-assembled sarcolemmal K_ATP channels. The effect of increased SUR2A to resistance of cardiomyocytes to hypoxia and physical endurance was retained in old mice. The magnitude of these effects was such that they were significantly increased even when compared to those in wild type young mice. We conclude that (1) the level of SUR2A expression in the heart is important factor in regulating physical endurance, (2) ageing-induced decrease in cardiac SUR2A is, at least in part, responsible for ageing-induced decline in physical fitness and (3) up-regulation of SUR2A could be a viable strategy to counteract ageing-induced decline in physical endurance.     

MgATP activates the β cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (K_ATP) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca²⁺ influx and exocytosis. Metabolic regulation of K_ATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell K_ATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate K_ATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg²⁺ caused an apparent reduction in the inhibitory action of ATP on wild-type K_ATP channels, and MgATP actually activated K_ATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates K_ATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg²⁺.     

Immuno-localization of sulphonylurea receptor 1 in rat pancreas

Aims/hypothesis. A sulphonylurea receptor, SUR1, and an inward rectifier potassium channel, Kir6.2, reconstitute the ATP-sensitive K⁺ channel that mediates glucose-induced insulin secretion in pancreatic beta cells. We reported previously that Kir6.2 were localized at insulin-, glucagon-, and somatostatin-producing cells. In this new study we aimed to determine the distribution of SUR1 in rat pancreatic islets and to suggest the location of the ATP-sensitive K⁺ channels in the islet. Methods. Western blot analysis was carried out using two anti-SUR1 antibodies, which had been raised against different portions of rat SUR1. SUR1, Kir 6.2, and islet hormones were then localized by indirect immunofluorescence staining of the cryosections of rat pancreas. Results. In Western blot analysis, each of the anti-SUR1 antibodies detected a band at 140 kDa, which is close to the predicted molecular weight of SUR1, in the homogenate of isolated pancreatic islets. Double immunofluorescence staining of cryosections showed that SUR1 occurred all over the islets, and that SUR1 colocalized with insulin, glucagon, somatostatin, and pancreatic polypeptide. Kir6.2 was also shown to be present in pancreatic polypeptide cells. Conclusion/interpretation. Together with our previously reported data, the above findings indicate that K_ATP channels comprising SUR1 and Kir6.2 occur not only in beta cells but also in the alpha, delta, and pancreatic polypeptide cells of the pancreatic islets, suggesting that therapeutic sulphonylureas could act on these cells directly. [Diabetologia (1999) 42: 1204–1211] Received: 21 January 1999 and in final revised form: 21 May 1999     

A soluble epoxide hydrolase inhibitor--8-HUDE increases pulmonary vasoconstriction through inhibition of K(ATP) channels

Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid, are endogenously produced epoxides that act as substrates for the soluble epoxide hydrolase (sEH). Recent studies indicate that EETs increase the tension of rat pulmonary arteries (PAs), and inhibition of sEH augments hypoxic pulmonary vasoconstriction. However, the mechanisms underlying the proconstrictive effects of sEH inhibitors in pulmonary artery smooth muscle cells (PASMCs) are unclear. In the present study, we used a sEH inhibitor, 12-(3-hexylureido) dodec-8-enoic acid (8-HUDE), to examine the ionic mechanisms underlying the constriction of PAs. 8-HUDE increased the tension of rat PAs to 145% baseline in a manner which was effectively eliminated by 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ (K_ATP) channels. Whole cell currents of HEK cells transfected with Kir6.1 or SUR2B were activated by K_ATP channel opener pinacidil, inhibited by K_ATP channel inhibitor glibenclamide or inhibited by 8-HUDE in a concentration-dependent manner with an IC50 value of 40 uM. In addition, 8-HUDE inhibited the expression of Kir6.1 and SUR2B at both mRNA and protein level in rat PASMCs. These observations suggest that 8-HUDE exerts acute effects on K_ATP channel activity as well as subacute effects through decreased channel expression, and these effects are, at least in part, via the Kir6.1/SUR2B channel.     

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