Permanent neonatal diabetes: combining sulfonylureas with insulin may be an effective treatment.

BACKGROUND: Permanent neonatal diabetes caused by mutations in the KCNJ11 gene may be managed with high-dose sulfonylureas. Complete transfer to sulfonylureas is not successful in all cases and can result in insulin monotherapy. In such cases, the outcomes of combining sulfonylureas with insulin have not been fully explored. We present the case of a woman with diabetes due to a KCNJ11 mutation, in whom combination therapy led to clinically meaningful improvements. CASE: A 22-year-old woman was found to have a KCNJ11 mutation (G334V) following diagnosis with diabetes at 3 weeks. She was treated with insulin-pump therapy, had hypoglycaemia unawareness and suboptimal glycaemic control. We assessed the in vitro response of the mutant channel to tolbutamide in Xenopus oocytes and undertook sulfonylurea dose-titration with C-peptide assessment and continuous glucose monitoring. In vitro studies predicted the G334V mutation would be sensitive to sulfonylurea therapy [91 ± 2% block (n = 6) with 0.5 mM tolbutamide]. C-peptide increased following a glibenclamide test dose (from 5 to 410 pmol/l). Glibenclamide dose-titration was undertaken: a lower glibenclamide dose did not reduce blood glucose levels, but at 1.2 mg/kg/day insulin delivery was reduced to 0.1 units/h. However, when insulin was stopped, hyperglycaemia ensued. Glibenclamide was further increased (2 mg/kg/day), but once-daily long-acting insulin was still required to maintain glycaemia. This resulted in improved HbA1c of 52 mmol/mol (6.9%), restoration of hypoglycaemia awareness and reduced glycaemic variability. CONCLUSION: In people with KCNJ11 mutations causing permanent neonatal diabetes, and where complete transfer is not possible, consideration should be given to dual insulin and sulfonylurea therapy. This article is protected by copyright. All rights reserved.

ß-Cell dysfunction in diabetes: a crisis of identity?

Type 2 diabetes is characterized by insulin resistance and a progressive loss of ß-cell function induced by a combination of both ß-cell loss and impaired insulin secretion from remaining ß-cells. Here, we review the fate of the ß-cell under chronic hyperglycaemic conditions with regard to ß-cell mass, gene expression, hormone content, secretory capacity and the ability to de- or transdifferentiate into other cell types. We compare data from various in vivo and in vitro models of diabetes with a novel mouse model of inducible, reversible hyperglycaemia (ßV59M mice). We suggest that insulin staining using standard histological methods may not always provide an accurate estimation of ß-cell mass or number. We consider how ß-cell identity is best defined, and whether expression of transcription factors normally found in islet progenitor cells, or in α-cells, implies that mature ß-cells have undergone dedifferentiation or transdifferentiation. We propose that even in long-standing diabetes, ß-cells predominantly remain ß-cells-but not as we know them.

Mice expressing a human K(ATP) channel mutation have altered channel ATP sensitivity but no cardiac abnormalities.

AIMS/HYPOTHESIS: Patients with severe gain-of-function mutations in the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, have neonatal diabetes, muscle hypotonia and mental and motor developmental delay-a condition known as iDEND syndrome. However, despite the fact that Kir6.2 forms the pore of the cardiac K(ATP) channel, patients show no obvious cardiac symptoms. The aim of this project was to use a mouse model of iDEND syndrome to determine whether iDEND mutations affect cardiac function and cardiac K(ATP) channel ATP sensitivity. METHODS: We performed patch-clamp and in vivo cine-MRI studies on mice in which the most common iDEND mutation (Kir6.2-V59M) was targeted to cardiac muscle using Cre-lox technology (m-V59M mice). RESULTS: Patch-clamp studies of isolated cardiac myocytes revealed a markedly reduced K(ATP) channel sensitivity to MgATP inhibition in m-V59M mice (IC(50) 62 µmol/l compared with 13 µmol/l for littermate controls). In vivo cine-MRI revealed there were no gross morphological differences and no differences in heart rate, end diastolic volume, end systolic volume, stroke volume, ejection fraction, cardiac output or wall thickening between m-V59M and control hearts, either under resting conditions or under dobutamine stress. CONCLUSIONS/INTERPRETATION: The common iDEND mutation Kir6.2-V59M decreases ATP block of cardiac K(ATP) channels but was without obvious effect on heart function, suggesting that metabolic changes fail to open the mutated channel to an extent that affects function (at least in the absence of ischaemia). This may have implications for the choice of sulfonylurea used to treat neonatal diabetes.

A mutation in KCNJ11 causing human hyperinsulinism (Y12X) results in a glucose-intolerant phenotype in the mouse.

AIMS/HYPOTHESIS: We identified a mouse with a point mutation (Y12STOP) in the Kcnj11 subunit of the K(ATP) channel. This point mutation is identical to that found in a patient with congenital hyperinsulinism of infancy (HI). We aimed to characterise the phenotype arising from this loss-of-function mutation and to compare it with that of other mouse models and patients with HI. METHODS: We phenotyped an N-ethyl-N-nitrosourea-induced mutation on a C3H/HeH background (Kcnj11 ( Y12STOP )) using intraperitoneal glucose tolerance testing to measure glucose and insulin plasma concentrations. Insulin secretion and response to incretins were measured on isolated islets. RESULTS: Homozygous male and female adult Kcnj11 ( Y12STOP ) mice exhibited impaired glucose tolerance and a defect in insulin secretion as measured in vivo and in vitro. Islets had an impaired incretin response and reduced insulin content. CONCLUSIONS/INTERPRETATION: The phenotype of homozygous Kcnj11 ( Y12STOP ) mice is consistent with that of other Kcnj11-knockout mouse models. In contrast to the patient carrying this mutation homozygously, the mice studied did not have hyperinsulinaemia or hypoglycaemia. It has been reported that HI patients may develop diabetes and our mouse model may reflect this clinical feature. The Kcnj11 ( Y12STOP ) model may thus be useful in further studies of K(ATP) channel function in various cell types and in investigation of the development of hyperglycaemia in HI patients.

The first clinical case of a mutation at residue K185 of Kir6.2 (KCNJ11): a major ATP-binding residue.

BACKGROUND: Closure of the adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel plays a key role in insulin secretion from the pancreatic beta-cells. Many mutations in KCNJ11 and ABCC8, which respectively encode the pore-forming (Kir6.2) and regulatory (SUR1) subunits of the K(ATP) channel, cause neonatal diabetes. All such mutations impair the ability of metabolically generated ATP to close the channel. Although lysine 185 is predicted to be a major contributor to the ATP-binding site of Kir6.2, no mutations at this residue have been found to cause neonatal diabetes to date. METHODS: We report a 3-year-old girl with permanent neonatal diabetes (PNDM) caused by a novel heterozygous mutation (K185Q) at residue K185 of KCNJ11. The patient presented with marked hyperglycaemia and ketoacidosis at 70 days after birth, and insulin therapy was commenced. RESULTS: Wild-type and mutant K(ATP) channels were expressed in Xenopus oocytes and the effects of intracellular ATP on macroscopic K(ATP) currents in inside-out membrane patches were measured. In the simulated heterozygous state, the K185Q mutation caused a substantial reduction in the ability of MgATP to inhibit the channel. Heterozygous K185Q channels were still blocked effectively by the sulphonylurea tolbutamide. CONCLUSIONS: We report the first clinical case of a PNDM caused by a mutation at K185. Functional studies indicate that the K185Q mutation causes PNDM by reducing the ATP sensitivity of the K(ATP) channel, probably via a reduction in ATP binding to Kir6.2. Based on the experimental data, the patient was successfully transferred to sulphonylurea therapy.

Variable phenotypic spectrum of diabetes mellitus in a family carrying a novel KCNJ11 gene mutation.

AIMS: Heterozygous activating mutations in KCNJ11, which encodes the Kir6.2 subunit of the pancreatic ATP-sensitive potassium (K(ATP)) channel, cause both permanent and transient neonatal diabetes. Identification of KCNJ11 mutations has important therapeutic implications, as many patients can replace insulin injections with sulphonylurea tablets. The aim was to determine if a KCNJ11 mutation was responsible for a dominantly inherited form of diabetes mellitus, showing variability in age at diagnosis, in an Italian family. METHODS: We sequenced KCNJ11 in members of a three-generation family with variable phenotypes of dominantly inherited diabetes mellitus. One had transient early-onset diabetes, one had impaired glucose tolerance during the second pregnancy, and two had young-onset diabetes. None of the subjects showed permanent neonatal diabetes or neurological symptoms. RESULTS: A novel heterozygous mutation (c. 679C-->G and c. 680A-->T) was identified, resulting in a GAG-->CTG (E227L) substitution in KCNJ11. Functional studies of recombinant heterozygous K(ATP) channels revealed a small reduction in channel inhibition by ATP (IC(50) of 15 micromol/l and 38 micromol/l for wild-type and heterozygous channels, respectively) and an increase in the resting K(ATP) current. This would be expected to impair insulin secretion. The results are in agreement with the mild phenotype of the patients. CONCLUSIONS: Our results broaden the spectrum of diabetes phenotypes resulting from KCNJ11 mutations. They indicate testing for KCNJ11 mutations should be considered not only for neonatal diabetes but also for other forms of dominantly inherited diabetes with later onset, especially where these are associated with a low body mass index and low birth weight.

A Kir6.2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications.

AIMS/HYPOTHESIS: Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects. METHODS: The patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg(-1) day(-1)). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes. RESULTS: Heterozygous (het) and homozygous L164P K(ATP) channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients. CONCLUSIONS/INTERPRETATION: Our results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response.

Functional analysis of two Kir6.2 (KCNJ11) mutations, K170T and E322K, causing neonatal diabetes.

Heterozygous activating mutations in Kir6.2 (KCNJ11), the pore-forming subunit of the adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel, are a common cause of neonatal diabetes (ND). We assessed the functional effects of two Kir6.2 mutations associated with ND: K170T and E322K. K(ATP) channels were expressed in Xenopus oocytes, and the heterozygous state was simulated by coexpression of wild-type and mutant Kir6.2 with SUR1 (the beta cell type of sulphonylurea receptor (SUR)). Both mutations reduced the sensitivity of the K(ATP) channel to inhibition by MgATP and enhanced whole-cell K(ATP) currents. In pancreatic beta cells, such an increase in the K(ATP) current is expected to reduce insulin secretion and thereby cause diabetes. The E322K mutation was without effect when Kir6.2 was expressed in the absence of SUR1, suggesting that this residue impairs coupling to SUR1. This is consistent with its predicted location on the outer surface of the tetrameric Kir6.2 pore. The kinetics of K170T channel opening and closing were altered by the mutation, which may contribute to the lower ATP sensitivity. Neither mutation affected the sensitivity of the channel to inhibition by the sulphonylurea tolbutamide, suggesting that patients carrying these mutations may respond to these drugs.

Nicotinamide nucleotide transhydrogenase: a link between insulin secretion, glucose metabolism and oxidative stress.

This paper reviews recent studies on the role of Nnt (nicotinamide nucleotide transhydrogenase) in insulin secretion and detoxification of ROS (reactive oxygen species). Glucose-stimulated insulin release from pancreatic beta-cells is mediated by increased metabolism. This elevates intracellular [ATP], thereby closing KATP channels (ATP-sensitive potassium channels) and producing membrane depolarization, activation of voltage-gated Ca2+ channels, Ca2+ influx and, consequently, insulin secretion. The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion, which results from a naturally occurring deletion in the Nnt gene. Transgenic expression of the wild-type Nnt gene in C57BL/6J mice rescues the phenotype. Knockdown of Nnt in the insulin-secreting cell line MIN6 with small interfering RNA dramatically reduced Ca2+ influx and insulin secretion. Similarly, mice carrying ENU (N-ethyl-N-nitrosourea)-induced loss-of-function mutations in Nnt were glucose intolerant and secreted less insulin during a glucose tolerance test. Islets isolated from these mice showed impaired insulin secretion in response to glucose, but not to the KATP channel blocker tolbutamide. This is explained by the fact that glucose failed to elevate ATP in Nnt mutant islets. Nnt is a nuclear-encoded mitochondrial protein involved in detoxification of ROS. beta-Cells isolated from Nnt mutant mice showed increased ROS production on glucose stimulation. We hypothesize that Nnt mutations enhance glucose-dependent ROS production and thereby impair beta-cell mitochondrial metabolism, possibly via activation of uncoupling proteins. This reduces ATP production and lowers KATP channel activity. Consequently, glucose-dependent electrical activity and insulin secretion are impaired.

K(ATP) channels and insulin secretion: a key role in health and disease.

This review summarizes advances in our understanding of the structure and function of the ATP-sensitive potassium (K(ATP)) channel of the pancreatic beta-cell that have been made over the last 5 years. It discusses recent structural studies of the octameric K(ATP) channel complex and studies of the regulation of K(ATP) channel activity by nucleotides. It then considers the molecular mechanism by which gain-of-function mutations in the Kir6.2 subunit of the K(ATP) channel reduce channel inhibition by ATP and thereby lead to neonatal diabetes, and how identification of these mutations has led to changes in therapy. Finally, it illustrates how mouse models of glucose intolerance or diabetes can provide fresh insight into beta-cell function, using the C57BL/6J mouse, whose glucose intolerance arises from mutations in nicotinamide nucleotide transhydrogenase, as an example.

Behavioral phenotyping of mice lacking the K ATP channel subunit Kir6.2.

ATP-sensitive potassium (K(ATP)) channels are expressed in various tissues and cell-types where they act as so-called metabolic sensors that couple metabolic state to cellular excitability. The pore of most K(ATP) channel types is built by Kir6.2 subunits. Analysis of a general Kir6.2 knockout (KO) mouse has identified a variety of different functional roles for central and peripheral K(ATP) channels in situations of metabolic demand. However, the widespread distribution of these channels suggests that they might influence cellular physiology and animal behavior under metabolic control conditions. As a comprehensive behavioral description of Kir6.2 KO mice under physiological control conditions has not yet been carried out, we subjected Kir6.2 KO and corresponding wild-type (WT) mice to a test battery to assess emotional behavior, motor activity and coordination, species-typical behaviors and cognition. The results indicated that in these test situations Kir6.2 KO mice were less active, had impaired motor coordination, and appeared to differ from controls in their emotional reactivity. Differences between KO and WT mice were generally attenuated in test situations that resembled the home cage environment. Moreover, in their home cages KO mice were more active than WT mice. Thus, our results suggest that loss of Kir6.2-containing K(ATP) channels does affect animal behavior under metabolic control conditions, especially in novel situations. These findings assign novel functional roles to K(ATP) channels beyond those previously described. However, according to the widespread expression of K(ATP) channels, these effects are complex, being dependent on details of test apparatus, procedure and prior experience.

A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice.

AIMS/HYPOTHESIS: C57BL/6J mice exhibit impaired glucose tolerance. The aims of this study were to map the genetic loci underlying this phenotype, to further characterise the physiological defects and to identify candidate genes. METHODS: Glucose tolerance was measured in an intraperitoneal glucose tolerance test and genetic determinants mapped in an F2 intercross. Insulin sensitivity was measured by injecting insulin and following glucose disposal from the plasma. To measure beta cell function, insulin secretion and electrophysiological studies were carried out on isolated islets. Candidate genes were investigated by sequencing and quantitative RNA analysis. RESULTS: C57BL/6J mice showed normal insulin sensitivity and impaired insulin secretion. In beta cells, glucose did not stimulate a rise in intracellular calcium and its ability to close KATP channels was impaired. We identified three genetic loci responsible for the impaired glucose tolerance. Nicotinamide nucleotide transhydrogenase (Nnt) lies within one locus and is a nuclear-encoded mitochondrial proton pump. Expression of Nnt is more than sevenfold and fivefold lower respectively in C57BL/6J liver and islets. There is a missense mutation in exon 1 and a multi-exon deletion in the C57BL/6J gene. Glucokinase lies within the Gluchos2 locus and shows reduced enzyme activity in liver. CONCLUSIONS/INTERPRETATION: The C57BL/6J mouse strain exhibits plasma glucose intolerance reminiscent of human type 2 diabetes. Our data suggest a defect in beta cell glucose metabolism that results in reduced electrical activity and insulin secretion. We have identified three loci that are responsible for the inherited impaired plasma glucose tolerance and identified a novel candidate gene for contribution to glucose intolerance through reduced beta cell activity.

Potent and selective activation of the pancreatic beta-cell type K(ATP) 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-4 H-thieno[3,2- e]-1,2,4-thiadiazine 1,1-dioxide (NNC 55-0118) and 6-chloro-3-(1-methylcyclopropyl)amino-4 H-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 EC(50) values of 0.33 micromol/l and 0.45 micromol/l, respectively, compared with that of 31 micro mol/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.

Characterisation of new KATP-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 micro mol/l to 120 micro mol/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.

Multiple sites of interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2.

The amino-terminal and carboxy-terminal domains of inwardly rectifying potassium channel (Kir) subunits are both intracellular. A direct physical interaction between these two domains is involved in the response of Kir channels to regulatory factors such as G-proteins, nucleotides and intracellular pH. We have previously mapped the region within the N-terminal domain of Kir6.2 that interacts with the C-terminus. In this study we use a similar in vitro protein-protein interaction assay to map the regions within the C-terminus which interact with the N-terminus. We find that multiple interaction domains exist within the C-terminus: CID1 (amino acids (aa) 279-323), CID2 (aa 214-222) and CID3 (aa 170-204). These domains correlate with regions previously identified as making important contributions to Kir channel assembly and function. The highly conserved nature of the C-terminus suggests that a similar association with the N-terminus may be a feature common to all members of the Kir family of potassium channels, and that it may be involved in gating of Kir channels by intracellular ligands.

Mutations within the P-loop of Kir6.2 modulate the intraburst kinetics of the ATP-sensitive potassium channel.

The ATP-sensitive potassium (K(ATP)) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 K(ATP) channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (tau(o)) and the short closed time (tauC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer tau(o), shorter tauC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the K(ATP) channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.

Structural basis for the interference between nicorandil and sulfonylurea action.

Nicorandil is a new antianginal agent that potentially may be used to treat the cardiovascular side effects of diabetes. It is both a nitric oxide donor and an opener of ATP-sensitive K(+) (K(ATP)) channels in muscle and thereby causes vasodilation of the coronary vasculature. The aim of this study was to investigate the domains of the K(ATP) channel involved in nicorandil activity and to determine whether nicorandil interacts with hypoglycemic sulfonylureas that target K(ATP) channels in pancreatic beta-cells. K(ATP) channels in muscle and beta-cells share a common pore-forming subunit, Kir6.2, but possess alternative sulfonylurea receptors (SURs; SUR1 in beta-cells, SUR2A in cardiac muscle, and SUR2B in smooth muscle). We expressed recombinant K(ATP) channels in Xenopus oocytes and measured the effects of drugs and nucleotides by recording macroscopic currents in excised membrane patches. Nicorandil activated Kir6.2/SUR2A and Kir6.2/SUR2B but not Kir6.2/SUR1 currents, consistent with its specificity for cardiac and smooth muscle K(ATP) channels. Drug activity depended on the presence of intracellular nucleotides and was impaired when the Walker A lysine residues were mutated in either nucleotide-binding domain of SUR2. Chimeric studies showed that the COOH-terminal group of transmembrane helices (TMs), especially TM 17, is responsible for the specificity of nicorandil for channels containing SUR2. The splice variation between SUR2A and SUR2B altered the off-rate of the nicorandil response. Finally, we showed that nicorandil activity was unaffected by gliclazide, which specifically blocks SUR1-type K(ATP) channels, but was severely impaired by glibenclamide and glimepiride, which target both SUR1 and SUR2-type K(ATP) channels.

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 (KATP) channels in the pancreatic beta-cell membrane. KATP 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 KATP channels (Kir6.2/SUR1) but not heart (Kir6.2/SUR2A) or smooth muscle (Kir6.2/SUR2B) KATP channels with high potency. In this study, we examined the specificity of gliclazide for the native (as opposed to recombinant) KATP channels in beta cells, heart and smooth muscle. METHODS: The action of the drug was studied by whole-cell current recordings of native KATP channels in isolated pancreatic beta-cells and myocytes from heart and smooth muscle. RESULTS: Gliclazide blocked whole-cell beta-cell KATP currents with an IC50 of 184 +/- 30 nmol/l (n = 6-10) but was much less effective in cardiac and smooth muscle (IC50s of 19.5 +/- 5.4 micromol/l (n = 6-12) and 37.9 +/- 1.0 micromol/l (n = 5-10), respectively). In all three tissues, the action of the drug on whole-cell KATP currents was rapidly reversible. In inside-out patches on beta-cells, gliclazide (1 micromol/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 KATP 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.

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 (KATP) channels in the plasma membrane of the pancreatic beta cell. In this paper we investigated. the specificity of repaglinide for three types of cloned (KATP) 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 KATP channel, respectively. METHODS: The action of the drug was studied by whole-cell current recordings of KATP 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 KATP 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 micromol/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 KATP 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.

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.