Absence Of SUR1 Research Articles

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

An In-Frame Deletion in Kir6.2 (KCNJ11) Causing Neonatal Diabetes Reveals a Site of Interaction between Kir6.2 and SUR1

Activating mutations in genes encoding the Kir6.2 (KCNJ11) and SUR1 (ABCC8) subunits of the pancreatic ATP-sensitive K+ channel are a common cause of permanent neonatal diabetes (PNDM). All Kir6.2 mutations identified to date are missense mutations. We describe here a novel in-frame deletion (residues 28–32) in Kir6.2 in a heterozygous patient with PNDM without neurological problems that are detected by standard evaluation. The aim of the study was to identify the mutation responsible for neonatal diabetes in this patient and characterize its functional effects. Wild-type and mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. The Kir6.2–28Δ32 mutation produced a significant decrease in ATP inhibition and an increase in whole-cell KATP currents, explaining the diabetes of the patient. Tolbutamide block was only slightly reduced in the simulated heterozygous state, suggesting that the patient should respond to sulfonylurea therapy. The mutation decreased ATP inhibition indirectly, by increasing the intrinsic (unliganded) channel open probability. Neither effect was observed when Kir6.2 was expressed in the absence of SUR1, suggesting that the mutation impairs coupling between SUR1 and Kir6.2. Coimmunoprecipitation studies further revealed that the mutation disrupted a physical interaction between Kir6.2 and residues 1–288 (but not residues 1–196) of SUR1. We report a novel KCNJ11 mutation causing PNDM. Our results show that residues 28–32 in the N terminus of Kir6.2 interact both physically and functionally with SUR1 and suggest that residues 196–288 of SUR1 are important in this interaction.

An In-Frame Deletion in Kir6.2 (KCNJ11) Causing Neonatal Diabetes Reveals a Site of Interaction between Kir6.2 and SUR1

Activating mutations in genes encoding the Kir6.2 (KCNJ11) and SUR1 (ABCC8) subunits of the pancreatic ATP-sensitive K(+) channel are a common cause of permanent neonatal diabetes (PNDM). All Kir6.2 mutations identified to date are missense mutations. We describe here a novel in-frame deletion (residues 28-32) in Kir6.2 in a heterozygous patient with PNDM without neurological problems that are detectable by standard evaluation. The aim of the study was to identify the mutation responsible for neonatal diabetes in this patient and characterize its functional effects. Wild-type and mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. The Kir6.2-28Delta32 mutation produced a significant decrease in ATP inhibition and an increase in whole-cell K(ATP) currents, explaining the diabetes of the patient. Tolbutamide block was only slightly reduced in the simulated heterozygous state, suggesting that the patient should respond to sulfonylurea therapy. The mutation decreased ATP inhibition indirectly, by increasing the intrinsic (unliganded) channel open probability. Neither effect was observed when Kir6.2 was expressed in the absence of SUR1, suggesting that the mutation impairs coupling between SUR1 and Kir6.2. Coimmunoprecipitation studies further revealed that the mutation disrupted a physical interaction between Kir6.2 and residues 1-288 (but not residues 1-196) of SUR1. We report a novel KCNJ11 mutation causing PNDM. Our results show that residues 28-32 in the N terminus of Kir6.2 interact both physically and functionally with SUR1 and suggest that residues 196-288 of SUR1 are important in this interaction.

Abstract 1515: Regional Effects of Katp Channel Openers Reflect Distinct Atrial and Ventricular Katp Channel Structure in the Murine Heart

Classically, cardiac sarcolemmal KATP channels are thought to be composed of Kir6.2 (inward-rectifier potassium channel 6.2, KCNJ11) and SUR2A (sulfonylurea receptor type 2A, ABCC9) subunits. However, the evidence is strong that SUR1 (sulfonylurea receptor type 1, ABCC8) subunits are also expressed in the heart and that they play a significant functional role in the atria. To examine this further, we have estimated the effects of potassium channel-opening drugs diazoxide (specific to SUR1 > SUR2A) and pinacidil (SUR2A > SUR1) in intact hearts from wild type (WT, n=6) and SUR1−/− mice (KO, n=3) that lack SUR1 subunits. Action potential duration (APD) in both atria and ventricles were estimated by optical mapping of posterior surface of Langendorff-perfused hearts using the voltage sensitive dye RH237 and high spatiotemporal resolution CMOS camera (100x100 pixels; 3,000 frames/sec). In WT hearts, diazoxide (300 μM) decreased APD in atria (from 33.6±2.1 ms to 25.5±1.0 ms, p<0.001) and did not change it in ventricles (60.0±8.5 ms vs 61.2±8.3 ms, NS). The absence of SUR1 in KO mice resulted in loss of efficacy of diazoxide in atria (37.5±0.7 ms vs 36.5±0.7 ms, NS). In contrast, pinacidil (300 μM) significantly decreased ventricular APD in both type of mice (from 60.0±8.5 ms to 30.5±4.2 ms in WT, p<0.001; and from 62.0±1.4 ms to 30.5±6.4 ms in KO, p<0.001) and did not change atrial APD in either WT or KO hearts. Glass microelectrode recordings from isolated superfused atria confirmed the optical data. In both WT and KO hearts, the APD in left ventricle was significantly longer and the effect of pinacidil was significantly greater than in right ventricle (APD decreasing by 56.3±4.2% and 62.3±12.0% in left ventricle vs 49.4±3.4% and 50.9±4.1% in right ventricle for WT and KO mice respectively, p<0.05). Similar differences between APDs in right and left atria were not observed. Collectively, these results indicate that in the intact mouse heart, significant differential KATP pharmacology in atria and ventricles results from SUR1 predominance in forming the atrial channel, highlighting an unappreciated heterogeneity of KATP function in the heart.

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.

Functional Effects of Mutations at F35 in the NH2-terminus of Kir6.2 (KCNJ11), Causing Neonatal Diabetes, and Response to Sulfonylurea Therapy

Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive K(+) channel (K(ATP) channel), cause neonatal diabetes. To date, all mutations increase whole-cell K(ATP) channel currents by reducing channel inhibition by MgATP. Here, we provide functional characterization of two mutations (F35L and F35V) at residue F35 of Kir6.2, which lies within the NH(2)-terminus. We further show that the F35V patient can be successfully transferred from insulin to sulfonylurea therapy. The patient has been off insulin for 24 months and shows improved metabolic control (mean HbA(1c) 7.58 before and 6.18% after sulfonylurea treatment; P < 0.007). Wild-type and mutant Kir6.2 were heterologously coexpressed with SUR1 in Xenopus oocytes. Whole-cell K(ATP) channel currents through homomeric and heterozygous F35V and F35L channels were increased due to a reduced sensitivity to inhibition by MgATP. The mutation also increased the open probability (P(O)) of homomeric F35 mutant channels in the absence of ATP. These effects on P(O) and ATP sensitivity were abolished in the absence of SUR1. Our results suggest that mutations at F35 cause permanent neonatal diabetes by affecting K(ATP) channel gating and thereby, indirectly, ATP inhibition. Heterozygous F35V channels were markedly inhibited by the sulfonylurea tolbutamide, accounting for the efficacy of sulfonylurea therapy in the patient.

ATP sensitivity of ATP‐sensitive K+ channels: role of the γ phosphate group of ATP and the R50 residue of mouse Kir6.2

ATP-sensitive K (K(ATP)) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the alpha and beta phosphate groups of ATP, respectively, to induce channel closure. A positively charged residue in the N terminus, R50, is also important, and has been proposed to interact with either the gamma or beta phosphate group of ATP. To examine this issue, we systematically mutated R50 to residues of different size, charge and hydropathy, and examined the effects on adenine nucleotide sensitivity in the absence and presence of SUR1. In the absence of SUR1, only the size of residue 50 significantly altered ATP sensitivity, with smaller side chains decreasing ATP sensitivity. In the presence of SUR1, however, hydrophathy and charge also played a role. Hydrophilic residues decreased ATP sensitivity more than hydrophobic residues for small size residues, and, surprisingly, negatively charged residues E and D preserved ATP sensitivity and increased ADP sensitivity relative to the wild-type residue R. These observations suggest that a negative charge near position 50, due to either mutation of R50 or the interaction of the gamma phosphate group of ATP with R50, facilitates closure of the ATP-dependent gate. Mutation of the nearby positively charged residue R54, known to be involved in stabilizing channel opening via electrostatic interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), also caused increased ADP sensitivity as compared with ATP, suggesting a loss of function of ATP's gamma phosphate. Based on these results, we propose that a phosphate group or a negative charge at position 50 initiates channel closure by destabilizing the electrostatic interactions between negative phosphate groups of PIP2 and residues such as R54.

Y-26763: ATP-sensitive K + channel activation and the inhibition of insulin release from human pancreatic β-cells

The effect of Y-26763 [(−)-(3 S,4 R)-4-( N-acetyl- N-hydroxyamino)-6-cyano-3,4-dihydro-2,2-dimethyl-2 H-1-benzopyran-3-ol], a novel ATP-sensitive K + (K ATP) channel activator, was tested on insulin secretion from human pancreatic islets in vitro. Y-26763 was able to inhibit both glucose- and tolbutamide-induced insulin secretion from islets as assessed by radioimmunoassay. The mechanism for inhibition of insulin secretion was characterised using patch clamp electrophysiology on dispersed human pancreatic β-cells which express K ATP channels comprised of Kir6.2 and SUR1, and the NES2Y human β-cell line, transfected with Kir6.2ΔC26. Y-26763 activated K ATP channels in a reversible manner with a similar activity to diazoxide. This required the presence of hydrolysable nucleotides and appeared to be mediated by interaction of Y-26763 with SUR1 since: (a) tolbutamide was able to reverse the actions of Y-26763 and (b) Y-26763 failed to activate Kir6.2ΔC26 in the absence of SUR1. We conclude that Y-26763-induced inhibition of insulin release is dependent upon the activation of K ATP channels in human β-cells.

Sulfonylurea Receptor Type 1 Knock-out Mice Have Intact Feeding-stimulated Insulin Secretion despite Marked Impairment in Their Response to Glucose

The ATP-sensitive potassium channel is a key molecular complex for glucose-stimulated insulin secretion in pancreatic beta cells. In humans, mutations in either of the two subunits for this channel, the sulfonylurea type 1 receptor (Sur1) or Kir6.2, cause persistent hyperinsulinemic hypoglycemia of infancy. We have generated and characterized Sur1 null mice. Interestingly, these animals remain euglycemic for a large portion of their life despite constant depolarization of membrane, elevated cytoplasmic free Ca(2+) concentrations, and intact sensitivity of the exocytotic machinery to Ca(2+). A comparison of glucose- and meal-stimulated insulin secretion showed that, although Sur1 null mice do not secrete insulin in response to glucose, they secrete nearly normal amounts of insulin in response to feeding. Because Sur1 null mice lack an insulin secretory response to GLP-1, even though their islets exhibit a normal rise in cAMP by GLP-1, we tested their response to cholinergic stimulation. We found that perfused Sur1 null pancreata secreted insulin in response to the cholinergic agonist carbachol in a glucose-dependent manner. Together, these findings suggest that cholinergic stimulation is one of the mechanisms that compensate for the severely impaired response to glucose and GLP-1 brought on by the absence of Sur1, thereby allowing euglycemia to be maintained.

The antimalarial agent mefloquine inhibits ATP-sensitive K-channels.

The aim of this study was to determine whether antimalarial agents inhibit ATP-sensitive potassium (K(ATP)) channels and thereby contribute to the observed side-effects of these drugs. Mefloquine (10 - 100 microM), but not artenusate (100 microM), stimulated insulin release from pancreatic islets in vitro. Macroscopic K(ATP) currents were studied in inside-out patches excised from Xenopus oocytes expressing cloned K(ATP) channels. Mefloquine (IC(50) approximately 3 microM), quinine (IC(50) approximately 3 microM), and chloroquine inhibited the pancreatic beta-cell type of K(ATP) channel Kir6.2/SUR1. Artenusate (100 microM) was without effect. Mefloquine and quinine also blocked a truncated form of Kir6.2 (Kir6. 2DeltaC36) when expressed in the absence of SUR1. The extent of block was similar to that observed for Kir6.2/SUR1 currents. Our results suggest that inhibition of the beta-cell K(ATP) channel accounts for the ability of quinoline-based antimalarial drugs to stimulate insulin secretion, and thereby produce hypoglycaemia. The results also indicate that quinoline-based antimalarial agents inhibit K(ATP) channels by interaction with the Kir6.2 subunit. This subunit is common to beta-cell, neuronal, cardiac, skeletal muscle, and some smooth muscle K(ATP) channels suggesting that K(ATP) channel inhibition may contribute to the other side effects of these drugs, which include cardiac conduction abnormalities and neuropsychiatric disturbances.

Identification of molecular regions responsible for the membrane trafficking of Kir6.2.

The subunits of the pancreatic ATP-sensitive potassium channel Kir6.2 and the sulphonylurea receptor (SUR1) contain endoplasmic reticulum (ER) retention signals (RKR), which prevent their plasma membrane expression when expressed individually. When co-expressed, however, these signals are masked and the complex traffics to the plasma membrane. To investigate this further, we have expressed epitope-tagged chimaeras between Kir6.2 and Kir2.1 (which traffics to the membrane independently of SUR1) in Xenopus oocytes alone and together with SUR1. By staining sections of the oocytes, we show that, in addition to the ER retention signal present in the distal C-terminus, the M2 transmembrane and the proximal C-terminal regions also contribute to the inability of Kir6.2 to traffic to the membrane in the absence of SUR1. Furthermore, by staining the whole oocytes for the hexa-histidine tag attached to the N-terminus of SUR1, we provide direct experimental evidence that the N-terminus of SUR1 is extracellular.

The role of lysine 185 in the kir6.2 subunit of the ATP-sensitive channel in channel inhibition by ATP.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, forms ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on Kir6.2. 2. Previous studies have shown that mutation of the lysine residue at position 185 (K185) in the C-terminus of Kir6.2 to glutamine, decreased the channel sensitivity to ATP without affecting the single-channel conductance or the intrinsic channel kinetics. This mutation also impaired 8-azido[32P]-ATP binding to Kir6.2. 3. To determine if K185 interacts directly with ATP, we made a range of mutations at this position, and examined the effect on the channel ATP sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2DeltaC26. 4. Substitution of K185 by a positively charged amino acid (arginine) had no substantial effect on the sensitivity of the channel to ATP. Mutation to a negatively charged residue markedly decreased the channel ATP sensitivity: the Ki for ATP inhibition increased from 85 microM to >30 mM when arginine was replaced with aspartic acid. Substitution of neutral residues had intermediate effects. 5. The inhibitory effects of ADP, ITP and GTP were also reduced when K185 was mutated to glutamine or glutamate. 6. The results indicate that a positively charged amino acid at position 185 is required for high-affinity ATP binding to Kir6.2. Our results demonstrate that ATP does not interact with the side-chain of K185. It remains unclear whether ATP interacts with the backbone of this residue, or whether its mutation influences ATP binding allosterically.

Involvement of the n-terminus of Kir6.2 in coupling to the sulphonylurea receptor.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. ATP inhibits the channel by interacting with Kir6.2, while sulphonylureas block channel activity by interaction with a high-affinity site on SUR1 and a low-affinity site on Kir6.2. MgADP and diazoxide interact with SUR1 to promote channel activity. 2. We examined the effect of N-terminal deletions of Kir6.2 on the channel open probability, ATP sensitivity and sulphonylurea sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2/SUR1. 3. A 14 amino acid N-terminal deletion (DeltaN14) did not affect the gating, ATP sensitivity or tolbutamide block of a truncated isoform of Kir6.2, Kir6.2DeltaC26, expressed in the absence of SUR1. Thus, the N-terminal deletion does not alter the intrinsic properties of Kir6.2. 4. When Kir6.2DeltaN14 was coexpressed with SUR1, the resulting KATP channels had a higher open probability (Po = 0.7) and a lower ATP sensitivity (Ki = 196 microM) than wild-type (Kir6.2/SUR1) channels (Po = 0.32, Ki = 28 microM). High-affinity tolbutamide block was also abolished. 5. Truncation of five or nine amino acids from the N-terminus of Kir6.2 also enhanced the open probability, and reduced both the ATP sensitivity and the fraction of high-affinity tolbutamide block, although to a lesser extent than for the DeltaN14 deletion. Site-directed mutagenesis suggests that hydrophobic residues in Kir6. 2 may be involved in this effect. 6. The reduced ATP sensitivity of Kir6.2DeltaN14 may be explained by the increased Po. However, when the Po was decreased (by ATP), tolbutamide was unable to block Kir6. 2DeltaN14/SUR1-K719A,K1385M currents, despite the fact that the drug inhibited Kir6.2-C166S/SUR1-K719A,K1385M currents (which in the absence of ATP have a Po of > 0.8 and are not blocked by tolbutamide). Thus the N-terminus of Kir6.2 may be involved in coupling sulphonylurea binding to SUR1 to closure of the Kir6.2 pore.

ATP inhibition of KATP channels: control of nucleotide sensitivity by the N-terminal domain of the Kir6.2 subunit.

1. To gain insight into the role of the cytoplasmic regions of the Kir6.2 subunit in regulating channel activity, we have expressed the sulphonylurea receptor SUR1 with Kir6.2 subunits containing systematic truncations of the N- and C-termini. Up to 30 amino acids could be truncated from the N-terminus, and up to 36 amino acids from the C-terminus without loss of functional channels in co-expression with SUR1. Furthermore, Kir6.2DeltaC25 and Kir6. 2DeltaC36 subunits expressed functional channels in the absence of SUR1. 2. In co-expression with SUR1, N-terminal truncations increased Ki,ATP ([ATP] causing half-maximal inhibition of channel activity) by as much as 10-fold, accompanied by an increase in the ATP-insensitive open probability, whereas the C-terminal truncations did not affect the ATP sensitivity of co-expressed channels. 3. A mutation in the near C-terminal region, K185Q, reduced ATP sensitivity of co-expressed channels by approximately 30-fold, and on the Kir6.2DeltaN2-30 background, this mutation decreased ATP sensitivity of co-expressed channels by approximately 400-fold. 4. Each of these mutations also reduced the sensitivity to inhibition by ADP, AMP and adenosine tetraphosphate. 5. The results can be quantitatively explained by assuming that the N-terminal deletions stabilize the ATP-independent open state, whereas the Kir6.2K185Q mutation may alter the stability of ATP binding. These two effects are energetically additive, causing the large reduction of ATP sensitivity in the double mutant channels.

Involvement of the N-terminus of Kir6.2 in the inhibition of the KATP channel by ATP.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, expresses ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on the Kir6. 2 subunit. 2. We examined the effect on the channel ATP sensitivity of mutating the arginine residue at position 50 (R50) in the N-terminus of Kir6.2, by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2DeltaC26. 3. Substitution of R50 by serine, alanine or glycine reduced the Ki for ATP inhibition from 117 microM to 800 microM, 1.1 mM and 3.8 mM, respectively. The single-channel conductance and kinetics were unaffected by any of these mutations. Mutation to glutamate, lysine, asparagine, glutamine or leucine had a smaller effect (Ki, approximately 300-400 microM). The results indicate that the side chain of the arginine residue at position 50 is unlikely to contribute directly to the binding site for ATP, and suggest it may affect ATP inhibition by allosteric interactions. 4. Mutation of the isoleucine residue at position 49 to glycine (I49G) reduced the channel ATP sensitivity, while the mutation of the glutamate residue at position 51 to glycine (E51G) did not. 5. When a mutation in the N-terminus of Kir6.2DeltaC26 that alters ATP sensitivity (R50S; Ki, 800 microM) was combined with one in the C-terminus (E179Q; Ki, 300 microM), the Ki for the apparent ATP sensitivity was increased to 2.8 mM. The Hill coefficient was also increased. This suggests that the N- and C-termini of Kir6.2 may co-operate to influence channel closure by ATP.

Mechanism of Cloned ATP-sensitive Potassium Channel Activation by Oleoyl-CoA

Insulin secretion from pancreatic beta cells is coupled to cell metabolism through closure of ATP-sensitive potassium (KATP) channels, which comprise Kir6.2 and sulfonylurea receptor (SUR1) subunits. Although metabolic regulation of KATP channel activity is believed to be mediated principally by the adenine nucleotides, other metabolic intermediates, including long chain acyl-CoA esters, may also be involved. We recorded macroscopic and single-channel currents from Xenopus oocytes expressing either Kir6.2/SUR1 or Kir6. 2DeltaC36 (which forms channels in the absence of SUR1). Oleoyl-CoA (1 microM) activated both wild-type Kir6.2/SUR1 and Kir6.2DeltaC36 macroscopic currents, approximately 2-fold, by increasing the number and open probability of Kir6.2/SUR1 and Kir6.2DeltaC36 channels. It was ineffective on the related Kir subunit Kir1.1a. Oleoyl-CoA also impaired channel inhibition by ATP, increasing the Ki values for both Kir6.2/SUR1 and Kir6.2DeltaC36 currents by approximately 3-fold. Our results indicate that activation of KATP channels by oleoyl-CoA results from an interaction with the Kir6.2 subunit, unlike the stimulatory effects of MgADP and diazoxide which are mediated through SUR1. The increased activity and reduced ATP sensitivity of KATP channels by oleoyl-CoA might contribute to the impaired insulin secretion observed in non-insulin-dependent diabetes mellitus.

Molecular determinants of KATP channel inhibition by ATP.

ATP-sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore-forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg-nucleotides complicates analysis of nucleotide inhibition of Kir6. 2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP-sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the beta-phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N-terminus preceding, and the C-terminus immediately following, the transmembrane domains. Some mutations in the C-terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single-channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C-terminus in KATP channel gating.

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

ATP-sensitive potassium (KATP) channels in the pancreatic beta 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 KATP 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 beta cell KATP 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 KATP 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 KATP channels, and MgATP actually activated KATP 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 KATP 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+.