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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.
A Kir6.2 mutation causing neonatal diabetes impairs electrical activity and insulin secretion from INS-1 beta-cells.
ATP-sensitive K(+) channels (K(ATP) channels) couple beta-cell metabolism to electrical activity and thereby play an essential role in the control of insulin secretion. Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of this channel, cause neonatal diabetes. We investigated the effect of the most common neonatal diabetes mutation (R201H) on beta-cell electrical activity and insulin secretion by stable transfection in the INS-1 cell line. Expression was regulated by placing the gene under the control of a tetracycline promoter. Transfection with wild-type Kir6.2 had no effect on the ATP sensitivity of the K(ATP) channel, whole-cell K(ATP) current magnitude, or insulin secretion. However, induction of Kir6.2-R201H expression strongly reduced K(ATP) channel ATP sensitivity (the half-maximal inhibitory concentration increased from approximately 20 mumol/l to approximately 2 mmol/l), and the metabolic substrate methyl succinate failed to close K(ATP) channels or stimulate electrical activity and insulin secretion. Thus, these results directly demonstrate that Kir6.2 mutations prevent electrical activity and insulin release from INS-1 cells by increasing the K(ATP) current and hyperpolarizing the beta-cell membrane. This is consistent with the ability of the R201H mutation to cause neonatal diabetes in patients. The relationship between K(ATP) current and the membrane potential reveals that very small changes in current amplitude are sufficient to prevent hormone secretion.
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.
Potassium channel regulation.
The sulphonylurea receptor (SUR) is a member of the ATP-binding cassette (ABC) family of membrane proteins. It functions as the regulatory subunit of the ATP-sensitive potassium (KATP) channel, which comprises SUR and Kir6.x proteins. Here, we review data demonstrating functional differences between the two nucleotide binding domains (NBDs) of SUR1. In addition, to explain the structural basis of these functional differences, we have constructed a molecular model of the NBD dimer of human SUR1. We discuss the experimental data in the context of this model, and show how the model can be used to design experiments aimed at elucidating the relationship between the structure and function of the KATP channel.
Direct interaction of Na-azide with the KATP channel.
1. The effects of the metabolic inhibitor sodium azide were tested on excised macropatches from Xenopus oocytes expressing cloned ATP-sensitive potassium (KATP) channels of the Kir6.2/SUR1 type. 2. In inside-out patches from oocytes expressing Kir6.2 delta C36 (a truncated form of Kir6.2 that expresses in the absence of SUR), intracellular Na-azide inhibited macroscopic currents with an IC50 of 11 mM. The inhibitory effect of Na-azide was mimicked by the same concentration of NaCl, but not by sucrose. 3. Na-azide and NaCl blocked Kir6.2/SUR1 currents with IC50 of 36 mM and 19 mM, respectively. Inhibition was abolished in the absence of intracellular Mg2+. In contrast, Kir6.2 delta C36 currents were inhibited by Na-azide both in the presence or absence of intracellular Mg2+. 4. Kir6.2/SUR1 currents were less sensitive to 3 mM Na-azide in the presence of MgATP. This apparent reduction in sensitivity is caused by a small activatory effect of Na-azide conferred by SUR. 5. We conclude that, in addition to its well-established inhibitory effect on cellular metabolism, which leads to activation of KATP channels in intact cells, intracellular Na-azide has direct effects on the KATP channel. Inhibition is intrinsic to Kir6.2, is mediated by Na+, and is modulated by SUR. There is also a small, ATP-dependent, stimulatory effect of Na-azide mediated by the SUR subunit. The direct effects of 3 mM Na-azide on KATP channels are negligible in comparison to the metabolic activation produced by the same Na-azide concentration.
New windows on the mechanism of action of K(ATP) channel openers.
K(ATP) channel openers are a diverse group of drugs with a wide range of potential therapeutic uses. Their molecular targets, the K(ATP) channels, exhibit tissue-specific responses because they possess different types of regulatory sulfonylurea receptor subunits. It is well recognized that complex interactions occur between K(ATP) channel openers and nucleotides, but the cloning of the K(ATP) channel has introduced a new dimension to the study of these events and has furthered our understanding of the molecular basis of the action of K(ATP) channel openers.
In vitro mechanism of action on insulin release of S-22068, a new putative antidiabetic compound.
1. The MIN6 cell line derived from in vivo immortalized insulin-secreting pancreatic beta cells was used to study the insulin-releasing capacity and the cellular mode of action of S-22068, a newly synthesized imidazoline compound known for its antidiabetic effect in vivo. 2. S-22068, was able to release insulin from MIN6 cells in a dose-dependent manner with a half-maximal stimulation at 100 micronM. Its efficacy (8 fold over the basal value), which did not differ whatever the glucose concentration (stimulatory or not), was intermediate between that of sulphonylurea and that of efaroxan. 3. Similarly to sulphonylureas and classical imidazolines, S-22068 blocked K(ATP) channels and, in turn, opened nifedipine-sensitive voltage-dependent Ca2+ channels, triggering Ca2+ entry. 4. Similarly to other imidazolines, S-22068 induced a closure of cloned K(ATP) channels injected to Xenopus oocytes by interacting with the pore-forming Kir6.2 moiety. 5. S-22068 did not interact with the sulphonylurea binding site nor with the non-I1 and non-I2 imidazoline site evidenced in the beta cells that is recognized by the imidazoline compounds efaroxan, phentolamine and RX821002. 6. We conclude that S-22068 is a novel imidazoline compound which stimulates insulin release via interaction with an original site present on the Kir6.2 moiety of the beta cell K(ATP) channels.
Amantadine and sparteine inhibit ATP-regulated K-currents in the insulin-secreting beta-cell line, HIT-T15.
1. The effects of pharmacological agents that potentiate insulin release were studied on ATP-regulated K-currents (K-ATP currents) in the insulin-secreting beta-cell line HIT-T15 by use of patch-clamp methods. 2. The tricyclic drug, 1-adamantanamine (amantadine), reversibly inhibited both whole-cell currents (with a Ki of 120 microM) and single channel currents in inside-out patches. This effect was principally due to an increase in a long closed state which reduced the channel open probability. The related compound, 1-adamantanol, in which the amino group is substituted by a hydroxyl one, did not inhibit K-ATP currents substantially. 3. The alkaloid, sparteine, reversibly inhibited both whole-cell K-ATP currents (Ki = 171 microM) and single channel currents in inside-out patches. 4. The results suggest that sparteine and amantadine can block the K-ATP channel from either side of the membrane and support the idea that at least part of the stimulatory effect of these agents on insulin secretion results from inhibition of this channel.
Voltage-activated calcium and potassium currents in human pancreatic beta-cells.
1. The whole-cell configuration of the patch clamp technique was used to study inward and delayed outward currents in beta-cells isolated from human pancreatic islets. 2. The delayed outward current activated at about -20 mV and increased linearly with further depolarization. The instantaneous current-voltage (I-V) relation, measured by tail current analysis, reversed at -70 mV. This is close to the K+ equilibrium potential and suggests the outward current is carried primarily by potassium ions. In support of this idea, outward currents were abolished when internal K+ was replaced by the impermeant cation N-methyl-D-glucamine (NMG). 3. The voltage dependence of K+ current activation could be fitted by a sigmoidal function with a mid-point at +1 mV. K+ currents showed voltage-dependent inactivation which was half-maximal at -25 mV. 4. Inward currents were studied after outward currents were suppressed by replacing internal potassium with NMG. In 5 mM [Ca2+]o, the inward current activated between -50 and -40 mV, had a peak amplitude at -10 mV and reversed at potentials positive to +60 mV. The voltage dependence of inward current activation was sigmoidal with half-maximal activation at -10 mV in 5 mM [Ca2+]o and at -22 mV in 5 mM [Ba2+]o. 5. Inward currents were unaffected by tetrodotoxin (TTX), but could be blocked by cadmium ions. Barium was also capable of carrying inward current. This pharmacology is consistent with inward currents flowing through Ca2+ channels. 6. The inactivation of the inward current was dependent on calcium entry. In two-pulse experiments, the voltage dependence of inactivation was U-shaped, and resembled that of the calcium current. Barium currents showed little inactivation. 7. In two-pulse experiments the degree of inward current inactivation during the test pulse was related to the amount of calcium entry during the first pulse. Calcium entering at more positive potentials was less effective at producing inactivation. 8. Calcium and barium currents also showed a slow, voltage-dependent inactivation when the holding potential was changed between -100 and -40 mV. This inactivation developed with a time course of seconds. 9. The Ca2+ and K+ currents described here are similar to those reported for rodent beta-cells and indicate the rodent beta-cell provides a good model for that of man.
The Walter B. Cannon Physiology in Perspective Lecture, 2007. ATP-sensitive K+ channels and disease: from molecule to malady.
This essay is based on a lecture given to the American Physiological Society in honor of Walter B. Cannon, an advocate of homeostasis. It focuses on the role of the ATP-sensitive potassium K(+) (K(ATP)) channel in glucose homeostasis and, in particular, on its role in insulin secretion from pancreatic beta-cells. The beta-cell K(ATP) channel comprises pore-forming Kir6.2 and regulatory SUR1 subunits, and mutations in either type of subunit can result in too little or too much insulin release. Here, I review the latest information on the relationship between K(ATP) channel structure and function, and consider how mutations in the K(ATP) channel genes lead to neonatal diabetes or congenital hyperinsulinism.
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.
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.
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.
Sulfonylurea sensitivity of adenosine triphosphate-sensitive potassium channels from beta cells and extrapancreatic tissues.
Sulfonylureas are widely used to stimulate insulin secretion in type 2 diabetic patients because they close adenosine triphosphate-sensitive potassium (K(ATP)) channels in the pancreatic beta-cell membrane. This action is mediated by binding of the drug to the sulfonylurea receptor (SUR1) subunit of the channel. K(ATP) channels are also present in a range of extrapancreatic tissues, but many of these contain an alternative type of SUR subunit (SUR2A in heart and SUR2B in smooth muscle). The sulfonylurea-sensitivity of K(ATP) channels containing the different types of SUR is variable: gliclazide and tolbutamide block the beta cell, but not the cardiac or smooth muscle types of K(ATP) channels with high affinity. Glibenclamide and glimepiride, on the other hand, block channels containing SUR1 and SUR2 with similar affinity. The reversibility of the different sulfonylureas also varies. Tolbutamide and gliclazide produce a reversible inhibition of Kir6.2/SUR1 and Kir6.2/SUR2 channels, whereas glibenclamide has a reversible effect on cardiac, but not beta-cell, K(ATP) channels. In this article, we summarize current knowledge of how sulfonylureas act on K(ATP) channels containing the different types of sulfonylurea receptor, and discuss the implications of these findings for the use of sulfonylureas in the treatment of diabetes mellitus.