Beta-cell KATP Channel Research Articles

Hyperinsulinism of the newborn

Neonatal hyperinsulinism (HI) is a clinical syndrome of pancreatic beta-cell dysfunction characterized by failure to suppress insulin secretion in the presence of hypoglycemia. Although rare, it is the most common cause for persistent hypoglycemia in the newborn period. Treatment can be extremely difficult, and partial pancreatectomy is frequently required to prevent recurrent hypoglycemia and irreversible brain damage. In the last 5 years much has been learned about the pathophysiology of this disease. In most patients, the disease is caused by recessive mutations in either of the 2 functional subunits of the beta-cell KATP channel (SUR1 or Kir6.2). Although in most families, the disease is transmitted as an autosomal recessive trait, a novel form of transmission, resulting in focal involvement of the pancreas has recently been described. Not all patients with HI have mutations in the KATP channel genes. An activating mutation in the "glucose sensor" glucokinase has recently been reported in one family with diazoxide-responsive autosomal dominant hyperinsulinemic hypoglycemia. Also, a new syndrome of hyperinsulinism associated with benign hyperammonemia was recently described and found to be caused by activating mutations in the glutamate dehydrogenase (GDH) gene (GLUD-1). Thus, the clinical syndrome of HI can be caused by mutations in 4 different genes and can be transmitted as either a recessive or a dominant trait. These findings aid in the therapeutic decision-making process and improve the accuracy and precision of genetic counseling. Despite these recent discoveries, however, the metabolic origin of the disease is still unknown in about 50% of cases.

Familial hyperinsulinism and pancreatic β-cell ATP-sensitive potassium channels

Familial hyperinsulinism, also known as persistent hyperinsulinemic hypoglycemia of infancy (PHHI), is a genetic disease characterized by mild to severe hypoglycemia in the presence of inappropriately high levels of insulin. The recessive form is caused by mutations in the adenosine 5'-triphosphate (ATP)-sensitive K+ channel (KATP channel) present in the plasma membrane of pancreatic beta-cells. This channel is formed by two subunits, the high-affinity sulfonylurea receptor, SUR1, and KIR6.2, a member of the inwardly rectifying family of K+ channels. KATP channels regulate insulin secretion by linking membrane excitability with glucose metabolism. Approximately 50 mutations, in both channel subunits, that abolish or alter the regulation of beta-cell KATP channels have been identified in patients with the recessive form of PHHI.

Interaction of Vanadate with the Cloned Beta Cell KATP Channel

Vanadate is used as a tool to trap magnesium nucleotides in the catalytic site of ATPases. However, it has also been reported to activate ATP-sensitive potassium (K(ATP)) channels in the absence of nucleotides. K(ATP) channels comprise Kir6.2 and sulfonylurea receptor subunits (SUR1 in pancreatic beta cells, SUR2A in cardiac and skeletal muscle, and SUR2B in smooth muscle). We explored the effect of vanadate (2 mM), in the absence and presence of magnesium nucleotides, on different types of cloned K(ATP) channels expressed in Xenopus oocytes. Currents were recorded from inside-out patches. Vanadate inhibited Kir6.2/SUR1 currents by approximately 50% but rapidly activated Kir6.2/SUR2A ( approximately 4-fold) and Kir6. 2/SUR2B ( approximately 2-fold) currents. Mutations in SUR that abolish channel activation by magnesium nucleotides did not prevent the effects of vanadate. Studies with chimeric SUR indicate that the first six transmembrane domains account for the difference in both the kinetics and the vanadate response of Kir6.2/SUR1 and Kir6. 2/SUR2A. Boiling the vanadate solution, which removes the decavanadate polymers, largely abolished both stimulatory and inhibitory actions of vanadate. Our results demonstrate that decavanadate modulates K(ATP) channel activity via the SUR subunit, that this modulation varies with the type of SUR, that it differs from that produced by magnesium nucleotides, and that it involves transmembrane domains 1-6 of SUR.

The C Terminus of SUR1 Is Required for Trafficking of KATP Channels

In beta cells from the pancreas, ATP-sensitive potassium channels, or KATP channels, are composed of two subunits, SUR1 and KIR6.2, assembled in a (SUR1/KIR6.2)4 stoichiometry. The correct stoichiometry of channels at the cell surface is tightly regulated by the presence of novel endoplasmic reticulum (ER) retention signals in SUR1 and KIR6.2; incompletely assembled KATP channels fail to exit the ER/cis-Golgi compartments. In addition to these retrograde signals, we show that the C terminus of SUR1 has an anterograde signal, composed in part of a dileucine motif and downstream phenylalanine, which is required for KATP channels to exit the ER/cis-Golgi compartments and transit to the cell surface. Deletion of as few as seven amino acids, including the phenylalanine, from SUR1 markedly reduces surface expression of KATP channels. Mutations leading to truncation of the C terminus of SUR1 are one cause of a severe, recessive form of persistent hyperinsulinemic hypoglycemia of infancy. We propose that the complete loss of beta cell KATP channel activity seen in this form of hyperinsulinism is a failure of KATP channels to traffic to the plasma membrane.

Molecular biology of adenosine triphosphate-sensitive potassium channels.

KATP channels are a newly defined class of potassium channels based on the physical association of an ABC protein, the sulfonylurea receptor, and a K+ inward rectifier subunit. The beta-cell KATP channel is composed of SUR1, the high-affinity sulfonylurea receptor with multiple TMDs and two NBFs, and KIR6.2, a weak inward rectifier, in a 1:1 stoichiometry. The pore of the channel is formed by KIR6.2 in a tetrameric arrangement; the overall stoichiometry of active channels is (SUR1/KIR6.2)4. The two subunits form a tightly integrated whole. KIR6.2 can be expressed in the plasma membrane either by deletion of an ER retention signal at its C-terminal end or by high-level expression to overwhelm the retention mechanism. The single-channel conductance of the homomeric KIR6.2 channels is equivalent to SUR/KIR6.2 channels, but they differ in all other respects, including bursting behavior, pharmacological properties, sensitivity to ATP and ADP, and trafficking to the plasma membrane. Coexpression with SUR restores the normal channel properties. The key role KATP channel play in the regulation of insulin secretion in response to changes in glucose metabolism is underscored by the finding that a recessive form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is caused by mutations in KATP channel subunits that result in the loss of channel activity. KATP channels set the resting membrane potential of beta-cells, and their loss results in a constitutive depolarization that allows voltage-gated Ca2+ channels to open spontaneously, increasing the cytosolic Ca2+ levels enough to trigger continuous release of insulin. The loss of KATP channels, in effect, uncouples the electrical activity of beta-cells from their metabolic activity. PHHI mutations have been informative on the function of SUR1 and regulation of KATP channels by adenine nucleotides. The results indicate that SUR1 is important in sensing nucleotide changes, as implied by its sequence similarity to other ABC proteins, in addition to being the drug sensor. An unexpected finding is that the inhibitory action of ATP appears to be through a site located on KIR6.2, whose affinity for ATP is modified by SUR1. A PHHI mutation, G1479R, in the second NBF of SUR1 forms active KATP channels that respond normally to ATP, but fail to activate with MgADP. The result implies that ATP tonically inhibits KATP channels, but that the ADP level in a fasting beta-cell antagonizes this inhibition. Decreases in the ADP level as glucose is metabolized result in KATP channel closure. Although KATP channels are the target for sulfonylureas used in the treatment of NIDDM, the available data suggest that the identified KATP channel mutations do not play a major role in diabetes. Understanding how KATP channels fit into the overall scheme of glucose homeostasis, on the other hand, promises insight into diabetes and other disorders of glucose metabolism, while understanding the structure and regulation of these channels offers potential for development of novel compounds to regulate cellular electrical activity.

The structure and function of the ATP-sensitive K+ channel in insulin-secreting pancreatic beta-cells.

ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. The KATP channels in pancreatic beta-cells are thought to be critical in the regulation of glucose-induced and sulfonylurea-induced insulin secretion. Until recently, however, the molecular structure of the KATP channel was not known. Cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the sulfonylurea receptors (SUR1 and SUR2) has clarified the molecular structure of KATP channels. The pancreatic beta-cell KATP channel comprises two subunits: a Kir6.2 subunit and an SUR1 subunit. Molecular biological and molecular genetic studies have provided insights into the physiological and pathophysiological roles of the pancreatic beta-cell KATP channel in insulin secretion.

A view of sur/KIR6.X, KATP channels.

ATP-sensitive potassium channels, termed KATP channels, link the electrical activity of cell membranes to cellular metabolism. These channels are heteromultimers of sulfonylurea receptor (SUR) and KIR6.X subunits associated with a 1:1 stoichiometry as a tetramer (SUR/KIR6.X forms the pores, whereas SUR regulates their activity. Changes in [ATP]i and [ADP]i gate the channel. The diversity of KATP channels results from the assembly of SUR and KIR6.X subtypes KIR6.1-based channels differ from KIR6.2 channels mainly by their smaller unitary conductance. SUR1- and SUR2-based channels are distinguished by their differential sensitivity to sulfonylureas, whereas SUR2A-based channels are distinguished from SUR2B channels by their differential sensitivity to diazoxide. Mutations that result in the loss of KATP channels in pancreatic beta-cells have been identified in SUR1 and KIR6.2. These mutations lead to familial hyperinsulinism. Understanding the mutations in SUR and KIR6.X is allowing insight into how these channels respond to nucleotides, sulfonylureas, and potassium channel openers, KCOs.

Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP.

The beta cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2DeltaC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2DeltaC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2DeltaC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 microM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.

MgADP Antagonism to Mg2+-independent ATP Binding of the Sulfonylurea Receptor SUR1

Pancreatic beta-cell ATP-sensitive potassium (KATP) channels play an important role in the regulation of glucose-induced insulin secretion. The beta-cell KATP channel comprises two subunits, the sulfonylurea receptor SUR1, a member of the ATP-binding cassette (ABC) superfamily, and Kir6.2, a member of the inward rectifier K+ channel family. The activity of the KATP channel is under complex regulation by the intracellular ATP and ADP. To understand the roles of the two nucleotide-binding folds (NBFs) of SUR1 in the regulation of KATP channel activity, we introduced point mutations into the core consensus sequence of the Walker A or B motif of each NBF of SUR1 and characterized ATP binding and ADP or MgADP antagonism to it. SUR1 was efficiently photolabeled with 8-azido-[alpha-32P]ATP and 8-azido-[gamma-32P]ATP in the presence or absence of Mg2+ or vanadate. NBF1 mutations impaired ATP binding, but NBF2 mutations did not. MgADP strongly antagonized ATP binding, and the NBF2 mutation reduced MgADP antagonism. These results show that SUR1, unlike other ABC proteins, strongly binds ATP at NBF1 even in the absence of Mg2+ and that MgADP, through binding at NBF2, antagonizes the Mg2+-independent high affinity ATP binding at NBF1.

Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor.

ATP-sensitive potassium channels (K-ATP channels) couple cell metabolism to electrical activity and are important in the physiology and pathophysiology of many tissues. In pancreatic beta-cells, K-ATP channels link changes in blood glucose concentration to insulin secretion. They are also the target for clinically important drugs such as sulphonylureas, which stimulate secretion, and the K+ channel opener diazoxide, which inhibits insulin release. Metabolic regulation of K-ATP channels is mediated by changes in intracellular ATP and Mg-ADP levels, which inhibit and activate the channel, respectively. The beta-cell K-ATP channel is a complex of two proteins: an inward-rectifier K+ channel subunit, Kir6.2, and the sulphonylurea receptor, SUR1. We show here that the primary site at which ATP acts to mediate K-ATP channel inhibition is located on Kir6.2, and that SUR1 is required for sensitivity to sulphonylureas and diazoxide and for activation by Mg-ADP.

Insulin secretion and its modulation by antiarrhythmic and sulfonylurea drugs.

Cardiovascular drugs such as antiarrhythmic agents with Vaughan Williams class Ia action have been found to induce a sporadic hypoglycemia. Recent investigation has revealed that these drugs induce insulin secretion from pancreatic beta-cells by inhibiting ATP-sensitive K+ (KATP) channels in a manner similar to sulfonylurea drugs. The mechanism underlying block of KATP channels by antiarrhythmic drugs was different, however, from that of sulfonylureas: firstly, because binding of radioactive glibenclamide could not be inhibited by unlabelled antiarrhythmic agents, and vice versa; secondly, because the two compounds differ in the kinetics and sidedness of drug action-antiarrhythmic drugs act on the channel from the inner surface of the cell membrane, whereas glibenclamide binds through the intramembrane pathway; finally, it was shown that functional KATP channels in beta-cells are composed of two distinct molecules-a sulfonylurea receptor (SUR) and a channel pore-forming subunit, an inwardly-rectifying K channel with two transmembrane regions (Kir6.2). Antiarrhythmic drugs reversibly inhibit the K+ conductance displayed by the Kir6.1 (a putative KATP channel clone)-transfected NIH3T3 cells. Therefore they appear to interact directly with the pore-forming subunit, thereby inhibiting KATP channel currents and exerting an insulinotrophic effect.

Loss of functional KATP channels in pancreatic beta-cells causes persistent hyperinsulinemic hypoglycemia of infancy.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a disorder of childhood associated with inappropriate hypersecretion of insulin by the pancreas. The pathogenesis of the condition has hitherto remained controversial. We show here that insulin-secreting cells from a homogeneous group of five infants with PHHI lack ATP-sensitive K+ channel (KATP) activity. As a consequence, PHHI beta-cells are spontaneously electrically active with high basal cytosolic Ca2+ concentrations due to Ca2+ influx. Our findings define the pathogenesis of this disease as a novel K+ channel disorder.

Mutation of the pancreatic islet inward rectifier Kir6.2 also leads to familial persistent hyperinsulinemic hypoglycemia of infancy.

Closure of ATP-sensitive potassium channels in pancreatic islet beta-cells initiates a cascade of events that leads to insulin secretion. beta-Cell ATP-sensitive potassium currents can be reconstituted by coexpression of the inward rectifier Kir6.2 and the sulfonylurea receptor (SUR), a member of the ATP-binding cassette superfamily. Mutations in SUR have been identified in individuals affected with familial persistent hyper-insulinemic hypoglycemia of infancy (PHHI), an autosomal recessive disorder of glucose metabolism which is linked to chromosome 11p15.1 and characterized by unregulated secretion of insulin and profound hypoglycemia. Because the Kir6.2 locus is within 5 kilobases (kb) of the SUR gene on chromosome 11p15.1 and it is a necessary member of the beta-cell KATP channel, we considered Kir6.2 as a candidate gene for PHHL we identified a homozygous point mutation in Kir6.2 in the genomic DNA of a child, severely affected with PHHI, from a consanguineous family. This mutation is predicted to disrupt the conserved alpha-helical second transmembrane (M2) domain of the inward rectifier by substitution of a proline for a leucine residue (L147P). Mutation of Kir6.2, like SUR, appears to lead to the PHHI phenotype suggesting that Kir6.2 is necessary, although not sufficient, for normal regulation of insulin release.

Dependence on NADH produced during glycolysis for beta-cell glucose signaling

An increase in cytosolic ATP following glucose metabolism by pancreatic beta-cells is the key signal initiating insulin secretion by causing blockade of ATP-dependent K+ channels (KATP). This induces membrane depolarization, leading to an elevation in cytosolic Ca2+ ([Ca2+]i) and insulin secretion. In this report we identify the critical metabolic step by which glucose initiates changes in beta-cell KATP channel activity, membrane potential, and [Ca2+]i. The signal stems from the glycolytic production of NADH during the oxidation of glyceraldehyde 3-phosphate, which is subsequently processed into ATP by mitochondria via the operation of discrete shuttle systems.