Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy (original) (raw)
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Journal of Biological Chemistry, 2002
Mutations in the pancreatic ATP-sensitive potassium (K(ATP)) channel subunits sulfonylurea receptor 1 (SUR1) and the inwardly rectifying potassium channel Kir6.2 cause persistent hyperinsulinemic hypoglycemia of infancy. We have identified a SUR1 mutation, L1544P, in a patient with the disease. Channels formed by co-transfection of Kir6.2 and the mutant SUR1 in COS cells have reduced response to MgADP ( approximately 10% that of the wild-type channels) and reduced surface expression ( approximately 19% that of the wild-type channels). However, the steady-state level of the SUR1 protein is unaffected. Treating cells with lysosomal or proteasomal inhibitors did not improve surface expression of the mutant channels, suggesting that increased degradation of mutant channels by either pathway is unlikely to account for the reduced surface expression. Removal of the RKR endoplasmic reticulum retention/retrieval trafficking motif in either SUR1 or Kir6.2 increased the surface expression of the mutant channel by approximately 35 and approximately 20%, respectively. The simultaneous removal of the RKR motif in both channel subunits restored surface expression of the mutant channel to the wild-type channel levels. Thus, the L1544P mutation may interfere with normal trafficking of K(ATP) channels by causing improper shielding of the RKR endoplasmic reticulum retention/retrieval trafficking signals in the two channel subunits.
Journal of Biological Chemistry, 2003
Mutations in the sulfonylurea receptor 1 (SUR1), a subunit of ATP-sensitive potassium (K ATP ) channels, cause familial hyperinsulinism. One such mutation, deletion of phenylalanine 1388 (⌬Phe-1388), leads to defects in both trafficking and MgADP response of K ATP channels. Here we investigated the biochemical features of Phe-1388 that control the proper trafficking and function of K ATP channels by substituting the residue with all other 19 amino acids. Whereas surface expression is largely dependent on hydrophobicity, channel response to MgADP is governed by multiple factors and involves the detailed architecture of the amino acid side chain. Thus, structural features in SUR1 required for proper channel function are distinct from those required for correct protein trafficking. Remarkably, replacing Phe-1388 by leucine profoundly alters the physiological and pharmacological properties of the channel. The F1388L-SUR1 channel has increased sensitivity to MgADP and metabolic inhibition, decreased sensitivity to glibenclamide, and responds to both diazoxide and pinacidil. Because this conservative amino acid substitution occurs in the SUR2A and SUR2B isoforms, the mutation provides a mechanism by which functional diversities in K ATP channels are generated.
Endocrine Reviews, 2010
Context: ATP-sensitive potassium (K ATP) channels regulate insulin secretion by coupling glucose metabolism to -cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect K ATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP 4Ϫ or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on -cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating.
Diabetes, 2011
OBJECTIVE Congenital hyperinsulinism in infancy (CHI) is characterized by unregulated insulin secretion from pancreatic β-cells; severe forms are associated with defects in ABCC8 and KCNJ11 genes encoding sulfonylurea receptor 1 (SUR1) and Kir6.2 subunits, which form ATP-sensitive K+ (KATP) channels in β-cells. Diazoxide therapy often fails in the treatment of CHI and may be a result of reduced cell surface expression of KATP channels. We hypothesized that conditions known to facilitate trafficking of cystic fibrosis transmembrane regulator (CFTR) and other proteins in recombinant expression systems might increase surface expression of KATP channels in native CHI β-cells. RESEARCH DESIGN AND METHODS Tissue was isolated during pancreatectomy from eight patients with CHI and from adult cadaver organ donors. Patients were screened for mutations in ABCC8 and KCNJ11. Isolated β-cells were maintained at 37°C or 25°C and in the presence of 1) phorbol myristic acid, forskolin and 3-isobutyl...
Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels
The EMBO Journal, 2006
ATP-sensitive potassium (K ATP ) channels couple the metabolic status of a cell to its membrane potential-a property that endows pancreatic b-cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that K ATP channels undergo rapid internalisation from the plasma membrane by clathrin-mediated endocytosis. We present several lines of evidence to demonstrate that endocytosis is mediated by a tyrosine based signal ( 330 YSKF 333 ) located in the carboxy-terminus of Kir6.2 and that SUR1 has no direct role. We show that genetic mutations, Y330C and F333I, which cause permanent neonatal diabetes mellitus, disrupt this motif and abrogate endocytosis of reconstituted mutant channels. The resultant increase in the surface density of K ATP channels would predispose b-cells to hyperpolarise and may account for reduced insulin secretion in these patients. The data imply that endocytosis of K ATP channels plays a crucial role in the (patho)-physiology of insulin secretion.
Journal of Biological Chemistry, 2010
The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (K(ATP)) channels that regulate insulin secretion in pancreatic beta-cells. Mutations in K(ATP) channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for K(ATP) channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues.
Journal of Biological Chemistry
Edited by Norma M. Allewell Neuroendocrine-type ATP-sensitive K ؉ (K ATP) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2) 4 , comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K ؉-selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward-or outward-facing conformations with low or high dissociation constant, K D , values for the conformation-sensitive channel antagonist [ 3 H]glibenclamide ([ 3 H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's K D for [ 3 H]GBM.ThisreductionrequiredtheKirNterminus(KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower K D for [ 3 H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases K ATP channels toward open states, consistent with SUR1 variants with lower K D values causing neonatal diabetes, whereas increased K D values cause congenital hyperinsulinism. Neuroendocrine-type ATP-sensitive K ϩ (K ATP) 2 channels comprise an ATP-binding cassette (ABC) protein (1), ABCC8/ SUR1, and a K ϩ-selective inward rectifier (2), KCNJ11/Kir6.2, assembled as heterooctamers (3-5), (SUR1/Kir6.2) 4. In pancreatic -cells, these channels are metabolite sensors that couple cellular metabolism with membrane electrical activity to link insulin secretion with blood glucose levels. This coupling is critical for normal physiology; loss of channel function is a cause of congenital hyperinsulinism (Ref. 6; for reviews, see Refs. 7 and 8), whereas gain-of-function mutations in Kir6.2 (9) and SUR1 (10) cause neonatal diabetes (ND) (for reviews, see Refs. 11 and 12). Gain of function is one cause of mature-onset diabetes of the young (13), whereas a ABCC8/SUR1 polymorphism, the Ala amino acid allele at p.A1369S, is associated with an increased risk for type 2 diabetes (14). Channel activity is regulated positively by ATP and ADP binding to SUR1 and negatively by nucleotide binding to Kir6.2 (15-20). Additionally, multiple metabolites, including phosphoinositides (21-23) and long-chain acyl-CoA esters (24-26), and phosphorylation (27-29) positively modulate channel activity. Pharmacologic modulation of SUR1 by channel antagonists, sulfonylureas like glibenclamide (GBM) and glinides like repaglinide (30-36), and by agonists like diazoxide (Refs. 31 and 37-39; for a review, see Ref. 40) is clinically important. These physiologic and pharmacologic modulators all affect channel gating via allosteric interactions in the sense that their binding sites on SUR1 are coupled to and known to be physically distinct from one another based on structural studies (41, 42). The available data are consistent with the idea that SUR1 exists in multiple conformations differing in affinity for ligands and ability to activate channel openings. How nucleotides regulate K ATP channels remains an open question. Early electrophysiological studies (15) showed that although ATP inhibited activity, MgADP, acting through SUR1, activated channel openings (16-18, 20). The finding that SUR1 was in the ABC protein family (1), whose members are ATPases that use the energy of ATP binding and hydrolysis to transport substrates across membranes (43-45), suggested that
Diabetes, 1998
The ATP-sensitive potassium channel, K AT P channel, a functional complex of the sulfonylurea receptor 1, SUR1, and an inward rectifier potassium channel subunit, Kir6.2, regulates insulin secretion in the pancreas. Mutations in both the Kir6.2 and SUR1 genes are associated with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a disorder of pancreatic -cell function characterized by excess insulin secretion and hypoglycemia. We have studied the functional properties of novel SUR1 mutations identified in PHHI patients, including H125Q, N188S, F591L, T1139M, R1215Q, G1382S, and R1394H. R1394H and F1388 SUR1, a previously identified PHHI mutation, resulted in no functional channels when coexpressed with Kir6.2 in COS cells, while H125Q, N188S, F591L, T1139M, R1215Q, and G1382S SUR1 generated functional channels in the absence of AT P. With the exception of N188S and H125Q, all mutants had reduced response to stimulation by MgADP. These results indicate that lack of, or reduction of, K AT P channel sensitivity to MgADP is a common molecular defect associated with the disease. The mutant channels also showed varied response to activation by the potassium channel opener diazoxide. Because these mutations are distributed throughout the molecule, our data have new implications for structurefunction relationships of the K ATP channel, suggesting that structural elements in SUR1 outside of the two nucleotide-binding folds are also important in regulating channel activity.