Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy: nucleotide-binding fold-2 mutation impairs cooperative binding of adenine nucleotides to sulfonylurea receptor 1.

Abstract

To elucidate the genetic etiology of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) in the Japanese population, we conducted a polymerase chain reaction-single-strand conformation polymorphism analysis of the sulfonylurea receptor 1 (SUR1) and Kir6.2 genes in 17 Japanese PHHI patients, including a pair of siblings from a consanguineous family. We also analyzed the glutamate dehydrogenase gene for the exons encoding an allosteric regulatory domain of the enzyme. In the SUR1 gene, we identified one frameshift (I446fsdelT) and two missense (R1420C, R1436Q) mutations. None of these mutations were found in control Japanese subjects. Siblings homozygous for the R1420C mutation had a mild form, whereas two patients heterozygous for the I446fsdelT and R1436Q mutations, respectively, exhibited a severe form of PHHI. Functional consequences of these mutations on K AT P function were evaluated using 8 6 R b + e fflux studies in COS-7 cells. SUR1-446fsdelT and SUR1-1436Q did not form a functional K AT P . Western blot analysis after transient expression in COS-7 cells revealed the expression of SUR1-1436Q protein to be markedly reduced, suggesting SUR1-1436Q to be unstable in these cells. K AT P ( S U R 1 -1 4 2 0 C ) showed reduced responses to metabolic inhibition by oligomycin and 2-deoxyglucose. K AT P c h a n n e l s are under complex regulation by intracellular ATP and ADP. AT P both inhibits and activates these channels. The inhibition is probably mediated through direct ATP interaction with a pore-forming subunit Kir6.2, whereas the activation is likely to be through a regulatory subunit SUR1. There is a cooperative regulation of ATP and ADP binding to SUR1, and this cooperativity may be involved in regulating the K AT P channel. 256450) is characterized by inappropriately elevated insulin secretion in the presence of hypoglycemia. Mutations have been identified in the genes encoding subunits of the ATP-sensitive potassium channel ( K AT P ), sulfonylurea receptor 1 (SUR1), and Kir6.2 genes in recessive and sporadic forms of PHHI (1-8). Mutations were also identified in the glucokinase gene (9) and in the glutamate dehydrogenase gene in patients with hyperinsulinemic hypoglycemia and hyperammonemia (10). However, the genetic etiology of PHHI in Japanese patients is unknown.The K AT P channel, a heterooctamer composed of two essential subunits, SUR1 and Kir6.2 (BIR) (11), plays a central role in glucose-induced insulin secretion in pancreatic -cells (12-15). Kir6.2, a pore-forming subunit and a member of the weak inward rectifier family (11), has been suggested

Functional Analysis of a Mutant Sulfonylurea Receptor, SUR1-R1420C, That Is Responsible for Persistent Hyperinsulinemic Hypoglycemia of Infancy

“…The mean current amplitude at Ϫ100 mV was 3.0 Ϯ 1.0 nA (n ϭ 10) for Kir6.2/SUR1 and 1.5 Ϯ 0.6 nA (n ϭ 15) for Kir6.2/SUR1-R1420C channels. The smaller currents observed with the mutant channel are probably due to reduced expression of the mutant SUR (31). A quantitatively similar reduction in SUR1-R1420C protein expression was observed in COS-7 cells, indicating that the lower expression of mutant SUR1 is confined to the oocyte expression system.…”

“…An 86 Rb ϩ efflux study revealed that K ATP ϩ channels composed of SUR1-R1420C and Kir6.2 are not activated by metabolic inhibition as much as wild-type channels. This may be related to the fact that the expression level of SUR1-R1420C was only about half that of the wild-type channel when it was transiently expressed in COS-7 cells (31). We also reported that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes the binding of 8-azido-ATP at NBF1 of wild-type SUR1 (19), and that the R1420C mutation impairs this effect without altering high affinity 8-azido-ATP binding to NBF1 (31).…”

“…This may be related to the fact that the expression level of SUR1-R1420C was only about half that of the wild-type channel when it was transiently expressed in COS-7 cells (31). We also reported that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes the binding of 8-azido-ATP at NBF1 of wild-type SUR1 (19), and that the R1420C mutation impairs this effect without altering high affinity 8-azido-ATP binding to NBF1 (31). However, it is not clear whether the mutation lowers the affinity of NBF2 for nucleotides or if it affects cooperative interactions between the two NBFs.…”

“…This might occur if, for example, channel activation is greater when MgADP binds to both NBFs than when MgATP binds to NBF1 and MgADP to NBF2. (31). Thus the R1420C mutation results in a reduced K ATP ϩ current whether expressed in oocytes or in mammalian cells.…”

“…Recently, we identified a missense SUR1 mutation (R1420C) in Japanese PHHI patients (31). They were siblings from a consanguineous family and homozygous for the mutation, and their clinical characteristics consisted of a mild form of PHHI.…”