Functional characterization of activating mutations in the sulfonylurea receptor 1 (ABCC8) causing neonatal diabetes mellitus in Asian Indian children

Kandasamy Balamurugan, Babu Kavitha, Zhongying Yang, Viswanathan Mohan, Venkatesan Radha, Show-Ling Shyng

Research output: Contribution to journalArticle

Abstract

Background: Gain-of-function of ATP-sensitive K + (K ATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in K ATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. Methods: Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known K ATP inhibitors in intact cells were determined by 86 Rb efflux assays. Results: Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP 4− inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the K ATP channel inhibitors glibenclamide and carbamazepine. Conclusions: The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect K ATP channel function to cause the disease. The reduced ATP 4− sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.

Original languageEnglish (US)
JournalPediatric Diabetes
DOIs
StatePublished - Jan 1 2019

Fingerprint

Sulfonylurea Receptors
Diabetes Mellitus
Adenosine Triphosphate
Mutation
Adenosine Diphosphate
Glyburide
Carbamazepine
Missense Mutation
Immunoblotting

Keywords

  • ABCC8
  • activating mutation
  • KCNJ11
  • neonatal diabetes mellitus
  • sulfonylurea

ASJC Scopus subject areas

  • Internal Medicine
  • Pediatrics, Perinatology, and Child Health
  • Endocrinology, Diabetes and Metabolism

Cite this

Functional characterization of activating mutations in the sulfonylurea receptor 1 (ABCC8) causing neonatal diabetes mellitus in Asian Indian children. / Balamurugan, Kandasamy; Kavitha, Babu; Yang, Zhongying; Mohan, Viswanathan; Radha, Venkatesan; Shyng, Show-Ling.

In: Pediatric Diabetes, 01.01.2019.

Research output: Contribution to journalArticle

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abstract = "Background: Gain-of-function of ATP-sensitive K + (K ATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in K ATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. Methods: Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known K ATP inhibitors in intact cells were determined by 86 Rb efflux assays. Results: Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP 4− inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the K ATP channel inhibitors glibenclamide and carbamazepine. Conclusions: The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect K ATP channel function to cause the disease. The reduced ATP 4− sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.",
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AU - Balamurugan, Kandasamy

AU - Kavitha, Babu

AU - Yang, Zhongying

AU - Mohan, Viswanathan

AU - Radha, Venkatesan

AU - Shyng, Show-Ling

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N2 - Background: Gain-of-function of ATP-sensitive K + (K ATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in K ATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. Methods: Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known K ATP inhibitors in intact cells were determined by 86 Rb efflux assays. Results: Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP 4− inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the K ATP channel inhibitors glibenclamide and carbamazepine. Conclusions: The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect K ATP channel function to cause the disease. The reduced ATP 4− sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.

AB - Background: Gain-of-function of ATP-sensitive K + (K ATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in K ATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. Methods: Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known K ATP inhibitors in intact cells were determined by 86 Rb efflux assays. Results: Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP 4− inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the K ATP channel inhibitors glibenclamide and carbamazepine. Conclusions: The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect K ATP channel function to cause the disease. The reduced ATP 4− sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.

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