Abstract
The beta-cell ATP-sensitive potassium (K(ATP)) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for K(ATP) channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants.
Highlights
sulfonylurea receptor 1 (SUR1) and Kir6.2 co-assemble in the endoplasmic reticulum (ER) to form channel complexes; successful assembly overcomes the arginine-lysine-arginine (RKR) ER retention motif in SUR1 and Kir6.2 to permit channel trafficking to the plasma membrane [3, 4]
We have previously shown that channel trafficking defects caused by congenital hyperinsulinism (CHI) mutations in the TMD0 of SUR1 can be overcome by the oral hypoglycemic drugs sulfonylureas, which act as chemical chaperones to increase the biogenesis efficiency of these mutant channels [13,14,15]
The R74W and E128K Mutations Reduce Channel Sensitivity to ATP—Previously, we reported several CHI-associated SUR1 mutations that reduce surface expression of KATP channels could be rescued to the cell surface efficiently by sulfonylureas such as glibenclamide and tolbutamide [13, 15]
Summary
SUR1 and Kir6.2 co-assemble in the endoplasmic reticulum (ER) to form channel complexes; successful assembly overcomes the arginine-lysine-arginine (RKR) ER retention motif in SUR1 and Kir6.2 to permit channel trafficking to the plasma membrane [3, 4]. Characterized by persistent insulin secretion despite severe hypoglycemia in neonates and infants, CHI frequently results from loss of function KATP channel mutations [12] These mutations typically reduce channel activity by preventing channel expression at the cell surface, diminishing channel response to MgADP stimulation and/or reducing channel Po [2]. We report two mutants, R74W and E128K, which, upon rescue to the cell surface, surprisingly revealed reduced ATP sensitivity-gating defects Such defects are typically associated with PNDM mutations. Unlike previously reported ATP-insensitive mutants, which tend to have increased intrinsic Po, the R74W and E128K mutants showed reduced intrinsic Po. The finding suggests R74W and E128K diminish channel ATP sensitivity by a distinct mechanism that likely involves functional uncoupling between SUR1 and Kir6.2
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