Abnormalities of pancreatic islets by targeted expression of a dominant-negative K _ATP  channel

Abstract

ATP-sensitive K ؉ (K ATP ) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of K ATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the K ATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. K ATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the K ATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion. ATP-sensitive Kϩ (K ATP ) channels are thought to regulate various cellular functions such as secretion and muscle and neural excitability by linking the cell's metabolic state to its membrane potential (1-7). We have shown previously that K ATP channels comprise at least two subunits, a sulfonylurea receptor (SUR), which belongs to the ATP-binding cassette superfamily (8), and the inward rectifier K ϩ channel member Kir6.2 (9). SUR is thought to confer the ATP and sulfonylurea sensitivities of K ATP channels, and Kir6.2 is thought to form the K ϩ ion-selective pore (9-12). However, a recent study (13)suggests that Kir6.2 alone might confer ATP-sensitivity. Although mutations of SUR1, the other subunit of pancreatic beta cell K ATP (SUR1͞Kir6.2) channels, is known to cause familial persistent hyperinsulinemic hypoglycemia of infancy (PHHI) due to a loss of functional K ATP channels (14-18), the consequences of inhibition of Kir6.2 function are unknown.The putative K ϩ ion permeable domain (H5) is highly conserved in K ϩ channels (19), and the motif Gly-Tyr (or Phe)-Gly in H5 is thought to be critical for K ϩ ion selectivity (19)(20)(21). A substitution of the first residue of the Gly-Tyr-Gly motif with serine (residue 156) is found in the G-protein-gated inward rectifier GIRK2 (Kir3.2) of weaver mice (22). This mutation is responsible for their abnormalities of neuronal differentiation and development (23)(24)(25)(26). In the present study, we have replaced the Gly-312 with Ser at the corresponding position of Kir6.2 (Kir6.2G132S) by in vitro mutagenesis. We found by 86 Rb ϩ flux measurements that Kir6.2G132S functions as a dominant-negative mutant. We then generated transgenic mice expressing Kir6.2G132S in pancreatic beta cells and studied the morphological and functional changes in the pancreatic islets.

Hyperinsulinism induced by targeted suppression of beta cell K ATP channels

“…Whereas fura-4F fluorescence is largely targeted to the cytosol, cytosolic fluorescence of EGFP-tagged K ATP channels, measured separately under the same conditions, was relatively low (J.D.J., unpublished observations). [4][5][6][7][8][9][10][11][12][13][14], there was also no difference in serum insulin levels between AAA-TG and control littermates (8). However, analysis of serum insulin levels of fasted and fed adult mice (4-8 months) indicated consistently 2.5-fold higher levels in AAA-TG than in control littermates (Table 1, Fig.…”

“…The G132S mutation perturbs the structure of the K ϩ -selectivity filter of the channel and renders channels nonfunctional or, perhaps, slightly Na ϩ -permeable (10). Beta cells from these transgenic mice show reduced K ATP currents, accompanied by a significant increase in both the resting membrane potential and [Ca 2ϩ ] i (5). Similar to PHHI phenotypes in humans, they also exhibit hypoglycemia with hyperinsulinemia as neonates.…”

A mechanism for ATP-sensitive potassium channel diversity: Functional coassembly of two pore-forming subunits

“…The dominant negative effect arises because a mutant nonfunctional subunit can inactivate the function of wild-type subunits in a tetramer. Thus, mutations were introduced into the conserved GFG sequence in the pore of Kir 6.1 (GFG to AFA; 61GA and GFG to SFG; 61GS) and Kir 6.2 (GFG to AFA; 62GA) (19,20). To ensure potentially equal expression of wild-type and mutant subunits, these constructs were transfected transiently at equal concentration together with SUR2B, and whole-cell membrane currents were recorded by using the patchclamp technique.…”

Hyperinsulinism in mice with heterozygous loss of KATP channels

“…Mice completely lacking Kir6.2 or SUR1 [12][13][14], as well as mice expressing a dominantnegative Kir6.2 transgene in beta cells (Kir6. 2[AAA] [15] or Kir6.2[G132S] [16]) have been generated. Kir6.2 [AAA] mice (which exhibit complete loss of K ATP channels in ∼70% of beta cells, but normal channel density in the remainder) show hyperinsulinaemia, enhanced glucose tolerance and increased glucose-stimulated insulin secretion (GSIS) [15].…”