Kir6.2 Mutations Associated With Neonatal Diabetes Reduce Expression of ATP-Sensitive K+ channels

Abstract

Heterozygous missense mutations in the pore-forming subunit Kir6.2 of ATP-sensitive K ؉ channels (K ATP channels) have recently been shown to cause permanent neonatal diabetes mellitus (PNDM). Functional studies demonstrated that PNDM mutations reduce K ATP channel sensitivity to ATP inhibition, resulting in gain of channel function. However, the impact of these mutations on channel expression has not been examined. Here, we show that PNDM mutations, including Q52R, V59G, V59M, R201C, R201H, and I296L, not only reduce channel ATP sensitivity but also impair channel expression at the cell surface to varying degrees. By tagging the PNDM Kir6.2 mutant V59G or R201H with an additional mutation, N160D, that confers voltage-dependent polyamine block of K ATP channels, we demonstrate that in simulated heterozygous state, all surface channels are either wild-type or heteromeric channels containing both wild-type and mutant Kir6.2 subunits. Comparison of the various PNDM mutations in their effects on channel nucleotide sensitivity and expression, as well as disease phenotype, suggests that both channel-gating defect and expression level may play a role in determining disease severity. Interestingly, sulfonylureas significantly increase surface expression of certain PNDM mutants, suggesting that the efficacy of sulfonylurea therapy may be compromised by the effect of these drugs on channel expression. Diabetes 55:1738 -1746, 2006 P ancreatic ATP-sensitive K ϩ channels (K ATP channels), each consisting of four pore-forming Kir6.2 subunits and four regulatory sulfonylurea receptor one (SUR1) subunits, link ␤-cell metabolism to insulin secretion (1-3). The activity of K ATP channels is governed mainly by the dynamics of intracellular adenine nucleotides ATP and ADP at the channel site during glucose metabolism (1,4). Both nucleotides can stimulate or inhibit channel activity depending on their relative concentrations and whether Mg 2ϩ is present. Inhibition of channels by nucleotides is mediated by the Kir6.2 subunit and does not require Mg 2ϩ (5,6), whereas nucleotide stimulation is conferred by the SUR1 subunit and requires Mg 2ϩ (7,8). The physiological activity of K ATP channels in ␤-cells is thus a balance between nucleotide inhibition and nucleotide stimulation. During glucose stimulation, ATP concentrations increase and ADP concentrations decrease, resulting in K ATP channel closure. Because K ATP channels carry the dominant conductance in high-input resistance ␤-cells at resting state, closure of K ATP channels leads to membrane depolarization, which in turn leads to opening of voltage-gated calcium channels, calcium influx, and insulin release.Recent studies have established heterozygous missense mutations in Kir6.2 as a major cause underlying permanent neonatal diabetes mellitus (PNDM) (9 -16). As Kir6.2 is also a constituent of K ATP channel subtypes expressed outside of pancreas, including cardiac muscle, skeletal muscle, and brain, some mutations have been reported to cause muscle weakness, dysmorphic features, ...

Dual role of K ATP channel C-terminal motif in membrane targeting and metabolic regulation

“…S8). In support of our findings, Shyng et al identified human Kir6.2 mutations with reduced membrane expression that also moderated severe gain-of-function phenotypes due to reduced ATP sensitivity (19). Specifically, Kir6.2 R201C and R201H display striking loss of ATP sensitivity (gain-of-function) compared with another PNDM mutation, Kir6.2 V59M.…”

“…Therefore, our data suggests that the dual molecular phenotype of defects in ATP sensitivity plus abnormal trafficking (i.e., fewer channels with gain-of-function properties) results in the ultimate PNDM clinical phenotype of E322K probands. These findings are consistent with recent findings of other neonatal diabetes mutations that show loss of channel density combined with reduced ATP sensitivity (19). While the mechanism(s) underlying dual phenotypes of other neonatal diabetes mutations are still unclear, our results strongly support dysfunction in ankyrin-based pathways as the mechanism underlying the Kir6.2 E322K human disease phenotype.…”

“…Specifically, Kir6.2 R201C and R201H display striking loss of ATP sensitivity (gain-of-function) compared with another PNDM mutation, Kir6.2 V59M. However, as both R201C and R201H also display reduced membrane expression phenotypes (loss-of-function), the two mutants manifest in much less severe PNDM disease phenotypes compared with V59M (19). Thus, defects in targeting may ameliorate the overall cellular phenotype associated with severe gain-of-function defects in channel gating.…”

Structurally Distinct Ligands Rescue Biogenesis Defects of the KATP Channel Complex via a Converging Mechanism

“…Mutant clones from multiple PCRs were sequenced and used to avoid undesired mutations introduced elsewhere during PCR. Construction of adenoviruses carrying K ATP subunit cDNA was as described previously (8).…”

“…Viral expression was done as described previously (8). Membranes were prepared 48 h postinfection according to the following procedure.…”